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Roach SN, Shepherd FK, Mickelson CK, Fiege JK, Thielen BK, Pross LM, Sanders AE, Mitchell JS, Robertson M, Fife BT, Langlois RA. Tropism for ciliated cells is the dominant driver of influenza viral burst size in the human airway. Proc Natl Acad Sci U S A 2024; 121:e2320303121. [PMID: 39008691 PMCID: PMC11295045 DOI: 10.1073/pnas.2320303121] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2023] [Accepted: 05/26/2024] [Indexed: 07/17/2024] Open
Abstract
Influenza viruses pose a significant burden on global human health. Influenza has a broad cellular tropism in the airway, but how infection of different epithelial cell types impacts replication kinetics and burden in the airways is not fully understood. Using primary human airway cultures, which recapitulate the diverse epithelial cell landscape of the human airways, we investigated the impact of cell type composition on virus tropism and replication kinetics. Cultures were highly diverse across multiple donors and 30 independent differentiation conditions and supported a range of influenza replication. Although many cell types were susceptible to influenza, ciliated and secretory cells were predominantly infected. Despite the strong tropism preference for secretory and ciliated cells, which consistently make up 75% or more of infected cells, only ciliated cells were associated with increased virus production. Surprisingly, infected secretory cells were associated with overall reduced virus output. The disparate response and contribution to influenza virus production could be due to different pro- and antiviral interferon-stimulated gene signatures between ciliated and secretory populations, which were interrogated with single-cell RNA sequencing. These data highlight the heterogeneous outcomes of influenza virus infections in the complex cellular environment of the human airway and the disparate impacts of infected cell identity on multiround burst size, even among preferentially infected cell types.
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Affiliation(s)
- Shanley N. Roach
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN55455
| | - Frances K. Shepherd
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN55455
| | - Clayton K. Mickelson
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN55455
| | - Jessica K. Fiege
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN55455
| | - Beth K. Thielen
- Division of Pediatric Infectious Diseases and Immunology, Department of Pediatrics, University of Minnesota, Minneapolis, MN55455
| | - Lauren M. Pross
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN55455
| | - Autumn E. Sanders
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN55455
| | - Jason S. Mitchell
- Center for Immunology, University of Minnesota, Minneapolis, MN55455
| | - Mason Robertson
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN55455
| | - Brian T. Fife
- Center for Immunology, University of Minnesota, Minneapolis, MN55455
- Department of Medicine, University of Minnesota, Minneapolis, MN55455
| | - Ryan A. Langlois
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN55455
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2
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Hamele CE, Spurrier MA, Leonard RA, Heaton NS. Segmented, Negative-Sense RNA Viruses of Humans: Genetic Systems and Experimental Uses of Reporter Strains. Annu Rev Virol 2023; 10:261-282. [PMID: 37774125 PMCID: PMC10795101 DOI: 10.1146/annurev-virology-111821-120445] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/01/2023]
Abstract
Negative-stranded RNA viruses are a large group of viruses that encode their genomes in RNA across multiple segments in an orientation antisense to messenger RNA. Their members infect broad ranges of hosts, and there are a number of notable human pathogens. Here, we examine the development of reverse genetic systems as applied to these virus families, emphasizing conserved approaches illustrated by some of the prominent members that cause significant human disease. We also describe the utility of their genetic systems in the development of reporter strains of the viruses and some biological insights made possible by their use. To conclude the review, we highlight some possible future uses of reporter viruses that not only will increase our basic understanding of how these viruses replicate and cause disease but also could inform the development of new approaches to therapeutically intervene.
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Affiliation(s)
- Cait E Hamele
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, North Carolina, USA;
| | - M Ariel Spurrier
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, North Carolina, USA;
| | - Rebecca A Leonard
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, North Carolina, USA;
| | - Nicholas S Heaton
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, North Carolina, USA;
- Duke Human Vaccine Institute, Duke University School of Medicine, Durham, North Carolina, USA
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3
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Xu L, Paine AC, Barbeau DJ, Alencastro F, Duncan AW, McElroy AK. Limiting viral replication in hepatocytes alters Rift Valley fever virus disease manifestations. J Virol 2023; 97:e0085323. [PMID: 37695055 PMCID: PMC10537571 DOI: 10.1128/jvi.00853-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2023] [Accepted: 07/13/2023] [Indexed: 09/12/2023] Open
Abstract
Rift Valley fever virus (RVFV) causes mild to severe disease in humans and livestock. Outbreaks of RVFV have been reported throughout Africa and have spread outside Africa since 2000, calling for urgent worldwide attention to this emerging virus. RVFV directly infects the liver, and elevated transaminases are a hallmark of severe RVFV infection. However, the specific contribution of viral replication in hepatocytes to pathogenesis of RVFV remains undefined. To address this, we generated a recombinant miRNA-targeted virus, RVFVmiR-122, to limit hepatocellular replication. MicroRNAs are evolutionarily conserved non-coding RNAs that regulate mRNA expression by targeting them for degradation. RVFVmiR-122 includes an insertion of four target sequences of the liver-specific miR-122. In contrast to control RVFVmiR-184, which contains four target sequences of mosquito-specific miR-184, RVFVmiR-122 has restricted replication in vitro in primary mouse hepatocytes. RVFVmiR-122-infected C57BL/6 mice survived acute hepatitis and instead developed late-onset encephalitis. This difference in clinical outcome was eliminated in Mir-122 KO mice, confirming the specificity of the finding. Interestingly, C57BL/6 mice infected with higher doses of RVFVmiR-122 had a higher survival rate which was correlated with faster clearance of virus from the liver, suggesting a role for activation of host immunity in the phenotype. Together, our data demonstrate that miR-122 can specifically restrict the replication of RVFVmiR-122 in liver tissue both in vitro and in vivo, and this restriction alters the clinical course of disease following RVFVmiR-122 infection. IMPORTANCE Rift Valley fever virus (RVFV) is a hemorrhagic fever virus that causes outbreaks in humans and livestock throughout Africa and has spread to continents outside Africa since 2000. However, no commercial vaccine or treatment is currently available for human use against RVFV. Although the liver has been demonstrated as a key target of RVFV, the contribution of viral replication in hepatocytes to overall RVFV pathogenesis is less well defined. In this study we addressed this question by using a recombinant miRNA-targeted virus with restricted replication in hepatocytes. We gained a better understanding of how this individual cell type contributes to the development of disease caused by RVFV. Techniques used in this study provide an innovative tool to the RVFV field that could be applied to study the consequences of limited RVFV replication in other target cells.
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Affiliation(s)
- Lingqing Xu
- Department of Pediatrics, Division of Pediatric Infectious Disease, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
- Center for Vaccine Research, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
| | - Alden C. Paine
- Department of Pediatrics, Division of Pediatric Infectious Disease, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
- Center for Vaccine Research, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
| | - Dominique J. Barbeau
- Department of Pediatrics, Division of Pediatric Infectious Disease, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
- Center for Vaccine Research, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
| | - Frances Alencastro
- Department of Pathology, McGowan Institute for Regenerative Medicine, Pittsburgh Liver Research Center, UPMC Hillman Cancer Center, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
- Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
| | - Andrew W. Duncan
- Department of Pathology, McGowan Institute for Regenerative Medicine, Pittsburgh Liver Research Center, UPMC Hillman Cancer Center, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
- Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
| | - Anita K. McElroy
- Department of Pediatrics, Division of Pediatric Infectious Disease, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
- Center for Vaccine Research, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
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4
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Tan S, Wu W, Ge X, Zhang Y, Han J, Guo X, Zhou L, Yang H. A novel strategy to attenuate porcine reproductive and respiratory syndrome virus by inhibiting viral replication in the target pulmonary alveolar macrophages via hematopoietic-specific miR-142. ONE HEALTH ADVANCES 2023; 1:3. [PMID: 37521530 PMCID: PMC10060136 DOI: 10.1186/s44280-023-00002-2] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 01/11/2023] [Revised: 01/18/2023] [Accepted: 01/30/2023] [Indexed: 08/01/2023]
Abstract
Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen for the global pork industry. Although modified live virus (MLV) vaccines are commonly used for PRRSV prevention and control, they still carry a risk of infecting the host and replicating in target cells, thereby increasing the likehood of virus recombination and reversion to virulence. In this study, we inserted the target sequence of miR-142 into the nsp2 hypervariable region of PRRSV to inhibit viral replication in its host cells of pigs, with the aim of achieving virus attenuation. The chimeric virus RvJX-miR-142t was successfully rescued and retained its growth characteristics in MARC-145 cells. Furthermore, it did not replicate in MARC-145 cells transfected with miRNA-142 mimic. We also observed limited replication ability of RvJX-miR-142t in pulmonary alveolar macrophages, which are the main cell types that PRRSV infects. Our animal inoculation study showed that pigs infected with RvJX-miR-142t displayed less severe clinical symptoms, lower viremia titers, lighter lung lesions, and significantly lower mortality rates during the first 7 days post-inoculation, in comparison to pigs infected with the backbone virus RvJXwn. We detected a partially deletion of the miR-142 target sequence in the RvJX-miR-142t genome at 14 dpi. It is highly possible that the reversion of viral virulence observed in the later timepoints of our animal experiment was caused by that. Our study provided a new strategy for attenuating PRRSV and confirmed its effectiveness. However, further studies are necessary to increase the stability of this virus under host selection pressure.
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Affiliation(s)
- Shaoyuan Tan
- Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, TN 38105 USA
| | - Weixin Wu
- National Key Laboratory of Veterinary Public Health Security, College of Veterinary Medicine, China Agricultural University, Beijing, China
- Key Laboratory of Animal Epidemiology of Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing, China
| | - Xinna Ge
- National Key Laboratory of Veterinary Public Health Security, College of Veterinary Medicine, China Agricultural University, Beijing, China
- Key Laboratory of Animal Epidemiology of Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing, China
| | - Yongning Zhang
- National Key Laboratory of Veterinary Public Health Security, College of Veterinary Medicine, China Agricultural University, Beijing, China
- Key Laboratory of Animal Epidemiology of Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing, China
| | - Jun Han
- National Key Laboratory of Veterinary Public Health Security, College of Veterinary Medicine, China Agricultural University, Beijing, China
- Key Laboratory of Animal Epidemiology of Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing, China
| | - Xin Guo
- National Key Laboratory of Veterinary Public Health Security, College of Veterinary Medicine, China Agricultural University, Beijing, China
- Key Laboratory of Animal Epidemiology of Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing, China
| | - Lei Zhou
- National Key Laboratory of Veterinary Public Health Security, College of Veterinary Medicine, China Agricultural University, Beijing, China
- Key Laboratory of Animal Epidemiology of Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing, China
| | - Hanchun Yang
- National Key Laboratory of Veterinary Public Health Security, College of Veterinary Medicine, China Agricultural University, Beijing, China
- Key Laboratory of Animal Epidemiology of Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing, China
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5
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Martin BE, Taylor EB, Attipoe EM, Wu W, Stec DE, Showmaker KC, Garrett MR. Sex and molecular differences in cardiovascular parameters at peak influenza disease in mice. Physiol Genomics 2023; 55:79-89. [PMID: 36645670 PMCID: PMC9925171 DOI: 10.1152/physiolgenomics.00146.2022] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2022] [Revised: 12/29/2022] [Accepted: 01/09/2023] [Indexed: 01/17/2023] Open
Abstract
There is a growing interest in the detection of subtle changes in cardiovascular physiology in response to viral infection to develop better disease surveillance strategies. This is not only important for earlier diagnosis and better prognosis of symptomatic carriers but also useful to diagnose asymptomatic carriers of the virus. Previous studies provide strong evidence of an association between inflammatory biomarker levels and both blood pressure (BP) and heart rate (HR) during infection. The identification of novel biomarkers during an inflammatory event could significantly improve predictions for cardiovascular events. Thus, we evaluated changes in cardiovascular physiology induced in A/Puerto Rico/8/34 (PR8) influenza infections in female and male C57BL/6J mice and compared them with the traditional method of influenza disease detection using body weight (BW). Using radiotelemetry, changes in BP, HR, and activity were studied. Change in BW of infected females was significantly decreased from 5 to 13 days postinfection (dpi), yet alterations in normal physiology including loss of diurnal rhythm and reduced activity was observed starting at about 3 dpi for HR and 4 dpi for activity and BP; continuing until about 13 dpi. In contrast, males had significantly decreased BW 8 to 12 dpi and demonstrated altered physiological measurements for a shorter period compared with females with a reduction starting at 5 dpi for activity, 6 dpi for BP, and 7 dpi for HR until about 12 dpi, 10 dpi, and 9 dpi, respectively. Finally, females and males exhibited different patterns of inflammatory maker expression in lungs at peak disease by analyzing bulk RNA-sequencing data for lungs and Bio-plex cytokine assay for blood collected from influenza-infected and naïve C57BL/6J female and male mice at 7 dpi. In total, this study provides insight into cardiovascular changes and molecular markers to distinguish sex differences in peak disease caused by influenza virus infection.NEW & NOTEWORTHY This study performed longitudinal cardiovascular measurements of influenza viral infection and identified sex difference in both physiological and molecular markers at peak disease.
