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Shirasaki T, Lenarcic E, Misumi I, Xie L, Fusco WG, Yonish B, Das A, Kim H, Cameron CE, Léger-Abraham M, Chen X, Cullen JM, Whitmire JK, Li Y, Duncan JA, Moorman NJ, Lemon SM. Hepatovirus translation requires PDGFA-associated protein 1, an eIF4E-binding protein regulating endoplasmic reticulum stress responses. SCIENCE ADVANCES 2024; 10:eadq6342. [PMID: 39565848 PMCID: PMC11578187 DOI: 10.1126/sciadv.adq6342] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/23/2024] [Accepted: 10/17/2024] [Indexed: 11/22/2024]
Abstract
The overexpression and misfolding of viral proteins in the endoplasmic reticulum (ER) may cause cellular stress, thereby inducing a cytoprotective, proteostatic host response involving phosphorylation of eukaryotic translation initiation factor 2 subunit alpha (eIF2α). Here, we show that hepatitis A virus, a positive-strand RNA virus responsible for infectious hepatitis, adopts a stress-resistant, eIF2α-independent mechanism of translation to ensure the synthesis of viral proteins within the infected liver. Cap-independent translation directed by the hepatovirus internal ribosome entry site and productive hepatovirus infection of mice both require platelet-derived growth factor subunit A (PDGFA)-associated protein 1 (PDAP1), a small phosphoprotein of unknown function with eIF4E-binding activity. PDAP1 also interacts with eIF1A and is essential for translating stress-resistant host messenger RNAs that evade the proteostatic response to ER stress and that encode proteins promoting the survival of stressed cells.
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Affiliation(s)
- Takayoshi Shirasaki
- Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Erik Lenarcic
- Department of Microbiology and Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Ichiro Misumi
- Department of Genetics, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Ling Xie
- Department of Biochemistry and Biophysics, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - William G. Fusco
- Department of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Bryan Yonish
- Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Anshuman Das
- Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Hyejeong Kim
- Department of Microbiology and Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Craig E. Cameron
- Department of Microbiology and Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Mélissa Léger-Abraham
- Division of Molecular Medicine, Harvard Medical School, Boston, MA, USA
- Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA
| | - Xian Chen
- Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
- Department of Biochemistry and Biophysics, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - John M. Cullen
- College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA
| | - Jason K. Whitmire
- Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
- Department of Genetics, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - You Li
- Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
- Department of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Joseph A. Duncan
- Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
- Department of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Nathaniel J. Moorman
- Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
- Department of Microbiology and Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Stanley M. Lemon
- Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
- Department of Microbiology and Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
- Department of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
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2
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Chavarria-Miró G, de Castellarnau M, Fuentes C, D'Andrea L, Pérez-Rodríguez FJ, Beguiristain N, Bosch A, Guix S, Pintó RM. Advances for the Hepatitis A Virus Antigen Production Using a Virus Strain With Codon Frequency Optimization Adjustments in Specific Locations. Front Microbiol 2021; 12:642267. [PMID: 33679679 PMCID: PMC7935560 DOI: 10.3389/fmicb.2021.642267] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2020] [Accepted: 01/26/2021] [Indexed: 12/13/2022] Open
Abstract
The available cell-adapted hepatitis A virus (HAV) strains show a very slow replication phenotype hampering the affordable production of antigen. A fast-growing strain characterized by the occurrence of mutations in the internal ribosome entry site (IRES), combined with changes in the codon composition has been selected in our laboratory. A characterization of the IRES activity of this fast-growing strain (HM175-HP; HP) vs. its parental strain (HM175; L0) was assessed in two cell substrates used in vaccine production (MRC-5 and Vero cells) compared with the FRhK-4 cell line in which its selection was performed. The HP-derived IRES was significantly more active than the L0-derived IRES in all cells tested and both IRES were more active in the FRhK-4 cells. The translation efficiency of the HP-derived IRES was also much higher than the L0-derived IRES, particularly, in genes with a HP codon usage background. These results correlated with a higher virus production in a shorter time for the HP strain compared to the L0 strain in any of the three cell lines tested, and of both strains in the FRhK-4 cells compared to Vero and MRC-5 cells. The addition of wortmannin resulted in the increase of infectious viruses and antigen in the supernatant of FRhK-4 infected cells, independently of the strain. Finally, the replication of both strains in a clone of FRhK-4 cells adapted to grow with synthetic sera was optimal and again the HP strain showed higher yields.
