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Ringlander J, Rydell GE, Kann M. From the Cytoplasm into the Nucleus-Hepatitis B Virus Travel and Genome Repair. Microorganisms 2025; 13:157. [PMID: 39858925 PMCID: PMC11767736 DOI: 10.3390/microorganisms13010157] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2024] [Revised: 01/03/2025] [Accepted: 01/08/2025] [Indexed: 01/27/2025] Open
Abstract
Hepatitis B virus (HBV) is a major global health concern, affecting millions of people worldwide. HBV is part of the hepadnaviridae family and one of the primary causes of acute and chronic liver infections, leading to conditions such as cirrhosis and hepatocellular carcinoma (HCC). Understanding the intracellular transport and genome repair mechanisms of HBV is crucial for developing new drugs, which-in combination with immune modulators-may contribute to potential cures. This review will explore the current knowledge of HBV intracytoplasmic and nuclear transport, as well as genome repair processes, while drawing comparisons to other viruses with nuclear replication.
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Affiliation(s)
- Johan Ringlander
- Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, 41346 Gothenburg, Sweden; (J.R.); (G.E.R.)
- Department of Clinical Microbiology, Region Västra Götaland, Sahlgrenska University Hospital, 41346 Gothenburg, Sweden
| | - Gustaf E. Rydell
- Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, 41346 Gothenburg, Sweden; (J.R.); (G.E.R.)
| | - Michael Kann
- Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, 41346 Gothenburg, Sweden; (J.R.); (G.E.R.)
- Department of Clinical Microbiology, Region Västra Götaland, Sahlgrenska University Hospital, 41346 Gothenburg, Sweden
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Reddy H, Srinivas MV, Vasu J, Prabavathy A, Dhodapkar R, Mukhopadhyay HK. Whole-genome sequence analysis of canine parvovirus reveals replacement with a novel CPV-2c strain throughout India. Arch Virol 2024; 169:189. [PMID: 39192096 DOI: 10.1007/s00705-024-06096-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2024] [Accepted: 05/27/2024] [Indexed: 08/29/2024]
Abstract
Canine parvovirus (CPV) infection causes severe gastroenteritis in canines, with high mortality in puppies. This virus evolved from feline panleukopenia virus by altering its transferrin receptor (TfR), followed by the emergence of CPV-2 variants in subsequent years with altered immunodominant amino acid residues in the VP2 protein. While previous studies have focused on the VP2 gene, there have been fewer studies on non-structural protein (NS1 and NS2) genes. In the present study, CPV genome sequences from clinical samples collected from canines throughout India in 2023, previous Indian CPV isolates from 2009-2019, and the current Indian CPV vaccine strain were compared. The study showed that the CPV-2c (N426E) variant had almost completely replaced the previously dominant CPV-2a variant (N426) in India. The Q370R mutation of VP2 was the most common change in the recent CPV-2c strain (CPV-2c 370Arg variant). Phylogenetic analysis showed the existence of three clades among the recent CPV-2c strains, and sequence analysis identified several new sites of positive selection in the VP1 (N-terminus), VP2, NS1, and NS2 protein-encoding genes in recent CPV strains, indicating the emergence of new CPV-2c variants with varied antigenic and replication properties. The predominant 'CPV-2c 370Arg variants' were grouped with the Chinese and Nigerian CPV-2c strains but were separate from the CPV vaccine strain and earlier isolates from our repository. VP2 epitope analysis predicted nine amino acid variations (including two new variations) in four potential linear B-cell epitopes in the CPV-2c 370Arg variants that might make vaccine failure more likely. This pan-Indian study lays the foundation for further research concerning the dynamics of virus evolution and understanding genetic mutations.