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Affiliation(s)
- Brigitte E Martin
- Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi
| | - Erin B Taylor
- Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, Mississippi
| | - Esinam M Attipoe
- Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi
| | - Wenjie Wu
- Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi
| | - David E Stec
- Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, Mississippi
| | | | - Michael R Garrett
- Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi
- Division of Nephrology, Department of Medicine, University of Mississippi Medical Center, Jackson, Mississippi
- Division of Genetics, Department of Pediatrics, University of Mississippi Medical Center, Jackson, Mississippi
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6
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Kenney AD, Aron SL, Gilbert C, Kumar N, Chen P, Eddy A, Zhang L, Zani A, Vargas-Maldonado N, Speaks S, Kawahara J, Denz PJ, Dorn L, Accornero F, Ma J, Zhu H, Rajaram MVS, Cai C, Langlois RA, Yount JS. Influenza virus replication in cardiomyocytes drives heart dysfunction and fibrosis. SCIENCE ADVANCES 2022; 8:eabm5371. [PMID: 35544568 PMCID: PMC9094651 DOI: 10.1126/sciadv.abm5371] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 09/24/2021] [Accepted: 03/24/2022] [Indexed: 05/04/2023]
Abstract
Cardiac dysfunction is a common complication of severe influenza virus infection, but whether this occurs due to direct infection of cardiac tissue or indirectly through systemic lung inflammation remains unclear. To test the etiology of this aspect of influenza disease, we generated a novel recombinant heart-attenuated influenza virus via genome incorporation of target sequences for miRNAs expressed in cardiomyocytes. Compared with control virus, mice infected with miR-targeted virus had significantly reduced heart viral titers, confirming cardiac attenuation of viral replication. However, this virus was fully replicative in the lungs and induced similar systemic inflammation and weight loss compared to control virus. The miR-targeted virus induced fewer cardiac conduction irregularities and significantly less fibrosis in mice lacking interferon-induced transmembrane protein 3 (IFITM3), which serve as a model for influenza-associated cardiac pathology. We conclude that robust virus replication in the heart is required for pathology, even when lung inflammation is severe.
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Affiliation(s)
- Adam D. Kenney
- Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH, USA
- Infectious Diseases Institute, Viruses and Emerging Pathogens Program, The Ohio State University, Columbus, OH, USA
| | - Stephanie L. Aron
- Department of Microbiology and Immunology, The University of Minnesota, Minneapolis, MN, USA
| | - Clara Gilbert
- Department of Microbiology and Immunology, The University of Minnesota, Minneapolis, MN, USA
| | - Naresh Kumar
- Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH, USA
| | - Peng Chen
- Department of Physiology and Cell Biology, The Ohio State University, Columbus, OH, USA
| | - Adrian Eddy
- Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH, USA
- Infectious Diseases Institute, Viruses and Emerging Pathogens Program, The Ohio State University, Columbus, OH, USA
| | - Lizhi Zhang
- Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH, USA
- Infectious Diseases Institute, Viruses and Emerging Pathogens Program, The Ohio State University, Columbus, OH, USA
| | - Ashley Zani
- Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH, USA
- Infectious Diseases Institute, Viruses and Emerging Pathogens Program, The Ohio State University, Columbus, OH, USA
| | - Nahara Vargas-Maldonado
- Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH, USA
| | - Samuel Speaks
- Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH, USA
| | - Jeffrey Kawahara
- Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH, USA
- Infectious Diseases Institute, Viruses and Emerging Pathogens Program, The Ohio State University, Columbus, OH, USA
| | - Parker J. Denz
- Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH, USA
- Infectious Diseases Institute, Viruses and Emerging Pathogens Program, The Ohio State University, Columbus, OH, USA
| | - Lisa Dorn
- Department of Physiology and Cell Biology, The Ohio State University, Columbus, OH, USA
| | - Federica Accornero
- Department of Physiology and Cell Biology, The Ohio State University, Columbus, OH, USA
| | - Jianjie Ma
- Department of Surgery, The Ohio State University, Columbus, OH, USA
| | - Hua Zhu
- Department of Surgery, The Ohio State University, Columbus, OH, USA
| | - Murugesan V. S. Rajaram
- Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH, USA
| | - Chuanxi Cai
- Department of Surgery, The Ohio State University, Columbus, OH, USA
| | - Ryan A. Langlois
- Department of Microbiology and Immunology, The University of Minnesota, Minneapolis, MN, USA
| | - Jacob S. Yount
- Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH, USA
- Infectious Diseases Institute, Viruses and Emerging Pathogens Program, The Ohio State University, Columbus, OH, USA
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7
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Characterization of influenza A virus induced transposons reveals a subgroup of transposons likely possessing the regulatory role as eRNAs. Sci Rep 2022; 12:2188. [PMID: 35140280 PMCID: PMC8828846 DOI: 10.1038/s41598-022-06196-6] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2021] [Accepted: 01/25/2022] [Indexed: 01/02/2023] Open
Abstract
Although many studies have observed genome-wide host transposon expression alteration during viral infection, the mechanisms of induction and the impact on the host remain unclear. Utilizing recently published influenza A virus (IAV) time series data and ENCODE functional genomics data, we characterized virus induced host differentially expressed transposons (virus-induced-TE) by investigating genome-wide spatial and functional relevance between the virus-induced-TEs and epigenomic markers (e.g. histone modification and chromatin remodelers). We found that a significant fraction of virus-induced-TEs are derived from host enhancer regions, where CHD4 binding and/or H3K27ac occupancy is high or H3K9me3 occupancy is low. By overlapping virus-induced-TEs to human enhancer RNAs (eRNAs), we discovered that a proportion of virus-induced-TEs are either eRNAs or part of enhancer RNAs. Upon further analysis of the eRNA targeted genes, we found that the virus-induced-TE related eRNA targets are overrepresented in differentially expressed host genes of IAV infected samples. Our results suggest that changing chromatin accessibility from repressive to permissive in the transposon docked enhancer regions to regulate host downstream gene expression is potentially one of the virus and host cell interaction mechanisms, where transposons are likely important regulatory genomic elements. Our study provides a new insight into the mechanisms of virus-host interaction and may lead to novel strategies for prevention and therapeutics of IAV and other virus infectious diseases.
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8
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Fiege JK, Block KE, Pierson MJ, Nanda H, Shepherd FK, Mickelson CK, Stolley JM, Matchett WE, Wijeyesinghe S, Meyerholz DK, Vezys V, Shen SS, Hamilton SE, Masopust D, Langlois RA. Mice with diverse microbial exposure histories as a model for preclinical vaccine testing. Cell Host Microbe 2021; 29:1815-1827.e6. [PMID: 34731647 DOI: 10.1016/j.chom.2021.10.001] [Citation(s) in RCA: 33] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2021] [Revised: 08/30/2021] [Accepted: 10/08/2021] [Indexed: 12/13/2022]
Abstract
Laboratory mice comprise an expeditious model for preclinical vaccine testing; however, vaccine immunogenicity in these models often inadequately translates to humans. Reconstituting physiologic microbial experience to specific pathogen-free (SPF) mice induces durable immunological changes that better recapitulate human immunity. We examined whether mice with diverse microbial experience better model human responses post vaccination. We co-housed laboratory mice with pet-store mice, which have varied microbial exposures, and then assessed immune responses to influenza vaccines. Human transcriptional responses to influenza vaccination are better recapitulated in co-housed mice. Although SPF and co-housed mice were comparably susceptible to acute influenza infection, vaccine-induced humoral responses were dampened in co-housed mice, resulting in poor control upon challenge. Additionally, protective heterosubtypic T cell immunity was compromised in co-housed mice. Because SPF mice exaggerated humoral and T cell protection upon influenza vaccination, reconstituting microbial experience in laboratory mice through co-housing may better inform preclinical vaccine testing.
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Affiliation(s)
- Jessica K Fiege
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN 55455, USA; Center for Immunology, University of Minnesota, Minneapolis, MN 55455, USA
| | - Katharine E Block
- Center for Immunology, University of Minnesota, Minneapolis, MN 55455, USA; Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455, USA
| | - Mark J Pierson
- Center for Immunology, University of Minnesota, Minneapolis, MN 55455, USA; Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455, USA
| | - Hezkiel Nanda
- Institute for Health Informatics, University of Minnesota, Minneapolis, MN 55455, USA
| | - Frances K Shepherd
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN 55455, USA; Center for Immunology, University of Minnesota, Minneapolis, MN 55455, USA
| | - Clayton K Mickelson
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN 55455, USA; Center for Immunology, University of Minnesota, Minneapolis, MN 55455, USA
| | - J Michael Stolley
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN 55455, USA; Center for Immunology, University of Minnesota, Minneapolis, MN 55455, USA
| | - William E Matchett
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN 55455, USA; Center for Immunology, University of Minnesota, Minneapolis, MN 55455, USA
| | - Sathi Wijeyesinghe
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN 55455, USA; Center for Immunology, University of Minnesota, Minneapolis, MN 55455, USA
| | - David K Meyerholz
- Department of Pathology, University of Iowa, Iowa City, IA 52242, USA
| | - Vaiva Vezys
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN 55455, USA; Center for Immunology, University of Minnesota, Minneapolis, MN 55455, USA
| | - Steven S Shen
- Institute for Health Informatics, University of Minnesota, Minneapolis, MN 55455, USA
| | - Sara E Hamilton
- Center for Immunology, University of Minnesota, Minneapolis, MN 55455, USA; Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455, USA.
| | - David Masopust
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN 55455, USA; Center for Immunology, University of Minnesota, Minneapolis, MN 55455, USA.
| | - Ryan A Langlois
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN 55455, USA; Center for Immunology, University of Minnesota, Minneapolis, MN 55455, USA.
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9
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Jenkins MM, Bachus H, Botta D, Schultz MD, Rosenberg AF, León B, Ballesteros-Tato A. Lung dendritic cells migrate to the spleen to prime long-lived TCF1 hi memory CD8 + T cell precursors after influenza infection. Sci Immunol 2021; 6:eabg6895. [PMID: 34516781 DOI: 10.1126/sciimmunol.abg6895] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
[Figure: see text].
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Affiliation(s)
- Meagan M Jenkins
- Division of Clinical Immunology and Rheumatology Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Holly Bachus
- Division of Clinical Immunology and Rheumatology Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Davide Botta
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Michael D Schultz
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Alexander F Rosenberg
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA.,Informatics Institute, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Beatriz León
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA
| | - André Ballesteros-Tato
- Division of Clinical Immunology and Rheumatology Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA
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10
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Felgenhauer JL, Brune JE, Long ME, Manicone AM, Chang MY, Brabb TL, Altemeier WA, Frevert CW. Evaluation of Nutritional Gel Supplementation in C57BL/6J Mice Infected with Mouse-Adapted Influenza A/PR/8/34 Virus. Comp Med 2020; 70:471-486. [PMID: 33323164 PMCID: PMC7754200 DOI: 10.30802/aalas-cm-20-990138] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2019] [Revised: 11/05/2019] [Accepted: 09/04/2020] [Indexed: 12/11/2022]
Abstract
Mice are a common animal model for the study of influenza virus A (IAV). IAV infection causes weight loss due to anorexia and dehydration, which can result in early removal of mice from a study when they reach a humane endpoint. To reduce the number of mice prematurely removed from an experiment, we assessed nutritional gel (NG) supplementation as a support strategy for mice infected with mouse-adapted Influenza A/Puerto Rico/8/34 (A/PR/8/34; H1N1) virus. We hypothesized that, compared with the standard of care (SOC), supplementation with NG would reduce weight loss and increase survival in mice infected with IAV without impacting the initial immune response to infection. To assess the effects of NG, male and female C57BL/6J mice were infected with IAV at low, intermediate, or high doses. When compared with SOC, mice given NG showed a significant decrease in the maximal percent weight loss at all viral doses in males and at the intermediate dose for females. Mice supplemented with NG had no deaths for either sex at the intermediate dose and a significant increase in survival in males at the high viral dose. Supplementation with NG did not alter the viral titer or the pulmonary recruitment of immune cells as measured by cell counts and flow cytometry of cells recovered in bronchoalveolar lavage (BAL) fluid in either sex. However, mice given NG had a significant reduction in IL6 and TNFα in BAL fluid and no significant differences in CCL2, IL4, IL10, CXCL1, CXCL2, and VEGF. The results of this study show that as compared with infected SOC mice, infected mice supplemented with NG have reduced weight loss and increased survival, with males showing a greater benefit. These results suggest that NG should be considered as a support strategy and indicate that sex is an important biologic variable in mice infected with IAV.