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Affiliation(s)
- Gemma Chavarria-Miró
- Enteric Virus Laboratory, Department of Genetics, Microbiology and Statistics, School of Biology, Institute of Nutrition and Food Safety, Campus Torribera, University of Barcelona, Barcelona, Spain
| | - Montserrat de Castellarnau
- Enteric Virus Laboratory, Department of Genetics, Microbiology and Statistics, School of Biology, Institute of Nutrition and Food Safety, Campus Torribera, University of Barcelona, Barcelona, Spain
| | - Cristina Fuentes
- Enteric Virus Laboratory, Department of Genetics, Microbiology and Statistics, School of Biology, Institute of Nutrition and Food Safety, Campus Torribera, University of Barcelona, Barcelona, Spain
| | - Lucía D'Andrea
- Enteric Virus Laboratory, Department of Genetics, Microbiology and Statistics, School of Biology, Institute of Nutrition and Food Safety, Campus Torribera, University of Barcelona, Barcelona, Spain
| | - Francisco-Javier Pérez-Rodríguez
- Enteric Virus Laboratory, Department of Genetics, Microbiology and Statistics, School of Biology, Institute of Nutrition and Food Safety, Campus Torribera, University of Barcelona, Barcelona, Spain
| | - Nerea Beguiristain
- Enteric Virus Laboratory, Department of Genetics, Microbiology and Statistics, School of Biology, Institute of Nutrition and Food Safety, Campus Torribera, University of Barcelona, Barcelona, Spain
| | - Albert Bosch
- Enteric Virus Laboratory, Department of Genetics, Microbiology and Statistics, School of Biology, Institute of Nutrition and Food Safety, Campus Torribera, University of Barcelona, Barcelona, Spain
| | - Susana Guix
- Enteric Virus Laboratory, Department of Genetics, Microbiology and Statistics, School of Biology, Institute of Nutrition and Food Safety, Campus Torribera, University of Barcelona, Barcelona, Spain
| | - Rosa M Pintó
- Enteric Virus Laboratory, Department of Genetics, Microbiology and Statistics, School of Biology, Institute of Nutrition and Food Safety, Campus Torribera, University of Barcelona, Barcelona, Spain
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Feng Z, Hirai-Yuki A, McKnight KL, Lemon SM. Naked Viruses That Aren't Always Naked: Quasi-Enveloped Agents of Acute Hepatitis. Annu Rev Virol 2014; 1:539-60. [PMID: 26958733 DOI: 10.1146/annurev-virology-031413-085359] [Citation(s) in RCA: 115] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Historically, viruses were considered to be either enveloped or nonenveloped. However, recent work on hepatitis A virus and hepatitis E virus challenges this long-held tenet. Whereas these human pathogens are shed in feces as naked nonenveloped virions, recent studies indicate that both circulate in the blood completely masked in membranes during acute infection. These membrane-wrapped virions are as infectious as their naked counterparts, although they do not express a virally encoded protein on their surface, thus distinguishing them from conventional enveloped viruses. The absence of a viral fusion protein implies that these quasi-enveloped virions have unique mechanisms for entry into cells. Like true enveloped viruses, however, these phylogenetically distinct viruses usurp components of the host ESCRT system to hijack host cell membranes and noncytolytically exit infected cells. The membrane protects these viruses from neutralizing antibodies, facilitating dissemination within the host, whereas nonenveloped virions shed in feces are stable in the environment, allowing for epidemic transmission.