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Affiliation(s)
- Harish Reddy
- Department of Veterinary Microbiology, Rajiv Gandhi Institute of Veterinary Education & Research, Puducherry, 605 009, India
| | - Mouttou Vivek Srinivas
- Department of Veterinary Microbiology, Rajiv Gandhi Institute of Veterinary Education & Research, Puducherry, 605 009, India.
| | - Jayalakshmi Vasu
- Department of Veterinary Microbiology, Rajiv Gandhi Institute of Veterinary Education & Research, Puducherry, 605 009, India
| | - Abiramy Prabavathy
- Department of Veterinary Medicine, Rajiv Gandhi Institute of Veterinary Education & Research, Puducherry, 605 009, India
| | - Rahul Dhodapkar
- Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education & Research, Puducherry, India
| | - Hirak Kumar Mukhopadhyay
- Department of Veterinary Microbiology, Rajiv Gandhi Institute of Veterinary Education & Research, Puducherry, 605 009, India
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Elizondo Quiroga D, De Los Santos Acuña MA, Gutierrez Ortega A, Galán Martinez C, Pedroza Roldán C. Genome Sequences of Canine Parvovirus Type 2c Prevalent in Western Mexico. ARCHIVES OF RAZI INSTITUTE 2024; 79:387-394. [PMID: 39463712 PMCID: PMC11512174 DOI: 10.32592/ari.2024.79.2.387] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Subscribe] [Scholar Register] [Received: 08/15/2023] [Accepted: 09/30/2023] [Indexed: 10/29/2024]
Abstract
Canine parvovirus type 2 (CPV-2) is one of the main etiologies of viral gastroenteritis in dogs across the globe. This disease is mainly characterized by the presence of diarrhea, abdominal pain, vomiting, anorexia, and dehydration. This virus is responsible for high mortality and morbidity rates in unvaccinated dogs and those younger than three months. The monitoring of viral variants in our region has demonstrated that in the last seven years, variant CPV-2c has been circulating exclusively, which is unusual if we consider that in the rest of the world, at least two variants co-circulate among dog populations. To the best of our knowledge, no studies in Mexico have reported genomic sequences of CPV-2, which are relevant for population comparisons at the genetic level. Therefore, the present study aimed to sequence genomes associated with CPV-2c. To meet this objective, rectal swab samples were collected from dogs with suspected CPV-2 infection. Five positive cases diagnosed by lateral flow testing and polymerase chain reaction were selected for viral genome sequencing. Comparative analyses illustrated that the obtained genome sequences were > 99% homologous to those reported for CPV-2 in the GenBank. On the other hand, 52 nucleotide mutations were identified in the vp1/vp2 gene, out of which three impacted amino acid transition (T226S, F267Y, and A440T). Phylogenetic analysis of the vp1/vp2 gene demonstrated that the five sequences clustered in a clade called "III", pertaining to sequences from USA and Uruguay. To our knowledge, this was the first report of genomic sequences associated with CPV-2 in Mexico, which is of great relevance for the epidemiological-molecular understanding and evolution of the virus.
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Affiliation(s)
- D Elizondo Quiroga
- Unidad de Biotecnología Médica y Farmacéutica, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, A.C. Av. Normalistas No. 800. Col. Colinas de la Normal, CP. 44270, Guadalajara, Jalisco, México
| | - M A De Los Santos Acuña
- Departmento de Medicina Veterinaria, Centro Universitario de Ciencias Biológicas Y Agropecuarias, Cam. Ramón Padilla Sánchez 2100, Las Agujas, 44600 Zapopan, Jal. Universidad de Guadalajara, Zapopan, Jalisco, México
| | - A Gutierrez Ortega
- Unidad de Biotecnología Médica y Farmacéutica, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, A.C. Av. Normalistas No. 800. Col. Colinas de la Normal, CP. 44270, Guadalajara, Jalisco, México
| | - C Galán Martinez
- Departmento de Medicina Veterinaria, Centro Universitario de Ciencias Biológicas Y Agropecuarias, Cam. Ramón Padilla Sánchez 2100, Las Agujas, 44600 Zapopan, Jal. Universidad de Guadalajara, Zapopan, Jalisco, México
| | - C Pedroza Roldán
- Departmento de Medicina Veterinaria, Centro Universitario de Ciencias Biológicas Y Agropecuarias, Cam. Ramón Padilla Sánchez 2100, Las Agujas, 44600 Zapopan, Jal. Universidad de Guadalajara, Zapopan, Jalisco, México
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4
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Zhang L, Li Y, Kuhn JH, Zhang K, Song Q, Liu F. Polyubiquitylated rice stripe virus NS3 translocates to the nucleus to promote cytosolic virus replication via miRNA-induced fibrillin 2 upregulation. PLoS Pathog 2024; 20:e1012112. [PMID: 38507423 PMCID: PMC10984529 DOI: 10.1371/journal.ppat.1012112] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2023] [Revised: 04/01/2024] [Accepted: 03/11/2024] [Indexed: 03/22/2024] Open
Abstract
Viruses are encapsidated mobile genetic elements that rely on host cells for replication. Several cytoplasmic RNA viruses synthesize proteins and/or RNAs that translocate to infected cell nuclei. However, the underlying mechanisms and role(s) of cytoplasmic-nuclear trafficking are unclear. We demonstrate that infection of small brown planthoppers with rice stripe virus (RSV), a negarnaviricot RNA virus, results in K63-linked polyubiquitylation of RSV's nonstructural protein 3 (NS3) at residue K127 by the RING ubiquitin ligase (E3) LsRING. In turn, ubiquitylation leads to NS3 trafficking from the cytoplasm to the nucleus, where NS3 regulates primary miRNA pri-miR-92 processing through manipulation of the microprocessor complex, resulting in accumulation of upregulated miRNA lst-miR-92. We show that lst-miR-92 regulates the expression of fibrillin 2, an extracellular matrix protein, thereby increasing RSV loads. Our results highlight the manipulation of intranuclear, cytoplasmic, and extracellular components by an RNA virus to promote its own replication in an insect vector.