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Key Words
- iav, influenza a virus
- soc, standard of care
- ng, nutritional gel
- eud50, euthanasia dose 50
- ld50, lethal-dose 50
- pfu, plaque forming unit
- dpi, days post infection
- il6, interleukin 6
- sem, standard error of mean
- ns, no significance
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Affiliation(s)
- Jessica L Felgenhauer
- Department of Comparative Medicine, University of Washington, Seattle, Washington; Center for Lung Biology, University of Washington at South Lake Union, Seattle, Washington
| | - Jourdan E Brune
- Department of Comparative Medicine, University of Washington, Seattle, Washington; Center for Lung Biology, University of Washington at South Lake Union, Seattle, Washington
| | - Matthew E Long
- Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, University of Washington, Seattle, Washington; Center for Lung Biology, University of Washington at South Lake Union, Seattle, Washington
| | - Anne M Manicone
- Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, University of Washington, Seattle, Washington; Center for Lung Biology, University of Washington at South Lake Union, Seattle, Washington
| | - Mary Y Chang
- Department of Comparative Medicine, University of Washington, Seattle, Washington; Center for Lung Biology, University of Washington at South Lake Union, Seattle, Washington
| | - Thea L Brabb
- Department of Comparative Medicine, University of Washington, Seattle, Washington; Center for Lung Biology, University of Washington at South Lake Union, Seattle, Washington
| | - William A Altemeier
- Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, University of Washington, Seattle, Washington; Center for Lung Biology, University of Washington at South Lake Union, Seattle, Washington
| | - Charles W Frevert
- Department of Comparative Medicine, University of Washington, Seattle, Washington; Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, University of Washington, Seattle, Washington; Center for Lung Biology, University of Washington at South Lake Union, Seattle, Washington;,
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11
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tenOever BR. Synthetic Virology: Building Viruses to Better Understand Them. Cold Spring Harb Perspect Med 2020; 10:cshperspect.a038703. [PMID: 31871242 DOI: 10.1101/cshperspect.a038703] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Generally comprised of less than a dozen components, RNA viruses can be viewed as well-designed genetic circuits optimized to replicate and spread within a given host. Understanding the molecular design that enables this activity not only allows one to disrupt these circuits to study their biology, but it provides a reprogramming framework to achieve novel outputs. Recent advances have enabled a "learning by building" approach to better understand virus biology and create valuable tools. Below is a summary of how modifying the preexisting genetic framework of influenza A virus has been used to track viral movement, understand virus replication, and identify host factors that engage this viral circuitry.
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Affiliation(s)
- Benjamin R tenOever
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA
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12
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Forsyth KS, Roy NH, Peauroi E, DeHaven BC, Wold ED, Hersperger AR, Burkhardt JK, Eisenlohr LC. Ectromelia-encoded virulence factor C15 specifically inhibits antigen presentation to CD4+ T cells post peptide loading. PLoS Pathog 2020; 16:e1008685. [PMID: 32745153 PMCID: PMC7425992 DOI: 10.1371/journal.ppat.1008685] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2020] [Revised: 08/13/2020] [Accepted: 06/06/2020] [Indexed: 01/02/2023] Open
Abstract
Smallpox and monkeypox pose severe threats to human health. Other orthopoxviruses are comparably virulent in their natural hosts, including ectromelia, the cause of mousepox. Disease severity is linked to an array of immunomodulatory proteins including the B22 family, which has homologs in all pathogenic orthopoxviruses but not attenuated vaccine strains. We demonstrate that the ectromelia B22 member, C15, is necessary and sufficient for selective inhibition of CD4+ but not CD8+ T cell activation by immunogenic peptide and superantigen. Inhibition is achieved not by down-regulation of surface MHC- II or co-stimulatory protein surface expression but rather by interference with antigen presentation. The appreciable outcome is interference with CD4+ T cell synapse formation as determined by imaging studies and lipid raft disruption. Consequently, CD4+ T cell activating stimulus shifts to uninfected antigen-presenting cells that have received antigen from infected cells. This work provides insight into the immunomodulatory strategies of orthopoxviruses by elucidating a mechanism for specific targeting of CD4+ T cell activation, reflecting the importance of this cell type in control of the virus.
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Affiliation(s)
- Katherine S. Forsyth
- Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America
| | - Nathan H. Roy
- Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America
| | - Elise Peauroi
- Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America
| | - Brian C. DeHaven
- Department of Biology, La Salle University, Philadelphia, Pennsylvania, United States of America
| | - Erik D. Wold
- Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America
| | - Adam R. Hersperger
- Department of Biology, Albright College, Reading, Pennsylvania, United States of America
| | - Janis K. Burkhardt
- Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America
- Children's Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania, United States of America
| | - Laurence C. Eisenlohr
- Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America
- Children's Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania, United States of America
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13
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Tsetsarkin KA, Acklin JA, Liu G, Kenney H, Teterina NL, Pletnev AG, Lim JK. Zika virus tropism during early infection of the testicular interstitium and its role in viral pathogenesis in the testes. PLoS Pathog 2020; 16:e1008601. [PMID: 32614902 PMCID: PMC7331987 DOI: 10.1371/journal.ppat.1008601] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2019] [Accepted: 05/05/2020] [Indexed: 12/18/2022] Open
Abstract
Sexual transmission and persistence of Zika virus (ZIKV) in the testes pose new challenges for controlling virus outbreaks and developing live-attenuated vaccines. It has been shown that testicular infection of ZIKV is initiated in the testicular interstitium, followed by spread of the virus in the seminiferous tubules. This leads to testicular damage and/or viral dissemination into the epididymis and eventually into semen. However, it remains unknown which cell types are targeted by ZIKV in the testicular interstitium, and what is the specific order of infectious events leading to ZIKV invasion of the seminiferous tubules. Here, we demonstrate that interstitial leukocytes expressing mir-511-3p microRNA are the initial targets of ZIKV in the testes, and infection of mir-511-3p-expressing cells in the testicular interstitium is necessary for downstream infection of the seminiferous tubules. Mir-511-3p is expressed concurrently with CD206, a marker of lineage 2 (M2) macrophages and monocyte derived dendritic cells (moDCs). Selective restriction of ZIKV infection of CD206-expressing M2 macrophages/moDCs results in the attenuation of macrophage-associated inflammatory responses in vivo and prevents the disruption of the Sertoli cell barrier in vitro. Finally, we show that targeting of viral genome for mir-511-3p significantly attenuates early ZIKV replication not only in the testes, but also in many peripheral organs, including spleen, epididymis, and pancreas. This incriminates M2 macrophages/moDCs as important targets for visceral ZIKV replication following hematogenous dissemination of the virus from the site of infection.
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Affiliation(s)
- Konstantin A. Tsetsarkin
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, United States of America
| | - Joshua A. Acklin
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America
- Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America
| | - Guangping Liu
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, United States of America
| | - Heather Kenney
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, United States of America
| | - Natalia L. Teterina
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, United States of America
| | - Alexander G. Pletnev
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, United States of America
| | - Jean K. Lim
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America
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14
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Wu W, Metcalf JP. The Role of Type I IFNs in Influenza: Antiviral Superheroes or Immunopathogenic Villains? J Innate Immun 2020; 12:437-447. [PMID: 32564033 PMCID: PMC7747089 DOI: 10.1159/000508379] [Citation(s) in RCA: 47] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2020] [Accepted: 05/03/2020] [Indexed: 12/29/2022] Open
Abstract
The important role of interferons (IFNs) in antiviral innate immune defense is well established. Although recombinant IFN-α was approved for cancer and chronic viral infection treatment by regulatory agencies in many countries starting in 1986, no IFNs are approved for treatment of influenza A virus (IAV) infection. This is partially due to the complex effects of IFNs in acute influenza infection. IAV attacks the human respiratory system and causes significant morbidity and mortality globally. During influenza infection, depending on the strain of IAV and the individual host, type I IFNs can have protective antiviral effects or can contribute to immunopathology. In the context of virus infection, the immune system has complicated mechanisms regulating the expression and effects of type I IFN to maximize the antiviral response by both activating and enhancing beneficial innate cell function, while limiting immunopathological responses that lead to exaggerated tissue damage. In this review, we summarize the complicated, but important, role of type I IFNs in influenza infections. This includes both protective and harmful effects of these important cytokines during infection.
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Affiliation(s)
- Wenxin Wu
- Department of Medicine, Pulmonary, Critical Care and Sleep Medicine, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA,
| | - Jordan P Metcalf
- Department of Medicine, Pulmonary, Critical Care and Sleep Medicine, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA
- Department of Microbiology and Immunology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA
- Pulmonary Section, Medicine Service, Veterans Affairs Medical Center, Oklahoma City, Oklahoma, USA
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15
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MiRNA Targeted NP Genome of Live Attenuated Influenza Vaccines Provide Cross-Protection against a Lethal Influenza Virus Infection. Vaccines (Basel) 2020; 8:vaccines8010065. [PMID: 32028575 PMCID: PMC7158662 DOI: 10.3390/vaccines8010065] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2019] [Revised: 01/31/2020] [Accepted: 02/01/2020] [Indexed: 12/20/2022] Open
Abstract
The miRNA-based strategy has been used to develop live attenuated influenza vaccines. In this study, the nucleoprotein (NP) genome segment of the influenza virus was inserted by different perfect miRNA-192-5p target sites, and the virus was rescued by standard reverse genetics method, so as to verify the virulence and protective efficacy of live attenuated vaccine in cells and mice. The results showed there was no significant attenuation in 192t virus with one perfect miRNA-192-5p target site, and 192t-3 virus with three perfect miRNA target sites. However, 192t-6 virus with 6 perfect miRNA target sites and 192t-9 virus with 9 perfect miRNA target sites were both significantly attenuated after infection, and their virulence were similar to that of temperature-sensitive (TS) influenza A virus (IAV) which is a temperature-sensitive live attenuated influenza vaccine. Mice were immunized with different doses of 192t-6, 192t-9, and TS IAV. Four weeks after immunization, the IgG in serum and IgA in lung homogenate were increased in the 192t-6, 192t-9, and TS IAV groups, and the numbers of IFN-γ secreting splenocytes were also increased in a dose-dependent manner. Finally, 192t-6, and 192t-9 can protect the mice against the challenge of homologous PR8 H1N1 virus and heterosubtypic H3N2 influenza virus. MiRNA targeted viruses 192t-6 and 192t-9 were significantly attenuated and showed the same virulence as TS IAV and played a role in the cross-protection.
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16
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Hemagglutinin Stability Regulates H1N1 Influenza Virus Replication and Pathogenicity in Mice by Modulating Type I Interferon Responses in Dendritic Cells. J Virol 2020; 94:JVI.01423-19. [PMID: 31694942 DOI: 10.1128/jvi.01423-19] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2019] [Accepted: 11/03/2019] [Indexed: 01/29/2023] Open
Abstract
Hemagglutinin (HA) stability, or the pH at which HA is activated to cause membrane fusion, has been associated with the replication, pathogenicity, transmissibility, and interspecies adaptation of influenza A viruses. Here, we investigated the mechanisms by which a destabilizing HA mutation, Y17H (activation pH, 6.0), attenuates virus replication and pathogenicity in DBA/2 mice compared to wild-type (WT) virus (activation pH, 5.5). The extracellular lung pH was measured to be near neutral (pH 6.9 to 7.5). WT and Y17H viruses had similar environmental stability at pH 7.0; thus, extracellular inactivation was unlikely to attenuate the Y17H virus. The Y17H virus had accelerated replication kinetics in MDCK, A549, and RAW 264.7 cells when inoculated at a multiplicity of infection (MOI) of 3 PFU/cell. The destabilizing mutation also increased early infectivity and type I interferon (IFN) responses in mouse bone marrow-derived dendritic cells (DCs). In contrast, the HA-Y17H mutation reduced virus replication in murine airway murine nasal epithelial cell and murine tracheal epithelial cell cultures and attenuated virus replication, virus spread, the severity of infection, and cellular infiltration in the lungs of mice. Normalizing virus infection and weight loss in mice by inoculating them with Y17H virus at a dose 500-fold higher than that of WT virus revealed that the destabilized mutant virus triggered the upregulation of more host genes and increased type I IFN responses and cytokine expression in DBA/2 mouse lungs. Overall, HA destabilization decreased virulence in mice by boosting early infection in DCs, resulting in the greater activation of antiviral responses, including the type I IFN response. These studies reveal that HA stability may regulate pathogenicity by modulating IFN responses.IMPORTANCE Diverse influenza A viruses circulate in wild aquatic birds, occasionally infecting farm animals. Rarely, an avian- or swine-origin influenza virus adapts to humans and starts a pandemic. Seasonal and many universal influenza vaccines target the HA surface protein, which is a key component of pandemic influenza viruses. Understanding the HA properties needed for replication and pathogenicity in mammals may guide response efforts to control influenza. Some antiviral drugs and broadly reactive influenza vaccines that target the HA protein have suffered resistance due to destabilizing HA mutations that do not compromise replicative fitness in cell culture. Here, we show that despite not compromising fitness in standard cell cultures, a destabilizing H1N1 HA stalk mutation greatly diminishes viral replication and pathogenicity in vivo by modulating type I IFN responses. This encourages targeting the HA stalk with antiviral drugs and vaccines as well as reevaluating previous candidates that were susceptible to destabilizing resistance mutations.