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Affiliation(s)
- Zongdi Feng
- Lineberger Comprehensive Cancer Center, Inflammatory Diseases Institute, and Departments of Medicine and Microbiology & Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7292;
| | - Asuka Hirai-Yuki
- Lineberger Comprehensive Cancer Center, Inflammatory Diseases Institute, and Departments of Medicine and Microbiology & Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7292;
| | - Kevin L McKnight
- Lineberger Comprehensive Cancer Center, Inflammatory Diseases Institute, and Departments of Medicine and Microbiology & Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7292;
| | - Stanley M Lemon
- Lineberger Comprehensive Cancer Center, Inflammatory Diseases Institute, and Departments of Medicine and Microbiology & Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7292;
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Hafrén A, Eskelin K, Mäkinen K. Ribosomal protein P0 promotes Potato virus A infection and functions in viral translation together with VPg and eIF(iso)4E. J Virol 2013; 87:4302-12. [PMID: 23365448 PMCID: PMC3624370 DOI: 10.1128/jvi.03198-12] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2012] [Accepted: 01/25/2013] [Indexed: 11/20/2022] Open
Abstract
We report here that the acidic ribosomal protein P0 is a component of the membrane-associated Potato virus A (PVA) ribonucleoprotein complex. As a constituent of the ribosomal stalk, P0 functions in translation. Although the ribosomal stalk proteins P0, P1, P2, and P3 are all important for PVA infection, P0 appears to have a distinct role from those of the other stalk proteins in infection. Our results indicate that P0 also regulates viral RNA functions as an extraribosomal protein. We reported previously that PVA RNA can be targeted by VPg to a specific gene expression pathway that protects the viral RNA from degradation and facilitates its translation. Here, we show that P0 is essential for this activity of VPg, similar to eIF4E/eIF(iso)4E. We also demonstrate that VPg, P0, and eIF(iso)4E synergistically enhance viral translation. Interestingly, the positive effects of VPg and P0 on viral translation were negatively correlated with the cell-to-cell spread of infection, suggesting that these processes may compete for viral RNA.
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Affiliation(s)
- Anders Hafrén
- Department of Food and Environmental Sciences, University of Helsinki, Helsinki, Finland
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Wales SQ, Ngo D, Hida K, Kulka M. Temperature and density dependent induction of a cytopathic effect following infection with non-cytopathic HAV strains. Virology 2012; 430:30-42. [PMID: 22608060 DOI: 10.1016/j.virol.2012.04.010] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2011] [Revised: 02/21/2012] [Accepted: 04/17/2012] [Indexed: 11/26/2022]
Abstract
Hepatitis A virus infection and growth in cultured cells is protracted, cell-type restricted, and generally not accompanied by the appearance of a cytopathic effect, with the exception of some culture-adapted strains. We demonstrate that the non-cytopathic HAV strain HM175/clone 1 can be induced to exhibit a cytopathic phenotype in both persistently or acutely infected cells under co-dependent conditions of lower incubation temperature (<34°C) and reduced cell density in both monkey (FRhK-4) and human (A549) cells. This phenotype is not virus-strain restricted, as it was also observed in cells infected with HAV strains, HAS-15 and LSH/S. Cytopathic effect was accompanied by rRNA cleavage, indicating activation of the RNase L pathway, viral negative strand synthesis, caspase-3 activation, and apoptosis. The results indicate that a cytopathic phenotype may be present in some HAV strains that can be induced under appropriate conditions, suggesting the potential for development of a plaque assay for this virus.
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Affiliation(s)
- Samantha Q Wales
- Division of Molecular Biology, Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 8301 Muirkirk Road, Laurel, MD 20708, USA.
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Goswami BB, Kulka M, Ngo D, Cebula TA. Apoptosis induced by a cytopathic hepatitis A virus is dependent on caspase activation following ribosomal RNA degradation but occurs in the absence of 2'-5' oligoadenylate synthetase. Antiviral Res 2004; 63:153-66. [PMID: 15451183 PMCID: PMC7127220 DOI: 10.1016/j.antiviral.2004.02.004] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2003] [Accepted: 02/23/2004] [Indexed: 12/26/2022]
Abstract
We have presented previously evidence that the cytopathogenic 18f strain of hepatitis A virus (HAV) induced degradation of ribosomal RNA (rRNA) in infected cells [Arch. Virol. 148 (2003) 1275–1300]. In contrast, the non-cytopathogenic parent virus HM175 clone 1 had no effect on rRNA integrity. We present here data showing that rRNA degradation is followed by apoptosis accompanied by characteristic DNA laddering in the cytoplasm of 18f infected cells. The DNA laddering coincided with the detection of caspase 3 and PARP-1 cleavage and was dependent upon activation of the caspase pathway, since treatment with Z-VAD-FMK, a pan-caspase inhibitor, inhibited both events. RNase L mRNA was present in both virus-infected and uninfected cells. Messenger RNA for the interferon inducible enzyme 2′–5′ oligoadenylate synthetase (2′–5′ OAS), which polymerizes ATP into 2′–5′ oligo adenylate (2–5A, the activator of RNase L) in the presence of double-stranded RNA, was not detected following virus infection. 2′–5′ OAS mRNA was induced by treatment of the cells with interferon-β (IFN-β). IFN-β mRNA was marginally induced following infection. However, phosphorylated STAT 1, a key regulator of interferon-stimulated gene transcription was not detected in virus infected cells. STAT 1 phosphorylation in response to IFN treatment was lower in virus-infected cells, compared to uninfected cells treated with interferon, suggesting that 18f virus infection interferes with interferon signaling. The results suggest that 18f infection causes the induction of a 2–5A independent RNase L like activity.