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Affiliation(s)
- Lu Zhang
- College of Plant Protection; Yángzhōu University; Yángzhōu, Jiāngsū Province; China
| | - Yao Li
- College of Plant Protection; Yángzhōu University; Yángzhōu, Jiāngsū Province; China
| | - Jens H. Kuhn
- Integrated Research Facility at Fort Detrick; National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick; Frederick, Maryland; United States of America
| | - Kun Zhang
- College of Plant Protection; Yángzhōu University; Yángzhōu, Jiāngsū Province; China
| | - Qisheng Song
- Division of Plant Science and Technology; College of Agriculture; Food and Natural Resources; University of Missouri; Columbia, Missouri; United States of America
| | - Fang Liu
- College of Plant Protection; Yángzhōu University; Yángzhōu, Jiāngsū Province; China
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López-Bueno A, Gil-Ranedo J, Almendral JM. Assembly of Structurally Simple Icosahedral Viruses. Subcell Biochem 2024; 105:403-430. [PMID: 39738953 DOI: 10.1007/978-3-031-65187-8_11] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2025]
Abstract
Icosahedral viruses exhibit elegant pathways of capsid assembly and maturation regulated by symmetry principles. Assembly is a dynamic process driven by consecutive and genetically programmed morphogenetic interactions between protein subunits. The non-symmetric capsid subunits are gathered by non-covalent contacts and interactions in assembly intermediates, which serve as blocks to build a symmetric capsid. In some virus examples, the assembly of the protein shell further requires non-symmetric interactions among intermediates to fold into specific conformations. In this chapter, the morphogenesis of some small and structurally simple icosahedral viruses, including representative members of the parvoviruses, picornaviruses, and polyomaviruses as paradigms, is described in some detail. Despite their small size, the assembly of these icosahedral viruses may follow rather complex pathways, as they may occur in different subcellular compartments, involve a panoply of cellular and viral factors, and regulatory protein post-translational modifications that challenge its comprehensive understanding. Mechanisms of viral genome encapsidation may imply direct interactions between the genome and the assembly intermediates, or active packaging into a preformed empty capsid. Further, membranes and factors at specific subcellular compartments may also be critically required for virus maturation. The high stability of intermediates and the process of viral maturation contribute to the overall irreversible character of the assembly process. These and other small, structurally less complex icosahedral viruses were pioneer models to understand basic principles of virus assembly, continue to be leading subjects of morphogenetic analyses, and have inspired ongoing studies on the assembly of larger, structurally more complex viruses as well as cellular and synthetic macromolecular complexes.
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Affiliation(s)
- Alberto López-Bueno
- Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM) and Department of Molecular Biology, Universidad Autónoma de Madrid, Madrid, Spain
| | - Jon Gil-Ranedo
- Faculty of Health: Medicine, Dentistry and Human Sciences, University of Plymouth, Plymouth, UK
| | - José M Almendral
- Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM) and Department of Molecular Biology, Universidad Autónoma de Madrid, Madrid, Spain.