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17
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Trobaugh DW, Sun C, Bhalla N, Gardner CL, Dunn MD, Klimstra WB. Cooperativity between the 3' untranslated region microRNA binding sites is critical for the virulence of eastern equine encephalitis virus. PLoS Pathog 2019; 15:e1007867. [PMID: 31658290 PMCID: PMC6936876 DOI: 10.1371/journal.ppat.1007867] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2019] [Revised: 12/30/2019] [Accepted: 09/20/2019] [Indexed: 11/18/2022] Open
Abstract
Eastern equine encephalitis virus (EEEV), a mosquito-borne RNA virus, is one of the most acutely virulent viruses endemic to the Americas, causing between 30% and 70% mortality in symptomatic human cases. A major factor in the virulence of EEEV is the presence of four binding sites for the hematopoietic cell-specific microRNA, miR-142-3p, in the 3’ untranslated region (3’ UTR) of the virus. Three of the sites are “canonical” with all 7 seed sequence residues complimentary to miR-142-3p while one is “non-canonical” and has a seed sequence mismatch. Interaction of the EEEV genome with miR-142-3p limits virus replication in myeloid cells and suppresses the systemic innate immune response, greatly exacerbating EEEV neurovirulence. The presence of the miRNA binding sequences is also required for efficient EEEV replication in mosquitoes and, therefore, essential for transmission of the virus. In the current studies, we have examined the role of each binding site by point mutagenesis of the seed sequences in all combinations of sites followed by infection of mammalian myeloid cells, mosquito cells and mice. The resulting data indicate that both canonical and non-canonical sites contribute to cell infection and animal virulence, however, surprisingly, all sites are rapidly deleted from EEEV genomes shortly after infection of myeloid cells or mice. Finally, we show that the virulence of a related encephalitis virus, western equine encephalitis virus, is also dependent upon miR-142-3p binding sites. Eastern equine encephalitis virus (EEEV) is one of the most acutely virulent mosquito-borne viruses in the Americas. A major determinant of EEEV virulence is a mammalian microRNA (miRNA) that is primarily expressed in hematopoietic cells, miR-142-3p. Like miRNA suppression of host mRNA, miR-142-3p binds to the 3’ untranslated region (UTR) of the EEEV genome only in myeloid cells suppressing virus replication and the induction of the innate immune response. In this study, we used point mutations in all four miR-142-3p binding sites in the EEEV 3’ UTR to understand the mechanism behind this miRNA suppression. We observed that decreasing the number of miR-142-3p binding sites leads to virus escape and ultimately attenuation in vivo. Furthermore, another virus, western equine encephalitis virus, also encodes miR-142-3p binding sites that contribute to virulence in vivo. These results provide insight into the mechanism of how cell-specific miRNAs can mediate suppression of virus replication.
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MESH Headings
- 3' Untranslated Regions/genetics
- Aedes
- Animals
- Binding Sites/genetics
- Cell Line
- Cricetinae
- Encephalitis Virus, Eastern Equine/genetics
- Encephalitis Virus, Eastern Equine/immunology
- Encephalitis Virus, Eastern Equine/pathogenicity
- Encephalitis Virus, Western Equine/genetics
- Encephalitis Virus, Western Equine/immunology
- Encephalitis Virus, Western Equine/pathogenicity
- Encephalomyelitis, Equine/immunology
- Encephalomyelitis, Equine/virology
- Female
- Immunity, Innate/immunology
- L Cells
- Mice
- Mice, Inbred C3H
- Mice, Inbred C57BL
- MicroRNAs/genetics
- RAW 264.7 Cells
- Virulence/genetics
- Virus Replication/genetics
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Affiliation(s)
- Derek W. Trobaugh
- Center for Vaccine Research, Department of Immunology and Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA United States of America
| | - Chengqun Sun
- Center for Vaccine Research, Department of Immunology and Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA United States of America
| | - Nishank Bhalla
- Center for Vaccine Research, Department of Immunology and Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA United States of America
| | - Christina L. Gardner
- Center for Vaccine Research, Department of Immunology and Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA United States of America
| | - Matthew D. Dunn
- Center for Vaccine Research, Department of Immunology and Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA United States of America
| | - William B. Klimstra
- Center for Vaccine Research, Department of Immunology and Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA United States of America
- * E-mail:
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18
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Fiege JK, Stone IA, Dumm RE, Waring BM, Fife BT, Agudo J, Brown BD, Heaton NS, Langlois RA. Long-term surviving influenza infected cells evade CD8+ T cell mediated clearance. PLoS Pathog 2019; 15:e1008077. [PMID: 31557273 PMCID: PMC6782110 DOI: 10.1371/journal.ppat.1008077] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2019] [Revised: 10/08/2019] [Accepted: 09/11/2019] [Indexed: 01/02/2023] Open
Abstract
Influenza A virus (IAV) is a seasonal pathogen with the potential to cause devastating pandemics. IAV infects multiple epithelial cell subsets in the respiratory tract, eliciting damage to the lungs. Clearance of IAV is primarily dependent on CD8+ T cells, which must balance control of the infection with immunopathology. Using a virus expressing Cre recombinase to permanently label infected cells in a Cre-inducible reporter mouse, we previously discovered infected club cells that survive both lytic virus replication and CD8+ T cell-mediated clearance. In this study, we demonstrate that ciliated epithelial cells, type I and type II alveolar cells can also become survivor cells. Survivor cells are stable in the lung long-term and demonstrate enhanced proliferation compared to uninfected cells. When we investigated how survivor cells evade CD8+ T cell killing we observed that survivor cells upregulated the inhibitory ligand PD-L1, but survivor cells did not use PD-L1 to evade CD8+ T cell killing. Instead our data suggest that survivor cells are not inherently resistant to CD8+ T cell killing, but instead no longer present IAV antigen and cannot be detected by CD8+ T cells. Finally, we evaluate the failure of CD8+ T cells to kill these previously infected cells. This work demonstrates that additional cell types can survive IAV infection and that these cells robustly proliferate and are stable long term. By sparing previously infected cells, the adaptive immune system may be minimizing pathology associated with IAV infection.
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Affiliation(s)
- Jessica K. Fiege
- University of Minnesota, Department of Microbiology and Immunology and the Center for Immunology, Minneapolis, Minnesota, United States of America
| | - Ian A. Stone
- University of Minnesota, Department of Microbiology and Immunology and the Center for Immunology, Minneapolis, Minnesota, United States of America
| | - Rebekah E. Dumm
- Duke University School of Medicine, Department of Molecular Genetics and Microbiology, Durham, North Carolina, United States of America
| | - Barbara M. Waring
- University of Minnesota, Department of Microbiology and Immunology and the Center for Immunology, Minneapolis, Minnesota, United States of America
| | - Brian T. Fife
- University of Minnesota, Department of Medicine and the Center for Immunology, Minneapolis, Minnesota, United States of America
| | - Judith Agudo
- Icahn School of Medicine at Mount Sinai, Department of Genetics and Genomic Sciences, New York City, New York, United States of America
| | - Brian D. Brown
- Icahn School of Medicine at Mount Sinai, Department of Genetics and Genomic Sciences, New York City, New York, United States of America
| | - Nicholas S. Heaton
- Duke University School of Medicine, Department of Molecular Genetics and Microbiology, Durham, North Carolina, United States of America
| | - Ryan A. Langlois
- University of Minnesota, Department of Microbiology and Immunology and the Center for Immunology, Minneapolis, Minnesota, United States of America
- * E-mail:
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19
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Hemann EA, Green R, Turnbull JB, Langlois RA, Savan R, Gale M. Interferon-λ modulates dendritic cells to facilitate T cell immunity during infection with influenza A virus. Nat Immunol 2019; 20:1035-1045. [PMID: 31235953 PMCID: PMC6642690 DOI: 10.1038/s41590-019-0408-z] [Citation(s) in RCA: 85] [Impact Index Per Article: 14.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2018] [Accepted: 04/29/2019] [Indexed: 12/12/2022]
Abstract
Type III interferon (IFN-λ) is important for innate immune protection at mucosal surfaces and has therapeutic benefit against influenza A virus (IAV) infection. However, the mechanisms by which IFN-λ programs adaptive immune protection against IAV are undefined. Here we found that IFN-λ signaling in dendritic cell (DC) populations was critical for the development of protective IAV-specific CD8+ T cell responses. Mice lacking the IFN-λ receptor (Ifnlr1-/-) had blunted CD8+ T cell responses relative to wild type and exhibited reduced survival after heterosubtypic IAV re-challenge. Analysis of DCs revealed IFN-λ signaling directed the migration and function of CD103+ DCs for development of optimal antiviral CD8+ T cell responses, and bioinformatic analyses identified IFN-λ regulation of a DC IL-10 immunoregulatory network. Thus, IFN-λ serves a critical role in bridging innate and adaptive immunity from lung mucosa to lymph nodes to program DCs to direct effective T cell immunity against IAV.
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Affiliation(s)
- Emily A Hemann
- Department of Immunology, Center for Innate Immunity and Immune Disease, University of Washington, Seattle, WA, USA
| | - Richard Green
- Department of Immunology, Center for Innate Immunity and Immune Disease, University of Washington, Seattle, WA, USA
| | - J Bryan Turnbull
- Department of Immunology, Center for Innate Immunity and Immune Disease, University of Washington, Seattle, WA, USA
| | - Ryan A Langlois
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN, USA
| | - Ram Savan
- Department of Immunology, Center for Innate Immunity and Immune Disease, University of Washington, Seattle, WA, USA
| | - Michael Gale
- Department of Immunology, Center for Innate Immunity and Immune Disease, University of Washington, Seattle, WA, USA.
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20
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Fiege JK, Stone IA, Fay EJ, Markman MW, Wijeyesinghe S, Macchietto MG, Shen S, Masopust D, Langlois RA. The Impact of TCR Signal Strength on Resident Memory T Cell Formation during Influenza Virus Infection. THE JOURNAL OF IMMUNOLOGY 2019; 203:936-945. [PMID: 31235552 DOI: 10.4049/jimmunol.1900093] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/23/2019] [Accepted: 06/08/2019] [Indexed: 02/01/2023]
Abstract
Resident memory T cells (TRM) in the lung are vital for heterologous protection against influenza A virus (IAV). Environmental factors are necessary to establish lung TRM; however, the role of T cell-intrinsic factors like TCR signal strength have not been elucidated. In this study, we investigated the impact of TCR signal strength on the generation and maintenance of lung TRM after IAV infection. We inserted high- and low-affinity OT-I epitopes into IAV and infected mice after transfer of OT-I T cells. We uncovered a bias in TRM formation in the lung elicited by lower affinity TCR stimulation. TCR affinity did not impact the overall phenotype or long-term maintenance of lung TRM Overall, these findings demonstrate that TRM formation is negatively correlated with increased TCR signal strength. Lower affinity cells may have an advantage in forming TRM to ensure diversity in the Ag-specific repertoire in tissues.