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Affiliation(s)
- Biswendu B Goswami
- Division of Molecular Biology, Office of Applied Research and Safety Assessment, Food and Drug Administration, HFS-025, OARSA, FDA, 8301 Muirkirk Road, Laurel, MD 20708, USA,
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8
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Ronfort C, De Breyne S, Sandrin V, Darlix JL, Ohlmann T. Characterization of two distinct RNA domains that regulate translation of the Drosophila gypsy retroelement. RNA (NEW YORK, N.Y.) 2004; 10:504-515. [PMID: 14970395 PMCID: PMC1370945 DOI: 10.1261/rna.5185604] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/12/2003] [Accepted: 11/19/2003] [Indexed: 05/24/2023]
Abstract
The genomic RNA of the gypsy retroelement from Drosophila melanogaster exhibits features similar to other retroviral RNAs because its 5' untranslated (5' UTR) region is unusually long (846 nucleotides) and potentially highly structured. Our initial aim was to search for an internal ribosome entry site (IRES) element in the 5' UTR of the gypsy genomic RNA by using various monocistronic and bicistronic RNAs in the rabbit reticulocyte lysate (RRL) system and in cultured cells. Results reported here show that two functionally distinct and independent RNA domains control the production of gypsy encoded proteins. The first domain corresponds to the 5' UTR of the env subgenomic RNA and exhibits features of an efficient IRES (IRES(E)) both in the reticulocyte lysate and in cells. The second RNA domain that encompasses the gypsy insulator can function as an IRES in the rabbit reticulocyte lysate but strongly represses translation in cultured cells. Taken together, these results suggest that expression of the gypsy encoded proteins from the genomic and subgenomic RNAs can be regulated at the level of translation.
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Affiliation(s)
- Corinne Ronfort
- LaboRetro, INSERM U 412, Ecole Normale Supérieure de Lyon, 69364 Lyon, France
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Losick VP, Schlax PE, Emmons RA, Lawson TG. Signals in hepatitis A virus P3 region proteins recognized by the ubiquitin-mediated proteolytic system. Virology 2003; 309:306-19. [PMID: 12758177 DOI: 10.1016/s0042-6822(03)00071-0] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
The hepatitis A virus 3C protease and 3D RNA polymerase are present in low concentrations in infected cells. The 3C protease was previously shown to be rapidly degraded by the ubiquitin/26S proteasome system and we present evidence here that the 3D polymerase is also subject to ubiquitination-mediated proteolysis. Our results show that the sequence (32)LGVKDDWLLV(41) in the 3C protease serves as a protein destruction signal recognized by the ubiquitin-protein ligase E3alpha and that the destruction signal for the RNA polymerase does not require the carboxyl-terminal 137 amino acids. Both the viral 3ABCD polyprotein and the 3CD diprotein were also found to be substrates for ubiquitin-mediated proteolysis. Attempts to determine if the 3C protease or the 3D polymerase destruction signals trigger the ubiquitination and degradation of these precursors yielded evidence suggesting, but not unequivocally proving, that the recognition of the 3D polymerase by the ubiquitin system is responsible.
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Affiliation(s)
- Vicki P Losick
- Department of Chemistry, Bates College, Lewiston, ME 04240, USA
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Hu NZ, Hu YZ, Shi HJ, Liu GD, Qu S. Mutational characteristics in consecutive passage of rapidly replicating variants of hepatitis A virus strain H2 during cell culture adaptation. World J Gastroenterol 2002; 8:872-8. [PMID: 12378633 PMCID: PMC4656578 DOI: 10.3748/wjg.v8.i5.872] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the molecular mechanism of cell adaptation and rapid replication of hepatitis A virus strain H2 in KBM17 cells.