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Chen DY, Tzang CC, Liu CM, Chiu TM, Lin JW, Chuang PH, Kuo CW, Tzang BS, Hsu TC. Effect of the Functional VP1 Unique Region of Human Parvovirus B19 in Causing Skin Fibrosis of Systemic Sclerosis. Int J Mol Sci 2023; 24:15294. [PMID: 37894973 PMCID: PMC10607574 DOI: 10.3390/ijms242015294] [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: 10/03/2023] [Revised: 10/15/2023] [Accepted: 10/16/2023] [Indexed: 10/29/2023] Open
Abstract
Human parvovirus B19 (B19V) is a single-stranded non-enveloped DNA virus of the family Parvoviridae that has been associated with various autoimmune disorders. Systemic sclerosis (SSc) is an autoimmune connective tissue disorder with high mortality and has been linked to B19V infection. However, the precise mechanism underlying the B19V contribution to the development of SSc remains uncertain. This study investigated the impacts of the functional B19V-VP1 unique region (VP1u) in macrophages and bleomycin (BLE)-induced SSc mice. Cell experimental data showed that significantly decreased viability and migration of both B19V-VP1u-treated U937 and THP-1 macrophages are detected in the presence of celastrol. Significantly increased MMP9 activity and elevated NF-kB, MMP9, IL-6, TNF-α, and IL-1β expressions were detected in both B19V-VP1u-treated U937 and THP-1 macrophages. Conversely, celastrol revealed an inhibitory effect on these molecules. Notably, celastrol intervened in this pathogenic process by suppressing the sPLA2 activity of B19V-VP1u and subsequently reducing the inflammatory response. Notably, the administration of B19V-VP1u exacerbated BLE-induced skin fibrosis in mice, with augmented expressions of TGF-β, IL-6, IL-17A, IL-18, and TNF-α, ultimately leading to α-SMA and collagen I deposits in the dermal regions of BLE-induced SSc mice. Altogether, this study sheds light on parvovirus B19 VP1u linked to scleroderma and aggravated dermal fibrosis.
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Affiliation(s)
- Der-Yuan Chen
- Institute of Medicine, Chung Shan Medical University, Taichung 402, Taiwan; (D.-Y.C.); (C.-M.L.); (T.-M.C.); (J.-W.L.); (P.-H.C.); (C.-W.K.)
- College of Medicine, China Medical University, Taichung 404, Taiwan
- Translational Medicine Laboratory, Rheumatology and Immunology Center, China Medical University Hospital, Taichung 404, Taiwan
| | - Chih-Chen Tzang
- School of Medicine, College of Medicine, National Taiwan University, Taipei City 100, Taiwan;
| | - Chuan-Ming Liu
- Institute of Medicine, Chung Shan Medical University, Taichung 402, Taiwan; (D.-Y.C.); (C.-M.L.); (T.-M.C.); (J.-W.L.); (P.-H.C.); (C.-W.K.)
| | - Tsu-Man Chiu
- Institute of Medicine, Chung Shan Medical University, Taichung 402, Taiwan; (D.-Y.C.); (C.-M.L.); (T.-M.C.); (J.-W.L.); (P.-H.C.); (C.-W.K.)
- Department of Dermatology, Chung Shan Medical University Hospital, Taichung 402, Taiwan
- School of Medicine, Chung Shan Medical University, Taichung 402, Taiwan
| | - Jhen-Wei Lin
- Institute of Medicine, Chung Shan Medical University, Taichung 402, Taiwan; (D.-Y.C.); (C.-M.L.); (T.-M.C.); (J.-W.L.); (P.-H.C.); (C.-W.K.)
| | - Pei-Hua Chuang
- Institute of Medicine, Chung Shan Medical University, Taichung 402, Taiwan; (D.-Y.C.); (C.-M.L.); (T.-M.C.); (J.-W.L.); (P.-H.C.); (C.-W.K.)
| | - Chia-Wei Kuo
- Institute of Medicine, Chung Shan Medical University, Taichung 402, Taiwan; (D.-Y.C.); (C.-M.L.); (T.-M.C.); (J.-W.L.); (P.-H.C.); (C.-W.K.)
| | - Bor-Show Tzang
- Institute of Medicine, Chung Shan Medical University, Taichung 402, Taiwan; (D.-Y.C.); (C.-M.L.); (T.-M.C.); (J.-W.L.); (P.-H.C.); (C.-W.K.)