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Affiliation(s)
- Jessica K Fiege
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN 55455.,Center for Immunology, University of Minnesota, Minneapolis, MN 55455
| | - Ian A Stone
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN 55455.,Center for Immunology, University of Minnesota, Minneapolis, MN 55455
| | - Elizabeth J Fay
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN 55455.,Center for Immunology, University of Minnesota, Minneapolis, MN 55455.,Biochemistry, Molecular Biology and Biophysics Graduate Program, University of Minnesota, Minneapolis, MN 55455; and
| | - Matthew W Markman
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN 55455.,Center for Immunology, University of Minnesota, Minneapolis, MN 55455
| | - Sathi Wijeyesinghe
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN 55455.,Center for Immunology, University of Minnesota, Minneapolis, MN 55455
| | - Marissa G Macchietto
- Institute for Health Informatics, University of Minnesota, Minneapolis, MN 55455
| | - Steven Shen
- Institute for Health Informatics, University of Minnesota, Minneapolis, MN 55455
| | - David Masopust
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN 55455.,Center for Immunology, University of Minnesota, Minneapolis, MN 55455
| | - Ryan A Langlois
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN 55455; .,Center for Immunology, University of Minnesota, Minneapolis, MN 55455.,Biochemistry, Molecular Biology and Biophysics Graduate Program, University of Minnesota, Minneapolis, MN 55455; and
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21
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Antiviral RNAi in Insects and Mammals: Parallels and Differences. Viruses 2019; 11:v11050448. [PMID: 31100912 PMCID: PMC6563508 DOI: 10.3390/v11050448] [Citation(s) in RCA: 61] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2019] [Revised: 05/14/2019] [Accepted: 05/15/2019] [Indexed: 12/26/2022] Open
Abstract
The RNA interference (RNAi) pathway is a potent antiviral defense mechanism in plants and invertebrates, in response to which viruses evolved suppressors of RNAi. In mammals, the first line of defense is mediated by the type I interferon system (IFN); however, the degree to which RNAi contributes to antiviral defense is still not completely understood. Recent work suggests that antiviral RNAi is active in undifferentiated stem cells and that antiviral RNAi can be uncovered in differentiated cells in which the IFN system is inactive or in infections with viruses lacking putative viral suppressors of RNAi. In this review, we describe the mechanism of RNAi and its antiviral functions in insects and mammals. We draw parallels and highlight differences between (antiviral) RNAi in these classes of animals and discuss open questions for future research.
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22
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Fay EJ, Aron SL, Stone IA, Waring BM, Plemper RK, Langlois RA. Engineered Small-Molecule Control of Influenza A Virus Replication. J Virol 2019; 93:e01677-18. [PMID: 30282710 PMCID: PMC6288343 DOI: 10.1128/jvi.01677-18] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2018] [Accepted: 09/26/2018] [Indexed: 01/01/2023] Open
Abstract
Influenza A virus (IAV) remains a global health concern despite the availability of a seasonal vaccine. It is difficult to predict which strains will circulate during influenza season, and therefore, it is extremely challenging to test novel vaccines in the human population. To overcome this obstacle, new vaccines must be tested in challenge studies. This approach poses significant safety problems, since current pharmacological interventions for IAV are poorly efficacious. New methods are needed to enhance the safety of these challenge studies. In this study, we have generated a virus expressing a small-molecule-assisted shutoff (SMASh) tag as a safety switch for IAV replication. The addition of the SMASh tag to an essential IAV protein allows for small-molecule-mediated inhibition of replication. Treatment with this drug controls the replication of a SMASh-tagged virus in vitro and in vivo This model for restriction of viral replication has potential for broad applications in vaccine studies, virotherapy, and basic virus research.IMPORTANCE Influenza A virus (IAV) causes significant morbidity and mortality annually worldwide, despite the availability of new formulations of the vaccine each season. There is a critical need to develop more-efficacious vaccines. However, testing novel vaccines in the human population in controlled studies is difficult due to the limited availability and efficacy of intervention strategies should the vaccine fail. There are also significant safety concerns for work with highly pathogenic IAV strains in the laboratory. Therefore, novel strategies are needed to improve the safety of vaccine studies and of research on highly pathogenic IAV. In this study, we developed an IAV strain engineered to contain a small-molecule-mediated safety switch. This tag, when attached to an essential viral protein, allows for the regulation of IAV replication in vitro and in vivo This strategy provides a platform for the regulation of virus replication without targeting viral proteins directly.
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Affiliation(s)
- Elizabeth J Fay
- Biochemistry, Molecular Biology, and Biophysics Graduate Program, University of Minnesota, Minneapolis, Minnesota, USA
- Center for Immunology, University of Minnesota Department of Microbiology and Immunology, University of Minnesota, Minneapolis, Minnesota, USA
| | - Stephanie L Aron
- Center for Immunology, University of Minnesota Department of Microbiology and Immunology, University of Minnesota, Minneapolis, Minnesota, USA
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, Minnesota, USA
| | - Ian A Stone
- Center for Immunology, University of Minnesota Department of Microbiology and Immunology, University of Minnesota, Minneapolis, Minnesota, USA
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, Minnesota, USA
| | - Barbara M Waring
- Center for Immunology, University of Minnesota Department of Microbiology and Immunology, University of Minnesota, Minneapolis, Minnesota, USA
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, Minnesota, USA
| | - Richard K Plemper
- Institute for Biomedical Sciences, Georgia State University, Atlanta, Georgia, USA
| | - Ryan A Langlois
- Biochemistry, Molecular Biology, and Biophysics Graduate Program, University of Minnesota, Minneapolis, Minnesota, USA
- Center for Immunology, University of Minnesota Department of Microbiology and Immunology, University of Minnesota, Minneapolis, Minnesota, USA
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, Minnesota, USA
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23
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Fay EJ, Langlois RA. MicroRNA-Attenuated Virus Vaccines. Noncoding RNA 2018; 4:E25. [PMID: 30279330 PMCID: PMC6316615 DOI: 10.3390/ncrna4040025] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2018] [Revised: 09/25/2018] [Accepted: 09/28/2018] [Indexed: 12/18/2022] Open
Abstract
Live-attenuated vaccines are the most effective way to establish robust, long-lasting immunity against viruses. However, the possibility of reversion to wild type replication and pathogenicity raises concerns over the safety of these vaccines. The use of host-derived microRNAs (miRNAs) to attenuate viruses has been accomplished in an array of biological contexts. The broad assortment of effective tissue- and species-specific miRNAs, and the ability to target a virus with multiple miRNAs, allow for targeting to be tailored to the virus of interest. While escape is always a concern, effective strategies have been developed to improve the safety and stability of miRNA-attenuated viruses. In this review, we discuss the various approaches that have been used to engineer miRNA-attenuated viruses, the steps that have been taken to improve their safety, and the potential use of these viruses as vaccines.
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Affiliation(s)
- Elizabeth J Fay
- Biochemistry, Molecular Biology, and Biophysics Graduate Program, University of Minnesota, Minneapolis, MN 55455, USA.
- Center for Immunology, University of Minnesota, Minneapolis, MN 55455, USA.
| | - Ryan A Langlois
- Biochemistry, Molecular Biology, and Biophysics Graduate Program, University of Minnesota, Minneapolis, MN 55455, USA.
- Center for Immunology, University of Minnesota, Minneapolis, MN 55455, USA.
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN 55455, USA.
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24
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Distinct antiviral signatures revealed by the magnitude and round of influenza virus replication in vivo. Proc Natl Acad Sci U S A 2018; 115:9610-9615. [PMID: 30181264 DOI: 10.1073/pnas.1807516115] [Citation(s) in RCA: 31] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Influenza virus has a broad cellular tropism in the respiratory tract. Infected epithelial cells sense the infection and initiate an antiviral response. To define the antiviral response at the earliest stages of infection we used a series of single-cycle reporter viruses. These viral probes demonstrated cells in vivo harbor a range in magnitude of virus replication. Transcriptional profiling of cells supporting different levels of replication revealed tiers of IFN-stimulated gene expression. Uninfected cells and cells with blunted replication expressed a distinct and potentially protective antiviral signature, while cells with high replication expressed a unique reserve set of antiviral genes. Finally, we used these single-cycle reporter viruses to determine the antiviral landscape during virus spread, which unveiled disparate protection of epithelial cell subsets mediated by IFN in vivo. Together these results highlight the complexity of virus-host interactions within the infected lung and suggest that magnitude and round of replication tune the antiviral response.
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25
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Genome-wide profiling of microRNAs reveals novel insights into the interactions between H9N2 avian influenza virus and avian dendritic cells. Oncogene 2018; 37:4562-4580. [DOI: 10.1038/s41388-018-0279-z] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2017] [Revised: 12/30/2017] [Accepted: 01/21/2018] [Indexed: 12/19/2022]
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26
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Li H, Bradley KC, Long JS, Frise R, Ashcroft JW, Hartgroves LC, Shelton H, Makris S, Johansson C, Cao B, Barclay WS. Internal genes of a highly pathogenic H5N1 influenza virus determine high viral replication in myeloid cells and severe outcome of infection in mice. PLoS Pathog 2018; 14:e1006821. [PMID: 29300777 PMCID: PMC5771632 DOI: 10.1371/journal.ppat.1006821] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2017] [Revised: 01/17/2018] [Accepted: 12/15/2017] [Indexed: 12/26/2022] Open
Abstract
The highly pathogenic avian influenza (HPAI) H5N1 influenza virus has been a public health concern for more than a decade because of its frequent zoonoses and the high case fatality rate associated with human infections. Severe disease following H5N1 influenza infection is often associated with dysregulated host innate immune response also known as cytokine storm but the virological and cellular basis of these responses has not been clearly described. We rescued a series of 6:2 reassortant viruses that combined a PR8 HA/NA pairing with the internal gene segments from human adapted H1N1, H3N2, or avian H5N1 viruses and found that mice infected with the virus with H5N1 internal genes suffered severe weight loss associated with increased lung cytokines but not high viral load. This phenotype did not map to the NS gene segment, and NS1 protein of H5N1 virus functioned as a type I IFN antagonist as efficient as NS1 of H1N1 or H3N2 viruses. Instead we discovered that the internal genes of H5N1 virus supported a much higher level of replication of viral RNAs in myeloid cells in vitro, but not in epithelial cells and that this was associated with high induction of type I IFN in myeloid cells. We also found that in vivo during H5N1 recombinant virus infection cells of haematopoetic origin were infected and produced type I IFN and proinflammatory cytokines. Taken together our data infer that human and avian influenza viruses are differently controlled by host factors in alternative cell types; internal gene segments of avian H5N1 virus uniquely drove high viral replication in myeloid cells, which triggered an excessive cytokine production, resulting in severe immunopathology. Some avian influenza viruses, including highly pathogenic H5N1 virus, cause severe disease in humans and in experimental animal models associated with excessive cytokine production. We aimed to understand the virological mechanism behind the cytokine storm, and particularly the contribution of internal gene segments that encode the viral polymerase and the non-structural proteins, since these might be retained in a pandemic virus. We found that the internal genes from an H5N1 avian influenza virus allowed virus to replicate to strikingly higher levels in myeloid cells compared to internal genes of human adapted strains. The higher viral RNA levels did not lead to higher viral load but drove excessive cytokine production and more severe outcome in infected mice. The remarkable difference in viral replication in myeloid cells was not observed in lung epithelial cells, suggesting that cell type specific differences in host factors were responsible. Understanding the molecular basis of excessive viral replication in myeloid cells may guide future therapeutic options for viruses that have recently crossed into humans from birds.