METHODS: Virus of strain H2 at passage 7 was consecutively passaged in KBM17 cells for 22 passages, every passage was incubated for 14 d. Antigenic and infectious titers of every passage and one-step growth dynamics of passage 22 were determined with ELISA. Genomes of passage 6, passage 12, passage 18 and passage 22 were sequenced and compared with H2K7.
RESULTS: During continuous passage of vaccine strain H2 at passage K7 in KMB17 cells, infectious and antigenic titers increased with the increase of passages, infectious titers at day 14 reached 6.77 LgCCID50ml-1 for passage 6 (P6), 7.0 LgCCID50ml-1 for passage 12 (P12), 7.33 LgCCID50ml-1 for passage 18 (P18) and 7.83 LgCCID50ml-1 for passage 22 (P22), respectively. The one-step growth dynamics showed that replicating peak of P22 appeared at day 14 with infectious titers of 7.83 LgCCID50ml-1 and antigenic titer of 1:1024. After passage 22 a new cell-adapted variant (P22) of H2K7 with rapid and shortened replication cycle from 28 d to 14 d was obtained. Sequencing and comparisons of genomes of P6, P12, P18 and P22 showed that mutational numbers in genomes of different passages increased with adaptive passages, and mutations scattered over the genome. In comparison with that of K7, P6 had only 6 nucleotides (nt) mutations, P12 had 7 mutational changes, in addition to 6 same mutations with P6, there appeared a new mutation in 5'NTR at nucleotide position 591 resulting in a nucleotide exchange from A to G. P18 had 10 nt mutations, among the 10 mutations, 7 mutational changes were same as with P12, three new mutational changes appeared in the genome, one in 5'NTR, one in 3C coding region, one in 3D coding region, at P22 there appeared 18 nucleotide changes in the genome, on the basis of P18, there occured additional 8 nucleotide mutations, two in 5'NTR, three in 2C, one in 3A, one in 3C and one in 3D. The results suggested that although H2K7 was already an attenuated strain, the mutations of genome is not sufficient to completely adapt the KMB17, further mutations caused rapid replication adaptation.
CONCLUSION: 18-nt changes scattering over the genome are cooperatively responsible for further adaptation characterized by rapid and shortened replication cycle from 28 d to 14 d in KMB17 cells. The mutations in 2C coding region play more important role in increase of infectious titer than other mutations, the mutations in 2B coding region show less important role than it usually does in cell adaptation, nucleotide changes in 5’NTR seem to be not relevant to cell adaptation during initial stages (before P6), but do in late stages.
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Affiliation(s)
- Ning-Zhu Hu
- Department of Vaccine Research, Institute of Medical Biology, Chinese Academy of Medical Sciences, Peking Union of Medical College, Kunming, 650118, Yunnan Province, China
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Kusov YY, Shatirishvili G, Klinger M, Gauss-Müller V. A vaccinia virus MVA-T7-mediated recovery of infectious hepatitis A virus from full-size cDNA or from two cDNAs, both by themselves unable to complete the virus life cycle. Virus Res 2002; 89:75-88. [PMID: 12367752 DOI: 10.1016/s0168-1702(02)00115-6] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
The replication-deficient vaccinia virus (VV) MVA-T7 produces large amounts of T7 RNA polymerase and permits efficient protein expression from cDNA of T7-promoted genes. Yet, unlike recombinant VV vTF7-3, (VV) MVA-T7 produces no cytopathic effect in primate cells, thus allowing the study of processes with slow kinetics. We have applied MVA-T7 to aid genome expression of HAV, a representative of the Picornaviridae family that is well known for its inefficient replication in mammalian cell cultures. After cDNA transfection and MVA-T7 infection, empty capsids and mature HAV particles were formed with different kinetics and were characterized by their morphology, protein content, and infectivity. The data suggests that HAV genome replication is initiated from RNA, which was transcribed in vivo by the MVA-T7-encoded T7 RNA polymerase. HAV genome replication was also demonstrated in a recombination assay. After co-expression of two subgenomic HAV cDNAs, both by themselves unable to complete the viral life cycle, infectious HAV was rescued, indicating that replication-dependent genetic recombination has occurred. We propose that the high-level genome expression mediated in vivo by the VV-encoded T7 RNA polymerase augments the amount of viral RNA, such that replication of viruses poorly replicating in cell cytoplasm is detectable.