- Department of Biochemistry, School of Medicine, Chung Shan Medical University, Taichung 402, Taiwan
- Department of Clinical Laboratory, Chung Shan Medical University Hospital, Taichung 402, Taiwan
- Immunology Research Center, Chung Shan Medical University, Taichung 402, Taiwan
| | - Tsai-Ching Hsu
- Institute of Medicine, Chung Shan Medical University, Taichung 402, Taiwan; (D.-Y.C.); (C.-M.L.); (T.-M.C.); (J.-W.L.); (P.-H.C.); (C.-W.K.)
- Department of Clinical Laboratory, Chung Shan Medical University Hospital, Taichung 402, Taiwan
- Immunology Research Center, Chung Shan Medical University, Taichung 402, Taiwan
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Früh SP, Adu OF, López-Astacio RA, Weichert WS, Wasik BR, Parrish CR. Isolation, cloning and analysis of parvovirus-specific canine antibodies from peripheral blood B cells. DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY 2023; 147:104894. [PMID: 37467826 PMCID: PMC10542859 DOI: 10.1016/j.dci.2023.104894] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/12/2023] [Revised: 07/05/2023] [Accepted: 07/16/2023] [Indexed: 07/21/2023]
Abstract
B-cell cloning methods enable the analysis of antibody responses against target antigens and can be used to reveal the host antibody repertoire, antigenic sites (epitopes), and details of protective immunity against pathogens. Here, we describe improved methods for isolation of canine peripheral blood B cells producing antibodies against canine parvovirus (CPV) capsids by fluorescence-activated cell sorting, followed by cell cloning. We cultured sorted B cells from an immunized dog in vitro and screened for CPV-specific antibody production. Updated canine-specific primer sets were used to amplify and clone the heavy and light chain immunoglobulin sequences directly from the B cells by reverse transcription and PCR. Monoclonal canine IgGs were produced by cloning heavy and light chain sequences into antibody expression vectors, which were screened for CPV binding. Three different canine monoclonal antibodies were analyzed, including two that shared the same heavy chain, and one that had distinct heavy and light chains. The antibodies showed broad binding to CPV variants, and epitopes were mapped to antigenic sites on the capsid. The methods described here are applicable for the isolation of canine B cells and monoclonal antibodies against many antigens.
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Affiliation(s)
- Simon P Früh
- Baker Institute for Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14853, USA; Department of Veterinary Sciences, Ludwig-Maximilians-Universität München, Munich, Germany
| | - Oluwafemi F Adu
- Baker Institute for Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14853, USA
| | - Robert A López-Astacio
- Baker Institute for Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14853, USA
| | - Wendy S Weichert
- Baker Institute for Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14853, USA
| | - Brian R Wasik
- Baker Institute for Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14853, USA
| | - Colin R Parrish
- Baker Institute for Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14853, USA.
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López-Astacio RA, Adu OF, Lee H, Hafenstein SL, Parrish CR. The Structures and Functions of Parvovirus Capsids and Missing Pieces: the Viral DNA and Its Packaging, Asymmetrical Features, Nonprotein Components, and Receptor or Antibody Binding and Interactions. J Virol 2023; 97:e0016123. [PMID: 37367301 PMCID: PMC10373561 DOI: 10.1128/jvi.00161-23] [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] [Indexed: 06/28/2023] Open
Abstract
Parvoviruses are among the smallest and superficially simplest animal viruses, infecting a broad range of hosts, including humans, and causing some deadly infections. In 1990, the first atomic structure of the canine parvovirus (CPV) capsid revealed a 26-nm-diameter T=1 particle made up of two or three versions of a single protein, and packaging about 5,100 nucleotides of single-stranded DNA. Our structural and functional understanding of parvovirus capsids and their ligands has increased as imaging and molecular techniques have advanced, and capsid structures for most groups within the Parvoviridae family have now been determined. Despite those advances, significant questions remain unanswered about the functioning of those viral capsids and their roles in release, transmission, or cellular infection. In addition, the interactions of capsids with host receptors, antibodies, or other biological components are also still incompletely understood. The parvovirus capsid's apparent simplicity likely conceals important functions carried out by small, transient, or asymmetric structures. Here, we highlight some remaining open questions that may need to be answered to provide a more thorough understanding of how these viruses carry out their various functions. The many different members of the family Parvoviridae share a capsid architecture, and while many functions are likely similar, others may differ in detail. Many of those parvoviruses have not been experimentally examined in detail (or at all in some cases), so we, therefore, focus this minireview on the widely studied protoparvoviruses, as well as the most thoroughly investigated examples of adeno-associated viruses.