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MESH Headings
- A549 Cells
- Animals
- Cells, Cultured
- Dogs
- Female
- Genes, Viral/physiology
- HEK293 Cells
- Humans
- Immunity, Innate/physiology
- Influenza A Virus, H5N1 Subtype/genetics
- Influenza A Virus, H5N1 Subtype/immunology
- Influenza A Virus, H5N1 Subtype/pathogenicity
- Influenza A Virus, H5N1 Subtype/physiology
- Influenza, Human/genetics
- Influenza, Human/immunology
- Influenza, Human/virology
- Madin Darby Canine Kidney Cells
- Mice
- Mice, Inbred BALB C
- Mice, Inbred C57BL
- Mice, Knockout
- Myeloid Cells/immunology
- Myeloid Cells/metabolism
- Myeloid Cells/virology
- Orthomyxoviridae Infections/genetics
- Orthomyxoviridae Infections/immunology
- Orthomyxoviridae Infections/mortality
- Orthomyxoviridae Infections/virology
- Severity of Illness Index
- Virus Replication/genetics
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Affiliation(s)
- Hui Li
- China-Japan Friendship Hospital, Capital Medical University, Beijing, China
- Section of Virology, Department of Medicine, Imperial College London, London, United Kingdom
| | - Konrad C. Bradley
- Section of Virology, Department of Medicine, Imperial College London, London, United Kingdom
| | - Jason S. Long
- Section of Virology, Department of Medicine, Imperial College London, London, United Kingdom
| | - Rebecca Frise
- Section of Virology, Department of Medicine, Imperial College London, London, United Kingdom
| | - Jonathan W. Ashcroft
- Section of Virology, Department of Medicine, Imperial College London, London, United Kingdom
| | - Lorian C. Hartgroves
- Section of Virology, Department of Medicine, Imperial College London, London, United Kingdom
| | - Holly Shelton
- Section of Virology, Department of Medicine, Imperial College London, London, United Kingdom
| | - Spyridon Makris
- Section of Respiratory Infections, National Heart and Lung Institute, Imperial College London
| | - Cecilia Johansson
- Section of Respiratory Infections, National Heart and Lung Institute, Imperial College London
| | - Bin Cao
- Department of Respiratory Medicine, Capital Medical University; Center for Respiratory Diseases, Department of Pulmonary and Critical Care Medicine, China-Japan Friendship Hospital, Beijing, China
- * E-mail: (WSB); (BC)
| | - Wendy S. Barclay
- Section of Virology, Department of Medicine, Imperial College London, London, United Kingdom
- * E-mail: (WSB); (BC)
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MicroRNA-Based Attenuation of Influenza Virus across Susceptible Hosts. J Virol 2018; 92:JVI.01741-17. [PMID: 29093096 DOI: 10.1128/jvi.01741-17] [Citation(s) in RCA: 38] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2017] [Accepted: 10/25/2017] [Indexed: 12/25/2022] Open
Abstract
Influenza A virus drives significant morbidity and mortality in humans and livestock. Annual circulation of the virus in livestock and waterfowl contributes to severe economic disruption and increases the risk of zoonotic transmission of novel strains into the human population, where there is no preexisting immunity. Seasonal vaccinations in humans help prevent infection and can reduce symptoms when infection does occur. However, current vaccination regimens available for livestock are limited in part due to safety concerns regarding reassortment/recombination with circulating strains. Therefore, inactivated vaccines are used instead of the more immunostimulatory live attenuated vaccines. MicroRNAs (miRNAs) have been used previously to generate attenuated influenza A viruses for use as a vaccine. Here, we systematically targeted individual influenza gene mRNAs using the same miRNA to determine the segment(s) that yields maximal attenuation potential. This analysis demonstrated that targeting of NP mRNA most efficiently ablates replication. We further increased the plasticity of miRNA-mediated attenuation of influenza A virus by exploiting a miRNA, miR-21, that is ubiquitously expressed across influenza-susceptible hosts. In order to construct this targeted virus, we used CRISPR/Cas9 to eliminate the universally expressed miR-21 from MDCK cells. miR-21-targeted viruses were attenuated in human, mouse, canine, and avian cells and drove protective immunity in mice. This strategy has the potential to enhance the safety of live attenuated vaccines in humans and zoonotic reservoirs.IMPORTANCE Influenza A virus circulates annually in both avian and human populations, causing significant morbidity, mortality, and economic burden. High incidence of zoonotic infections greatly increases the potential for transmission to humans, where no preexisting immunity or vaccine exists. There is a critical need for new vaccine strategies to combat emerging influenza outbreaks. MicroRNAs were used previously to attenuate influenza A viruses. We propose the development of a novel platform to produce live attenuated vaccines that are highly customizable, efficacious across a broad species range, and exhibit enhanced safety over traditional vaccination methods. This strategy exploits a microRNA that is expressed abundantly in influenza virus-susceptible hosts. By eliminating this ubiquitous microRNA from a cell line, targeted viruses that are attenuated across susceptible strains can be generated. This approach greatly increases the plasticity of the microRNA targeting approach and enhances vaccine safety.
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28
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Fino KK, Yang L, Silveyra P, Hu S, Umstead TM, DiAngelo S, Halstead ES, Cooper TK, Abraham T, Takahashi Y, Zhou Z, Wang HG, Chroneos ZC. SH3GLB2/endophilin B2 regulates lung homeostasis and recovery from severe influenza A virus infection. Sci Rep 2017; 7:7262. [PMID: 28779131 PMCID: PMC5544693 DOI: 10.1038/s41598-017-07724-5] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2017] [Accepted: 07/03/2017] [Indexed: 12/17/2022] Open
Abstract
New influenza A viruses that emerge frequently elicit composite inflammatory responses to both infection and structural damage of alveolar-capillary barrier cells that hinders regeneration of respiratory function. The host factors that relinquish restoration of lung health to enduring lung injury are insufficiently understood. Here, we investigated the role of endophilin B2 (B2) in susceptibility to severe influenza infection. WT and B2-deficient mice were infected with H1N1 PR8 by intranasal administration and course of influenza pneumonia, inflammatory, and tissue responses were monitored over time. Disruption of B2 enhanced recovery from severe influenza infection as indicated by swift body weight recovery and significantly better survival of endophilin B2-deficient mice compared to WT mice. Compared to WT mice, the B2-deficient lungs exhibited induction of genes that express surfactant proteins, ABCA3, GM-CSF, podoplanin, and caveolin mRNA after 7 days, temporal induction of CCAAT/enhancer binding protein CEBPα, β, and δ mRNAs 3-14 days after infection, and differences in alveolar extracellular matrix integrity and respiratory mechanics. Flow cytometry and gene expression studies demonstrated robust recovery of alveolar macrophages and recruitment of CD4+ lymphocytes in B2-deficient lungs. Targeting of endophilin B2 alleviates adverse effects of IAV infection on respiratory and immune cells enabling restoration of alveolar homeostasis.
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Affiliation(s)
- Kristin K Fino
- Department of Pediatrics, Pulmonary Immunology and Physiology Laboratory, Pennsylvania State University College of Medicine, Pennsylvania, USA
| | - Linlin Yang
- Department of Pediatrics, Pulmonary Immunology and Physiology Laboratory, Pennsylvania State University College of Medicine, Pennsylvania, USA
| | - Patricia Silveyra
- Department of Pediatrics, Pulmonary Immunology and Physiology Laboratory, Pennsylvania State University College of Medicine, Pennsylvania, USA
- Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Pennsylvania, USA
| | - Sanmei Hu
- Department of Pediatrics, Pulmonary Immunology and Physiology Laboratory, Pennsylvania State University College of Medicine, Pennsylvania, USA
| | - Todd M Umstead
- Department of Pediatrics, Pulmonary Immunology and Physiology Laboratory, Pennsylvania State University College of Medicine, Pennsylvania, USA
| | - Susan DiAngelo
- Department of Pediatrics, Pulmonary Immunology and Physiology Laboratory, Pennsylvania State University College of Medicine, Pennsylvania, USA
| | - E Scott Halstead
- Department of Pediatrics, Pulmonary Immunology and Physiology Laboratory, Pennsylvania State University College of Medicine, Pennsylvania, USA
- Department of Pediatrics, Critical Care Medicine, Pennsylvania State University College of Medicine, Pennsylvania, USA
- Children's Hospital, Penn State Health Milton S. Hershey Medical Center, Pennsylvania, USA
| | - Timothy K Cooper
- Department of Comparative Medicine, Pennsylvania State University College of Medicine, Pennsylvania, USA
- Department Pathology, Pennsylvania State University College of Medicine, Pennsylvania, USA
| | - Thomas Abraham
- Department of Neural and Behavioral Sciences, and the Microscopy Imaging Facility, Pennsylvania, USA
| | - Yoshinori Takahashi
- Department of Pediatrics, Hematology Oncology, Pennsylvania State University College of Medicine, Pennsylvania, USA
| | - Zhixiang Zhou
- The College of Life Science and Bioengineering, Beijing University of Technology, Beijing, China
| | - Hong Gang Wang
- Department of Pediatrics, Hematology Oncology, Pennsylvania State University College of Medicine, Pennsylvania, USA.
- Department of Pharmacology, Pennsylvania State University College of Medicine, Pennsylvania, USA.
| | - Zissis C Chroneos
- Department of Pediatrics, Pulmonary Immunology and Physiology Laboratory, Pennsylvania State University College of Medicine, Pennsylvania, USA.
- Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Pennsylvania, USA.
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29
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Affiliation(s)
- Benjamin R tenOever
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA
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30
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Zhao HM, Han F, Xu R, Huang XY, Cheng SM, Huang MF, Yue HY, Wang X, Zou Y, Xu HL, Liu DY. Therapeutic effect of curcumin on experimental colitis mediated by inhibiting CD8 +CD11c + cells. World J Gastroenterol 2017; 23:1804-1815. [PMID: 28348486 PMCID: PMC5352921 DOI: 10.3748/wjg.v23.i10.1804] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/22/2016] [Revised: 12/25/2016] [Accepted: 01/17/2017] [Indexed: 02/06/2023] Open
Abstract
AIM To verify whether curcumin (Cur) can treat inflammatory bowel disease by regulating CD8+CD11c+ cells.
METHODS We evaluated the suppressive effect of Cur on CD8+CD11c+ cells in spleen and Peyer’s patches (PPs) in colitis induced by trinitrobenzene sulfonic acid. Mice with colitis were treated by 200 mg/kg Cur for 7 d. On day 8, the therapeutic effect of Cur was evaluated by visual assessment and histological examination, while co-stimulatory molecules of CD8+CD11c+ cells in the spleen and PPs were measured by flow cytometry. The levels of interleukin (IL)-10, interferon (IFN)-γ and transforming growth factor (TGF)-β1 in spleen and colonic mucosa were determined by ELISA.
RESULTS The disease activity index, colon weight, weight index of colon and histological score of experimental colitis were obviously decreased after Cur treatment, while the body weight and colon length recovered. After treatment with Cur, CD8+CD11c+ cells were decreased in the spleen and PPs, and the expression of major histocompatibility complex II, CD205, CD40, CD40L and intercellular adhesion molecule-1 was inhibited. IL-10, IFN-γ and TGF-β1 levels were increased compared with those in mice with untreated colitis.
CONCLUSION Cur can effectively treat experimental colitis, which is realized by inhibiting CD8+CD11c+ cells.
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Tundup S, Kandasamy M, Perez JT, Mena N, Steel J, Nagy T, Albrecht RA, Manicassamy B. Endothelial cell tropism is a determinant of H5N1 pathogenesis in mammalian species. PLoS Pathog 2017; 13:e1006270. [PMID: 28282445 PMCID: PMC5362246 DOI: 10.1371/journal.ppat.1006270] [Citation(s) in RCA: 44] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2016] [Revised: 03/22/2017] [Accepted: 03/02/2017] [Indexed: 12/19/2022] Open
Abstract
The cellular and molecular mechanisms underpinning the unusually high virulence of highly pathogenic avian influenza H5N1 viruses in mammalian species remains unknown. Here, we investigated if the cell tropism of H5N1 virus is a determinant of enhanced virulence in mammalian species. We engineered H5N1 viruses with restricted cell tropism through the exploitation of cell type-specific microRNA expression by incorporating microRNA target sites into the viral genome. Restriction of H5N1 replication in endothelial cells via miR-126 ameliorated disease symptoms, prevented systemic viral spread and limited mortality, despite showing similar levels of peak viral replication in the lungs as compared to control virus-infected mice. Similarly, restriction of H5N1 replication in endothelial cells resulted in ameliorated disease symptoms and decreased viral spread in ferrets. Our studies demonstrate that H5N1 infection of endothelial cells results in excessive production of cytokines and reduces endothelial barrier integrity in the lungs, which culminates in vascular leakage and viral pneumonia. Importantly, our studies suggest a need for a combinational therapy that targets viral components, suppresses host immune responses, and improves endothelial barrier integrity for the treatment of highly pathogenic H5N1 virus infections. In healthy individuals, the symptoms of seasonal influenza virus infection are mild and the infection is cleared within 4–7 days. However, infection with highly pathogenic avian influenza virus (H5N1) can be severe and often results in fatal pneumonia even in healthy adults. While it is known that both viral and host factors play a role in enhanced disease progression, the molecular mechanisms for the high virulence of H5N1 virus are not completely understood. In this study, we engineered avian influenza H5N1 viruses incapable of replicating in endothelial cells and evaluated disease symptoms in mice and ferrets. Our studies show that H5N1 infection of endothelial cells causes severe disease and death of infected animals in part due to the damage of endothelial cells lining the blood vessels, which results in leakage of fluid into the lungs (pneumonia).