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Affiliation(s)
- Yuri Y Kusov
- Institute of Medical Molecular Biology, Medical University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany.
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12
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Jones LR, Zandomeni R, Weber EL. Quasispecies in the 5' untranslated genomic region of bovine viral diarrhoea virus from a single individual. J Gen Virol 2002; 83:2161-2168. [PMID: 12185269 DOI: 10.1099/0022-1317-83-9-2161] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
The variability of the 5' untranslated genomic region (5'UTR) of bovine viral diarrhoea virus (BVDV) RNA obtained from a single individual was analysed. Lung, kidney and spleen tissues from a naturally infected foetus were used as the source of viral RNA. A fragment of 288 bases of the internal ribosome entry site from the BVDV 5'UTR was amplified by RT-PCR using a proofreading DNA polymerase. PCR products were cloned into pGem and, subsequently, transformed into Escherichia coli. The single-strand conformational polymorphisms of 158 lung-derived clones were analysed; a total of 11 banding patterns was observed. DNAs corresponding to all patterns were sequenced. Of the randomly selected clones, 11 and 10 clones derived from the kidney and spleen, respectively, were also sequenced. All sequences presented differences ranging from 1 to 6 nt substitutions. Analysis of the secondary structure of the variant sequences and comparisons to variant nucleotide sites from the 5'UTR of several BVDV isolates showed that the observed changes were almost free of randomness. Clustering and phylogenetic analyses suggested the existence of low-kinetic variants. BVDV quasispecies may be involved in establishing persistent infections by means of eluding maternal antibodies. The methods described here may be adapted easily both to analyse large numbers of samples from other genomic regions and for the study of BVDV quasispecies evolution in other systems.
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Affiliation(s)
- Leandro Roberto Jones
- Instituto de Virología, CICVyA, INTA-Castelar, CC77 (1708) Morón, Buenos Aires, Argentina1
| | - Rubén Zandomeni
- Consejo Nacional de Investigaciones Científicas, Argentina3
- Instituto de Microbiología y Zoología Agrícola, CICVyA, INTA-Castelar, CC77 (1708) Morón, Buenos Aires, Argentina2
| | - E Laura Weber
- Consejo Nacional de Investigaciones Científicas, Argentina3
- Instituto de Virología, CICVyA, INTA-Castelar, CC77 (1708) Morón, Buenos Aires, Argentina1
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Gauss-Müller V, Kusov YY. Replication of a hepatitis A virus replicon detected by genetic recombination in vivo. J Gen Virol 2002; 83:2183-2192. [PMID: 12185272 DOI: 10.1099/0022-1317-83-9-2183] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Unlike other picornaviruses, hepatitis A virus (HAV) replicates so inefficiently in cell culture that the study of its RNA biosynthesis presents a major experimental challenge. To assess viral RNA replication independent of particle formation, a subgenomic replicon representing a self-replicating RNA was constructed by replacing the P1 domain encoding the capsid proteins with the firefly luciferase sequence. Although translation of the HAV replicon was as efficient as a similar poliovirus replicon, the luciferase activity derived from replication of the HAV construct was more than 100-fold lower than that of poliovirus. The replication capacity of the HAV replicon was clearly demonstrated by its ability to recombine genetically with a non-viable, full-length HAV genome that served as capsid donor and thus to rescue a fully infectious virus. In contrast to a replication-deficient replicon, co-expression of the genetically marked and replication-competent HAV replicon with several lethally mutated HAV genomes resulted in the successful rescue of infectious HAV with a unique genetic marker. Our data suggest: (i) that autonomous HAV RNA replication does not require sequences for the HAV structural proteins; and (ii) that low-level genome replication can unequivocally be demonstrated by the rescue of infectious virus after co-expression with non-viable genomes.