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Affiliation(s)
- Robert A. López-Astacio
- Baker Institute for Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA
| | - Oluwafemi F. Adu
- Baker Institute for Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA
| | - Hyunwook Lee
- Department of Biochemistry and Molecular Biology, Penn State University, University Park, Pennsylvania, USA
| | - Susan L. Hafenstein
- Department of Biochemistry and Molecular Biology, Penn State University, University Park, Pennsylvania, USA
| | - Colin R. Parrish
- Baker Institute for Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA
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9
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Sarabandi S, Pourtaghi H. Whole genome sequence analysis of CPV-2 isolates from 1998 to 2020. Virol J 2023; 20:138. [PMID: 37400901 DOI: 10.1186/s12985-023-02102-2] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2023] [Accepted: 06/14/2023] [Indexed: 07/05/2023] Open
Abstract
Canine parvovirus-2 (CPV-2) is a virus with worldwide spread causing canine gastroenteritis. New strains of this virus have unique characteristics and are resistant to some vaccine strains. Therefore, understanding the root causes of resistance has proven to be of increasing concern to many scientists. This study collected 126 whole genome sequences of CPV-2 subtypes with specific collection dates from the NCBI data bank. The whole genome sequences of CPV-2 collected from different countries were analyzed to detect the new substitutions and update these mutations. The result indicated 12, 7, and 10 mutations in NS1, VP1, and VP2, in that respective order. Moreover, the A5G and Q370R mutations of VP2 are the most common changes in the recent isolates of the CPV-2C subtype, and the new N93K residue of VP2 is speculated to be the cause of vaccine failure. To summarize, the observed mutations, which are increasing over time, causes several changes in viral characteristic. A comprehensive understanding of these mutations can lead us to control potential future epidemics associated with this virus more efficiently.
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Affiliation(s)
- Sajed Sarabandi
- Department of Pathobiology, Islamic Azad University, Karaj Branch, Karaj, Iran
| | - Hadi Pourtaghi
- Department of Microbiology, Islamic Azad University, Karaj Branch, Karaj, Iran.
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10
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Calvo-López T, Grueso E, Sánchez-Martínez C, Almendral JM. Intracellular virion traffic to the endosome driven by cell type specific sialic acid receptors determines parvovirus tropism. Front Microbiol 2023; 13:1063706. [PMID: 36756201 PMCID: PMC9899843 DOI: 10.3389/fmicb.2022.1063706] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2022] [Accepted: 12/28/2022] [Indexed: 01/24/2023] Open
Abstract
Parvoviruses are promising anticancer and gene therapy agents, but a deep knowledge of the entry process is crucial to exploit their therapeutic potential. We addressed this issue while attempting to retarget the oncolytic parvovirus minute virus of mice (MVMp) to the tumor vasculature. Residues at three functional domains of the icosahedral capsid were substituted by rational design with peptides competing with the vascular endothelial growth factor. Most substitutions impaired virus maturation, though some yielded infectious chimeric virions, and substitutions in a dimple at the twofold axis that allocates sialic acid (SIA) receptors altered viral tropism. One dimple-modified chimeric virion was efficiently attached as MVMp to α2-linked SIA moieties, but the infection was impaired by the binding to some inhibitory α2-3,-6,-8 SIA pseudoreceptors, which hampers intracellular virus traffic to the endosome in a cell type-dependent manner. Infectious from nonproductive traffic could be mechanistically discriminated by an endosomal drastic capsid structural transition comprising the cleavage of some VP2-Nt sequences and its associated VP1-Nt exposure. Correspondingly, neuraminidase removal of inhibitory SIA moieties enhanced the infection quantitatively, correlating to the restored virus traffic to the endosome and the extent of VP2-Nt cleavage/VP1-Nt exposure. This study illustrates (i) structural constraints to retarget parvoviruses with evolutionary adopted narrow grooves allocating small SIA receptors, (ii) the possibility to enhance parvovirus oncolysis by relaxing the glycan network on the cancer cell surface, and (iii) the major role played by the attachment to cell type-specific SIAs in the intracellular virus traffic to the endosome, which may determine parvovirus tropism and host range.