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Affiliation(s)
- Smanla Tundup
- Department of Microbiology, University of Chicago, Chicago, IL, United States of America
- Howard Taylor Ricketts Laboratory, University of Chicago, Argonne, IL, United States of America
| | - Matheswaran Kandasamy
- Department of Microbiology, University of Chicago, Chicago, IL, United States of America
- Howard Taylor Ricketts Laboratory, University of Chicago, Argonne, IL, United States of America
| | - Jasmine T. Perez
- Department of Microbiology, University of Chicago, Chicago, IL, United States of America
| | - Nacho Mena
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, United States of America
| | - John Steel
- Department of Microbiology, Emory University, Atlanta, GA, United States of America
| | - Tamas Nagy
- Comparative Pathology Laboratory, University of Georgia, Athens, GA, United States of America
| | - Randy A. Albrecht
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, United States of America
| | - Balaji Manicassamy
- Department of Microbiology, University of Chicago, Chicago, IL, United States of America
- Howard Taylor Ricketts Laboratory, University of Chicago, Argonne, IL, United States of America
- * E-mail:
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Abstract
Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and understanding the biological properties of viruses. There are several mechanisms for regulating viral tropism, however they tend to be virus class specific and many result in virus attenuation. Additionally, many viruses, including picornaviruses, exhibit size constraints that do not allow for incorporation of large amounts of foreign genetic material required for some targeting methods. MicroRNAs are short, non-coding RNAs that regulate gene expression in eukaryotic cells by binding complementary target sequences in messenger RNAs, preventing their translation or accelerating their degradation. Different cells exhibit distinct microRNA signatures and many microRNAs serve as biomarkers. These differential expression patterns can be exploited for restricting gene expression in cells that express specific microRNAs while maintaining expression in cells that do not. In regards to regulating viral tropism, sequences complementary to specific microRNAs are incorporated into the viral genome, generally in the 3' non-coding regions, targeting them for destruction in the presence of the cognate microRNAs thus preventing viral gene expression and/or replication. MicroRNA-targeting is a technique that theoretically can be applied to all viral vectors without altering the potency of the virus in the absence of the corresponding microRNAs. Here we describe experimental methods associated with generating a microRNA-targeted picornavirus and evaluating the efficacy and specificity of that targeting in vitro. This protocol is designed for a rapidly replicating virus with a lytic replication cycle, however, modification of the time points analyzed and the specific virus titration readouts used will aid in the adaptation of this protocol to many different viruses.
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Affiliation(s)
- Autumn J Ruiz
- Department of Molecular Medicine, Mayo Clinic College of Medicine;
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33
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Herbert KM, Nag A. A Tale of Two RNAs during Viral Infection: How Viruses Antagonize mRNAs and Small Non-Coding RNAs in The Host Cell. Viruses 2016; 8:E154. [PMID: 27271653 PMCID: PMC4926174 DOI: 10.3390/v8060154] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2016] [Revised: 05/12/2016] [Accepted: 05/20/2016] [Indexed: 02/06/2023] Open
Abstract
Viral infection initiates an array of changes in host gene expression. Many viruses dampen host protein expression and attempt to evade the host anti-viral defense machinery. Host gene expression is suppressed at several stages of host messenger RNA (mRNA) formation including selective degradation of translationally competent messenger RNAs. Besides mRNAs, host cells also express a variety of noncoding RNAs, including small RNAs, that may also be subject to inhibition upon viral infection. In this review we focused on different ways viruses antagonize coding and noncoding RNAs in the host cell to its advantage.
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Affiliation(s)
- Kristina M Herbert
- Department of Experimental Microbiology, Center for Scientific Research and Higher Education of Ensenada (CICESE), Ensenada, Baja California 22860, Mexico.
| | - Anita Nag
- Department of Chemistry, Florida A&M University, Tallahassee, FL 32307, USA.
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Abstract
MicroRNAs (miRNAs) are small, non-protein-coding, single-stranded RNAs. They function as posttranscriptional regulators of gene expression by interacting with target mRNAs. This process prevents translation of target mRNAs into a functional protein. miRNAs are considered to be functionally involved in virtually all physiologic processes, including differentiation and proliferation, metabolism, hemostasis, apoptosis, and inflammation. Many of these functions have important implications for anesthesiology and critical care medicine. Studies indicate that miRNA expression levels can be used to predict the risk for eminent organ injury or sepsis. Pharmacologic approaches targeting miRNAs for the treatment of human diseases are currently being tested in clinical trials. The present review highlights the important biological functions of miRNAs and their usefulness as perioperative biomarkers and discusses the pharmacologic approaches that modulate miRNA functions for disease treatment. In addition, the authors discuss the pharmacologic interactions of miRNAs with currently used anesthetics and their potential to impact anesthetic toxicity and side effects.
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Zhou K, Wang J, Li A, Zhao W, Wang D, Zhang W, Yan J, Gao GF, Liu W, Fang M. Swift and Strong NK Cell Responses Protect 129 Mice against High-Dose Influenza Virus Infection. THE JOURNAL OF IMMUNOLOGY 2016; 196:1842-54. [DOI: 10.4049/jimmunol.1501486] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/01/2015] [Accepted: 12/15/2015] [Indexed: 11/19/2022]
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36
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Plasmacytoid Dendritic Cells Require Direct Infection To Sustain the Pulmonary Influenza A Virus-Specific CD8 T Cell Response. J Virol 2015; 90:2830-7. [PMID: 26719269 DOI: 10.1128/jvi.02546-15] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2015] [Accepted: 12/20/2015] [Indexed: 11/20/2022] Open
Abstract
UNLABELLED Following influenza A virus (IAV) infection, development of a robust IAV-specific CD8 T cell response is required for clearance of primary infection and enhances memory protection. Following IAV infection, plasmacytoid dendritic cells (pDC) or CD8α(+) DC regulate pulmonary effector CD8 T cell responses within the lung. Without this DC-T cell interaction, insufficient effector CD8 T cells are maintained in the lungs, leading to enhanced morbidity and mortality. Previous studies have demonstrated that pDC are capable of classical presentation or cross-presentation of IAV antigens and could potentially regulate IAV-specific CD8 T cell responses through either mechanism. Our results demonstrate that pDC from the lungs of donor mice infected with an IAV that is not able to replicate in hematopoietic cells (142t-IAV), unlike donor pDC isolated from the lungs of control infected mice, are not able to rescue the host IAV-specific CD8 T cell response from apoptosis. This indicates that pDC must utilize the direct presentation pathway for this rescue. This inability of pDC from 142t-IAV donors to rescue the IAV-specific CD8 T cell response is not due to differences in the overall ability of 142t-IAV to replicate within the lungs or generate defective viral genomes or to differences in levels of costimulatory molecules required for this interaction. We further demonstrate that bypassing the antigen presentation pathway by coating the 142t-IAV pDC with IAV peptide epitopes restores their ability to rescue the IAV-specific CD8 T cell response. IMPORTANCE IAV continues to be a global health burden, infecting 5 to 20% of the global population annually. Continued investigation into the mechanisms that mediate protective immune responses against IAV is important to improving current vaccination and antiviral strategies antagonistic toward IAV. Our findings presented herein demonstrate a key requirement for pDC promotion of effector CD8 T cell survival: that rather than utilizing cross-presentation, pDC must be infected and utilize the endogenous pathway for presentation of antigens to CD8 T cells during in vivo IAV infections. This suggests that targeting presentation via the endogenous pathway in pDC could be important for the development of unique antiviral cellular therapies.
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Benitez AA, Spanko LA, Bouhaddou M, Sachs D, tenOever BR. Engineered Mammalian RNAi Can Elicit Antiviral Protection that Negates the Requirement for the Interferon Response. Cell Rep 2015; 13:1456-1466. [PMID: 26549455 DOI: 10.1016/j.celrep.2015.10.020] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2015] [Revised: 09/10/2015] [Accepted: 10/07/2015] [Indexed: 12/20/2022] Open
Abstract
Although the intrinsic antiviral cell defenses of many kingdoms utilize pathogen-specific small RNAs, the antiviral response of chordates is primarily protein based and not uniquely tailored to the incoming microbe. In an effort to explain this evolutionary bifurcation, we determined whether antiviral RNAi was sufficient to replace the protein-based type I interferon (IFN-I) system of mammals. To this end, we recreated an RNAi-like response in mammals and determined its effectiveness to combat influenza A virus in vivo in the presence and absence of the canonical IFN-I system. Mammalian antiviral RNAi, elicited by either host- or virus-derived small RNAs, effectively attenuated virus and prevented disease independently of the innate immune response. These data find that chordates could have utilized RNAi as their primary antiviral cell defense and suggest that the IFN-I system emerged as a result of natural selection imposed by ancient pathogens.
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Affiliation(s)
- Asiel Arturo Benitez
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Laura Adrienne Spanko
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Mehdi Bouhaddou
- Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - David Sachs
- Department of Genetics and Genomics, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
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Miller MA, Ganesan APV, Luckashenak N, Mendonca M, Eisenlohr LC. Endogenous antigen processing drives the primary CD4+ T cell response to influenza. Nat Med 2015; 21:1216-22. [PMID: 26413780 PMCID: PMC4629989 DOI: 10.1038/nm.3958] [Citation(s) in RCA: 59] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2015] [Accepted: 08/28/2015] [Indexed: 12/12/2022]
Abstract
By convention, CD4+ T lymphocytes recognize foreign and self peptides derived from internalized antigens in combination with MHC class II molecules. Alternative pathways of epitope production have been identified but their contributions to host defense have not been established. We show here in a mouse infection model that the CD4+ T cell response to influenza, critical for durable protection from the virus, is driven principally by unconventional processing of antigen synthesized within the infected antigen-presenting cell, not by classical processing of endocytosed virions or material from infected cells. Investigation of the cellular components involved, including the H2-M molecular chaperone, the proteasome, and gamma-interferon inducible lysosomal thiol reductase revealed considerable heterogeneity in the generation of individual epitopes, an arrangement that ensures peptide diversity and broad CD4+ T cell engagement. These results could fundamentally revise strategies for rational vaccine design and may lead to key insights into the induction of autoimmune and anti-tumor responses.
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Affiliation(s)
- Michael A Miller
- Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA
| | - Asha Purnima V Ganesan
- Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA
| | - Nancy Luckashenak
- Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA
| | - Mark Mendonca
- Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA
| | - Laurence C Eisenlohr
- Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA
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Feng C, Tan M, Sun W, Shi Y, Xing Z. Attenuation of the influenza virus by microRNA response element in vivo and protective efficacy against 2009 pandemic H1N1 virus in mice. Int J Infect Dis 2015; 38:146-52. [PMID: 26163223 DOI: 10.1016/j.ijid.2015.07.002] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2015] [Revised: 06/29/2015] [Accepted: 07/02/2015] [Indexed: 11/28/2022] Open
Abstract
BACKGROUND The 2009 influenza pandemics underscored the need for effective vaccines to block the spread of influenza virus infection. Most live attenuated vaccines utilize cold-adapted, temperature-sensitive virus. An alternative to live attenuated virus is presented here, based on microRNA-induced gene silencing. METHODS In this study, miR-let-7b target sequences were inserted into the H1N1 genome to engineer a recombinant virus - miRT-H1N1. Female BALB/c mice were vaccinated intranasally with the miRT-H1N1 and challenged with a lethal dose of homologous virus. RESULTS This miRT-H1N1 virus was attenuated in mice, while it exhibited wild-type characteristics in chicken embryos. Mice vaccinated intranasally with the miRT-H1N1 responded with robust immunity that protected the vaccinated mice from a lethal challenge with the wild-type 2009 pandemic H1N1 virus. CONCLUSIONS These results indicate that the influenza virus containing microRNA response elements (MREs) is attenuated in vivo and can be used to design a live attenuated vaccine.
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Affiliation(s)
- Chunlai Feng
- Department of Respiratory and Critical Care Medicine, Jinling Hospital affiliated to Southern Medical University, Nanjing, China
| | - Mingming Tan
- Department of Respiratory and Critical Care Medicine, Jinling Hospital affiliated to Southern Medical University, Nanjing, China
| | - Wenkui Sun
- Department of Respiratory and Critical Care Medicine, Jinling Hospital affiliated to Southern Medical University, Nanjing, China
| | - Yi Shi
- Department of Respiratory and Critical Care Medicine, Jinling Hospital affiliated to Southern Medical University, Nanjing, China.
| | - Zheng Xing
- The Key Laboratory of Pharmaceutical Biotechnology and Medical School, Nanjing University, Nanjing, China; Department of Veterinary Biomedical Sciences, College of Veterinary Medicine, University of Minnesota at Twin Cities, Saint Paul, Minnesota, USA.
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40
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Benitez AA, Panis M, Xue J, Varble A, Shim JV, Frick AL, López CB, Sachs D, tenOever BR. In Vivo RNAi Screening Identifies MDA5 as a Significant Contributor to the Cellular Defense against Influenza A Virus. Cell Rep 2015; 11:1714-26. [PMID: 26074083 DOI: 10.1016/j.celrep.2015.05.032] [Citation(s) in RCA: 60] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2014] [Revised: 02/09/2015] [Accepted: 05/18/2015] [Indexed: 11/15/2022] Open
Abstract
Responding to an influenza A virus (IAV) infection demands an effective intrinsic cellular defense strategy to slow replication. To identify contributing host factors to this defense, we exploited the host microRNA pathway to perform an in vivo RNAi screen. To this end, IAV, lacking a functional NS1 antagonist, was engineered to encode individual siRNAs against antiviral host genes in an effort to rescue attenuation. This screening platform resulted in the enrichment of strains targeting virus-activated transcription factors, specific antiviral effectors, and intracellular pattern recognition receptors (PRRs). Interestingly, in addition to RIG-I, the PRR for IAV, a virus with the capacity to silence MDA5 also emerged as a dominant strain in wild-type, but not in MDA5-deficient mice. Transcriptional profiling of infected knockout cells confirmed RIG-I to be the primary PRR for IAV but implicated MDA5 as a significant contributor to the cellular defense against influenza A virus.