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Affiliation(s)
- Verena Gauss-Müller
- Institute of Medical Molecular Biology, Medical University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany1
| | - Yuri Y Kusov
- Institute of Medical Molecular Biology, Medical University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany1
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Yi M, Lemon SM. Replication of subgenomic hepatitis A virus RNAs expressing firefly luciferase is enhanced by mutations associated with adaptation of virus to growth in cultured cells. J Virol 2002; 76:1171-80. [PMID: 11773393 PMCID: PMC135777 DOI: 10.1128/jvi.76.3.1171-1180.2002] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Replication of hepatitis A virus (HAV) in cultured cells is inefficient and difficult to study due to its protracted and generally noncytopathic cycle. To gain a better understanding of the mechanisms involved, we constructed a subgenomic HAV replicon by replacing most of the P1 capsid-coding sequence from an infectious cDNA copy of the cell culture-adapted HM175/18f virus genome with sequence encoding firefly luciferase. Replication of this RNA in transfected Huh-7 cells (derived from a human hepatocellular carcinoma) led to increased expression of luciferase relative to that in cells transfected with similar RNA transcripts containing a lethal premature termination mutation in 3D(pol) (RNA polymerase). However, replication could not be confirmed in either FrhK4 cells or BSC-1 cells, cells that are typically used for propagation of HAV. Replication was substantially slower than that observed with replicons derived from other picornaviruses, as the basal luciferase activity produced by translation of input RNA did not begin to increase until 24 to 48 h after transfection. Replication of the RNA was reversibly inhibited by guanidine. The inclusion of VP4 sequence downstream of the viral internal ribosomal entry site had no effect on the basal level of luciferase or subsequent increases in luciferase related to its amplification. Thus, in this system this sequence does not contribute to viral translation or replication, as suggested previously. Amplification of the replicon RNA was profoundly enhanced by the inclusion of P2 (but not 5' noncoding sequence or P3) segment mutations associated with adaptation of wild-type virus to growth in cell culture. These results provide a simple reporter system for monitoring the translation and replication of HAV RNA and show that critical mutations that enhance the growth of virus in cultured cells do so by promoting replication of viral RNA in the absence of encapsidation, packaging, and cellular export of the viral genome.
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Affiliation(s)
- MinKyung Yi
- Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019, USA
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Beard MR, Cohen L, Lemon SM, Martin A. Characterization of recombinant hepatitis A virus genomes containing exogenous sequences at the 2A/2B junction. J Virol 2001; 75:1414-26. [PMID: 11152515 PMCID: PMC114048 DOI: 10.1128/jvi.75.3.1414-1426.2001] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
Hepatitis A virus (HAV) differs from other members of the family Picornaviridae in that the cleavage of the polyprotein at the 2A/2B junction, commonly considered to be the primary polyprotein cleavage by analogy with other picornaviruses, is mediated by 3C(pro), the only proteinase encoded by the virus. However, it has never been formally demonstrated that the 2A/2B junction is the site of primary cleavage, and the actual function of the 2A sequence, which lacks homology with sequence of other picornaviruses, remains unknown. To determine whether 2A functions in cis as a precursor with the nonstructural proteins, we constructed dicistronic HAV genomes in which a heterologous picornaviral internal ribosome entry site was inserted at the 2A/2B junction. Transfection of permissive FRhK-4 cells with these dicistronic RNAs failed to result in the rescue of infectious virus, indicating a possible cis replication function spanning the 2A/2B junction. However, infectious virus was recovered from recombinant HAV genomes containing exogenous protein-coding sequences inserted in-frame at the 2A/2B junction and flanked by consensus 3C(pro) cleavage sites. The replication of these recombinants was less efficient than that of the parent virus but was variable and not dependent upon the length of the inserted sequence. An HAV recombinant containing a 420-nt insertion encoding the bleomycin resistance protein Zeo was stable for up to five passages in cell culture. Inserted sequences were deleted from replicating viruses, but this did not result from homologous recombination at the flanking 3C(pro) cleavage sites, since the 5' and 3' segments of the inserted sequence were retained in the deletion mutants. These results indicate that the HAV polyprotein can tolerate an insertion at the 2A/2B junction and that the 2A polypeptide does not function in cis as a 2AB precursor. Recombinant HAV genomes containing foreign protein-coding sequences inserted at the 2A/2B junction are novel and potentially useful protein expression vectors.
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Affiliation(s)
- M R Beard
- Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019, USA.