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Affiliation(s)
- Asiel A Benitez
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Maryline Panis
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Jia Xue
- Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Andrew Varble
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Jaehee V Shim
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Amy L Frick
- Department of Neurosciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Carolina B López
- Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - David Sachs
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Benjamin R tenOever
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
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41
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Investigating influenza A virus infection: tools to track infection and limit tropism. J Virol 2015; 89:6167-70. [PMID: 25855737 DOI: 10.1128/jvi.00462-15] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
Influenza A viruses display a broad cellular tropism within the respiratory tracts of mammalian hosts. Uncovering the relationship between tropism and virus immunity, pathogenesis, and transmission will be critical for the development of therapeutic interventions. Here we discuss recent developments of several recombinant strains of influenza A virus. These viruses have inserted reporters to track tropism, microRNA target sites to restrict tropism, or barcodes to assess transmission dynamics, expanding our understanding of pathogen-host interactions.
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42
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Khanna M, Saxena L, Rajput R, Kumar B, Prasad R. Gene silencing: a therapeutic approach to combat influenza virus infections. Future Microbiol 2015; 10:131-40. [DOI: 10.2217/fmb.14.94] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
ABSTRACT Selective gene silencing technologies such as RNA interference (RNAi) and nucleic acid enzymes have shown therapeutic potential for treating viral infections. Influenza virus is one of the major public health concerns around the world and its management is challenging due to a rapid increase in antiviral resistance. Influenza vaccine also has its limitations due to the emergence of new strains that may escape the immunity developed by the previous year's vaccine. Antiviral drugs are the primary mode of prevention and control against a pandemic and there is an urgency to develop novel antiviral strategies against influenza virus. In this review, we discuss the potential utility of several gene silencing mechanisms and their prophylactic and therapeutic potential against the influenza virus.
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Affiliation(s)
- Madhu Khanna
- Department of Respiratory Virology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India
| | - Latika Saxena
- Department of Respiratory Virology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India
| | - Roopali Rajput
- Department of Respiratory Virology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India
| | - Binod Kumar
- Department of Respiratory Virology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India
| | - Rajendra Prasad
- Department of Pulmonary Medicine, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India
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Heaton NS, Langlois RA, Sachs D, Lim JK, Palese P, tenOever BR. Long-term survival of influenza virus infected club cells drives immunopathology. ACTA ACUST UNITED AC 2014; 211:1707-14. [PMID: 25135297 PMCID: PMC4144728 DOI: 10.1084/jem.20140488] [Citation(s) in RCA: 80] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
Respiratory infection of influenza A virus (IAV) is frequently characterized by extensive immunopathology and proinflammatory signaling that can persist after virus clearance. In this report, we identify cells that become infected, but survive, acute influenza virus infection. We demonstrate that these cells, known as club cells, elicit a robust transcriptional response to virus infection, show increased interferon stimulation, and induce high levels of proinflammatory cytokines after successful viral clearance. Specific depletion of these surviving cells leads to a reduction in lung tissue damage associated with IAV infection. We propose a model in which infected, surviving club cells establish a proinflammatory environment aimed at controlling virus levels, but at the same time contribute to lung pathology.
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Affiliation(s)
- Nicholas S Heaton
- Department of Microbiology, Global Health and Emerging Pathogens Institute, and Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - Ryan A Langlois
- Department of Microbiology, Global Health and Emerging Pathogens Institute, and Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029 Department of Microbiology, Global Health and Emerging Pathogens Institute, and Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - David Sachs
- Department of Microbiology, Global Health and Emerging Pathogens Institute, and Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - Jean K Lim
- Department of Microbiology, Global Health and Emerging Pathogens Institute, and Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - Peter Palese
- Department of Microbiology, Global Health and Emerging Pathogens Institute, and Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - Benjamin R tenOever
- Department of Microbiology, Global Health and Emerging Pathogens Institute, and Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029 Department of Microbiology, Global Health and Emerging Pathogens Institute, and Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029
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44
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Abstract
Virus-host interactions highlight key regulatory steps in the control of gene expression. MicroRNAs (miRNAs) are small noncoding RNAs that regulate protein production via base pairing with mRNAs. Both DNA and RNA viruses have evolved mechanisms to degrade, boost, or hijack cellular miRNAs to benefit the viral life cycle. This minireview focuses on recent discoveries in virus-host miRNA interactions.
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45
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The Mammalian response to virus infection is independent of small RNA silencing. Cell Rep 2014; 8:114-25. [PMID: 24953656 DOI: 10.1016/j.celrep.2014.05.038] [Citation(s) in RCA: 62] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2014] [Revised: 04/24/2014] [Accepted: 05/14/2014] [Indexed: 01/18/2023] Open
Abstract
A successful cellular response to virus infection is essential for evolutionary survival. In plants, arthropods, and nematodes, cellular antiviral defenses rely on RNAi. Interestingly, the mammalian response to virus is predominantly orchestrated through interferon (IFN)-mediated induction of antiviral proteins. Despite the potency of the IFN system, it remains unclear whether mammals also have the capacity to employ antiviral RNAi. Here, we investigated this by disabling IFN function, small RNA function, or both activities in the context of virus infection. We find that loss of small RNAs in the context of an in vivo RNA virus infection lowers titers due to reduced transcriptional repression of the host antiviral response. In contrast, enabling a virus with the capacity to inhibit the IFN system results in increased titers. Taken together, these results indicate that small RNA silencing is not a physiological contributor to the IFN-mediated cellular response to virus infection.
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46
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Iwasaki A, Pillai PS. Innate immunity to influenza virus infection. Nat Rev Immunol 2014; 14:315-28. [PMID: 24762827 DOI: 10.1038/nri3665] [Citation(s) in RCA: 841] [Impact Index Per Article: 76.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Influenza viruses are a major pathogen of both humans and animals. Recent studies using gene-knockout mice have led to an in-depth understanding of the innate sensors that detect influenza virus infection in a variety of cell types. Signalling downstream of these sensors induces distinct sets of effector mechanisms that block virus replication and promote viral clearance by inducing innate and adaptive immune responses. In this Review, we discuss the various ways in which the innate immune system uses pattern recognition receptors to detect and respond to influenza virus infection. We consider whether the outcome of innate sensor stimulation promotes antiviral resistance or disease tolerance, and propose rational treatment strategies for the acute respiratory disease that is caused by influenza virus infection.
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Affiliation(s)
- Akiko Iwasaki
- Department of Immunobiology, Yale University School of Medicine, 300 Cedar Street, New Haven, Connecticut 06520, USA
| | - Padmini S Pillai
- Department of Immunobiology, Yale University School of Medicine, 300 Cedar Street, New Haven, Connecticut 06520, USA
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Shapiro JS, Schmid S, Aguado LC, Sabin LR, Yasunaga A, Shim JV, Sachs D, Cherry S, tenOever BR. Drosha as an interferon-independent antiviral factor. Proc Natl Acad Sci U S A 2014; 111:7108-13. [PMID: 24778219 PMCID: PMC4024876 DOI: 10.1073/pnas.1319635111] [Citation(s) in RCA: 53] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Utilization of antiviral small interfering RNAs is thought to be largely restricted to plants, nematodes, and arthropods. In an effort to determine whether a physiological interplay exists between the host small RNA machinery and the cellular response to virus infection in mammals, we evaluated antiviral activity in the presence and absence of Dicer or Drosha, the RNase III nucleases responsible for generating small RNAs. Although loss of Dicer did not compromise the cellular response to virus infection, Drosha deletion resulted in a significant increase in virus levels. Here, we demonstrate that diverse RNA viruses trigger exportin 1 (XPO1/CRM1)-dependent Drosha translocation into the cytoplasm in a manner independent of de novo protein synthesis or the canonical type I IFN system. Additionally, increased virus infection in the absence of Drosha was not due to a loss of viral small RNAs but, instead, correlated with cleavage of viral genomic RNA and modulation of the host transcriptome. Taken together, we propose that Drosha represents a unique and conserved arm of the cellular defenses used to combat virus infection.
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Affiliation(s)
- Jillian S Shapiro
- Department of Microbiology,Icahn Graduate School of Biomedical Sciences, and
| | | | - Lauren C Aguado
- Department of Microbiology,Icahn Graduate School of Biomedical Sciences, and
| | - Leah R Sabin
- Department of Microbiology, University of Pennsylvania, Philadelphia, PA 19104
| | - Ari Yasunaga
- Department of Microbiology, University of Pennsylvania, Philadelphia, PA 19104
| | | | - David Sachs
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029; and
| | - Sara Cherry
- Department of Microbiology, University of Pennsylvania, Philadelphia, PA 19104
| | - Benjamin R tenOever
- Department of Microbiology,Icahn Graduate School of Biomedical Sciences, and
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48
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Teterina NL, Liu G, Maximova OA, Pletnev AG. Silencing of neurotropic flavivirus replication in the central nervous system by combining multiple microRNA target insertions in two distinct viral genome regions. Virology 2014; 456-457:247-58. [PMID: 24889244 DOI: 10.1016/j.virol.2014.04.001] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2014] [Revised: 02/28/2014] [Accepted: 04/01/2014] [Indexed: 01/23/2023]
Abstract
In recent years, microRNA-targeting has become an effective strategy for selective control of tissue-tropism and pathogenesis of both DNA and RNA viruses. Here, using a neurotropic flavivirus as a model, we demonstrate that simultaneous miRNA targeting of the viral genome in the open reading frame and 3'-noncoding regions for brain-expressed miRNAs had an additive effect and produced a more potent attenuation of the virus compared to separate targeting of those regions. Multiple miRNA co-targeting of these two distantly located regions completely abolished the virus neurotropism as no viral replication was detected in the developing brain of neonatal mice. Furthermore, no viral antigens were detected in neurons, and neuronal integrity in the brain of mice was well preserved. This miRNA co-targeting approach can be adapted for other viruses in order to minimize their replication in a cell- or tissue-type specific manner, but most importantly, to prevent virus escape from miRNA-mediated silencing.
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Affiliation(s)
- Natalya L Teterina
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, United States
| | - Guangping Liu
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, United States
| | - Olga A Maximova
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, United States
| | - Alexander G Pletnev
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, United States.
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49
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Kroeker AL, Coombs KM. Systems biology unravels interferon responses to respiratory virus infections. World J Biol Chem 2014; 5:12-25. [PMID: 24600511 PMCID: PMC3942539 DOI: 10.4331/wjbc.v5.i1.12] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/10/2013] [Revised: 12/11/2013] [Accepted: 01/06/2014] [Indexed: 02/05/2023] Open
Abstract
Interferon production is an important defence against viral replication and its activation is an attractive therapeutic target. However, it has long been known that viruses perpetually evolve a multitude of strategies to evade these host immune responses. In recent years there has been an explosion of information on virus-induced alterations of the host immune response that have resulted from data-rich omics technologies. Unravelling how these systems interact and determining the overall outcome of the host response to viral infection will play an important role in future treatment and vaccine development. In this review we focus primarily on the interferon pathway and its regulation as well as mechanisms by which respiratory RNA viruses interfere with its signalling capacity.
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Trobaugh DW, Gardner CL, Sun C, Haddow AD, Wang E, Chapnik E, Mildner A, Weaver SC, Ryman KD, Klimstra WB. RNA viruses can hijack vertebrate microRNAs to suppress innate immunity. Nature 2013; 506:245-8. [PMID: 24352241 PMCID: PMC4349380 DOI: 10.1038/nature12869] [Citation(s) in RCA: 182] [Impact Index Per Article: 15.2] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2013] [Accepted: 11/05/2013] [Indexed: 12/17/2022]
Affiliation(s)
- Derek W Trobaugh
- Center for Vaccine Research and Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA
| | - Christina L Gardner
- Center for Vaccine Research and Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA
| | - Chengqun Sun
- Center for Vaccine Research and Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA
| | - Andrew D Haddow
- Institute for Human Infections and Immunity, Center for Biodefense and Emerging Infectious Diseases, and Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555, USA
| | - Eryu Wang
- Institute for Human Infections and Immunity, Center for Biodefense and Emerging Infectious Diseases, and Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555, USA
| | - Elik Chapnik
- Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel
| | - Alexander Mildner
- Department of Immunology, Weizmann Institute of Science, Rehovot 7610001, Israel
| | - Scott C Weaver
- Institute for Human Infections and Immunity, Center for Biodefense and Emerging Infectious Diseases, and Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555, USA
| | - Kate D Ryman
- Center for Vaccine Research and Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA
| | - William B Klimstra
- Center for Vaccine Research and Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA
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