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Lerat H, Shimizu YK, Lemon SM. Cell type-specific enhancement of hepatitis C virus internal ribosome entry site-directed translation due to 5' nontranslated region substitutions selected during passage of virus in lymphoblastoid cells. J Virol 2000; 74:7024-31. [PMID: 10888641 PMCID: PMC112219 DOI: 10.1128/jvi.74.15.7024-7031.2000] [Citation(s) in RCA: 45] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Low-level replication of hepatitis C virus (HCV) in cultured lymphoblastoid cells inoculated with H77 serum inoculum led to the appearance of new virus variants containing identical substitutions at three sites within the viral 5' nontranslated RNA (5'NTR): G(107)-->A, C(204)-->A, and G(243)-->A (N. Nakajima, M. Hijikata, H. Yoshikura, and Y. K. Shimizu, J. Virol. 70:3325-3329, 1996). These results suggest that virus with this 5'NTR sequence may have a greater capacity for replication in such cells, possibly due to more efficient cap-independent translation, since these nucleotide substitutions reside within the viral internal ribosome entry site (IRES). To test this hypothesis, we examined the translation of dicistronic RNAs containing upstream and downstream reporter sequences (Renilla and firefly luciferases, respectively) separated by IRES sequences containing different combinations of these substitutions. The activity of the IRES was assessed by determining the relative firefly and Renilla luciferase activities expressed in transfected cells. Compared with the IRES present in the dominant H77 quasispecies, an IRES containing all three nucleotide substitutions had significantly greater translational activity in three of five human lymphoblastoid cell lines (Raji, Bjab, and Molt4 but not Jurkat or HPBMa10-2 cells). In contrast, these substitutions did not enhance IRES activity in cell lines derived from monocytes or granulocytes (HL-60, KG-1, or THP-1) or hepatocytes (Huh-7) or in cell-free translation assays carried out with rabbit reticulocyte lysates. Each of the three substitutions was required for maximally increased translational activity in the lymphoblastoid cells. The 2- to 2.5-fold increase in translation observed with the modified IRES sequence may facilitate the replication of HCV, possibly accounting for differences in quasispecies variants recovered from liver tissue and peripheral blood mononuclear cells of the same patient.
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Affiliation(s)
- H Lerat
- Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019, USA
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Gosert R, Chang KH, Rijnbrand R, Yi M, Sangar DV, Lemon SM. Transient expression of cellular polypyrimidine-tract binding protein stimulates cap-independent translation directed by both picornaviral and flaviviral internal ribosome entry sites In vivo. Mol Cell Biol 2000; 20:1583-95. [PMID: 10669736 PMCID: PMC85342 DOI: 10.1128/mcb.20.5.1583-1595.2000] [Citation(s) in RCA: 109] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
The regulation of cap-independent translation directed by the internal ribosome entry sites (IRESs) present in some viral and cellular RNAs is poorly understood. Polypyrimidine-tract binding protein (PTB) binds specifically to several viral IRESs. IRES-directed translation may be reduced in cell-free systems that are depleted of PTB and restored by reconstitution of lysates with recombinant PTB. However, there are no data concerning the effects of PTB on IRES-directed translation in vivo. We transfected cells with plasmids expressing dicistronic transcripts in which the upstream cistron encoded PTB or PTB deletion mutants (including a null mutant lacking amino acid residues 87 to 531). The downstream cistron encoded a reporter protein (chloramphenicol acetyltransferase [CAT]) under translational control of the poliovirus IRES which was placed within the intercistronic space. In transfected BS-C-1 cells, transcripts expressing wild-type PTB produced 12-fold more reporter protein than similar transcripts encoding the PTB null mutant. There was a 2.4-fold difference in CAT produced from these transcripts in HeLa cells, which contain a greater natural abundance of PTB. PTB similarly stimulated CAT production from transcripts containing the IRES of hepatitis A virus or hepatitis C virus in BS-C-1 cells and Huh-7 cells (37- to 44-fold increase and 5 to 5.3-fold increase, respectively). Since PTB had no quantitative or qualitative effect on transcription from these plasmids, we conclude that PTB stimulates translation of representative picornaviral and flaviviral RNAs in vivo. This is likely to reflect the stabilization of higher ordered RNA structures within the IRES and was not observed with PTB mutants lacking RNA recognition motifs located in the C-terminal third of the molecule.
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Affiliation(s)
- R Gosert
- Department of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7030, USA
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