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Affiliation(s)
- Tania Calvo-López
- Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain,Departamento de Biología Molecular, Universidad Autónoma de Madrid, Madrid, Spain
| | - Esther Grueso
- Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain,Departamento de Biología Molecular, Universidad Autónoma de Madrid, Madrid, Spain
| | - Cristina Sánchez-Martínez
- Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain,Departamento de Biología Molecular, Universidad Autónoma de Madrid, Madrid, Spain
| | - José M. Almendral
- Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain,Departamento de Biología Molecular, Universidad Autónoma de Madrid, Madrid, Spain,*Correspondence: José M. Almendral ✉
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Fu P, He D, Cheng X, Niu X, Wang C, Fu Y, Li K, Zhu H, Lu W, Wang J, Chu B. Prevalence and Characteristics of Canine Parvovirus Type 2 in Henan Province, China. Microbiol Spectr 2022; 10:e0185622. [PMID: 36377944 PMCID: PMC9769957 DOI: 10.1128/spectrum.01856-22] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2022] [Accepted: 09/09/2022] [Indexed: 11/16/2022] Open
Abstract
To investigate the epidemic profile and genetic diversity of canine parvovirus type 2 (CPV-2), a total of 111 clinical samples collected from dogs suspected of CPV-2 infection in 10 cities of Henan province of China during 2020 to 2021 were screened by PCR. The results showed a CPV-2-positive rate of 88.29% (98/111). Nearly full-length genomes of 98 CPV-2 strains were sequenced and analyzed. CPV-2c strains (91.84%, 90/98) were significantly higher than that of new CPV-2a strains (8.16%, 8/98) in Henan province without detecting other CPV genotypes, indicating that CPV-2c has become the dominant genotype in Henan province. A phylogenetic analysis of NS1 and VP2 amino acids grouped the strains in this study with Asian strains, which clustered into an identical branch. Based on the CPV-2 VP2 sequences in this study and available in the NCBI database, the adaptation analyses showed that 17 positive selection sites and 10 parallel evolution sites were identified in the VP2 protein of CPV-2, of which three sites (sites 5, 370, and 426) were both under positive selection pressure and parallel evolution. Interestingly, two amino acid mutations (A5G and Q370R) were also observed in the VP2 proteins of 82 CPV-2c strains in this study, which differed from the earlier CPV-2c strain (GU380303) in China. In addition, a unique mutation (I447M) was observed in the VP2 protein of five CPV-2c strains, which was first reported in China. This study provides powerful insight to further our understanding of the epidemic status and evolution of CPV-2 in China. IMPORTANCE CPV-2 was the original virus strain identified in dogs, which cause an acute and lethal disease in dogs. Subsequently, the original CPV-2 was replaced throughout the world by novel antigenic variants (e.g., CPV-2a, CPV-2b, new CPV-2a, new CPV-2b, and CPV-2c). Currently, the epidemiological characteristics of CPV-2 in Henan province of China is still unclear. In our study, a total of 98 nearly full-length genomes of CPV-2 strains were obtained to explore prevalence and genetic evolution of CPV-2 in Henan Province. Moreover, the epidemiological and genetic evolution of CPV-2 in China since its discovery was also investigated. The results of this study will provide valuable information regarding the evolution of CPV-2 strains in China.
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Affiliation(s)
- Pengfei Fu
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan Province, China
- Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affairs of the People’s Republic of China, Zhengzhou, Henan Province, China
- Key Laboratory of Animal Growth and Development, The Education Department of Henan Province, Zhengzhou, Henan Province, China
| | - Dongchang He
- Animal Infectious Disease Laboratory, College of Veterinary Medicine, Yangzhou University, Yangzhou, China
| | - Xuan Cheng
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan Province, China
- Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affairs of the People’s Republic of China, Zhengzhou, Henan Province, China
- Key Laboratory of Animal Growth and Development, The Education Department of Henan Province, Zhengzhou, Henan Province, China
| | - Xinrui Niu
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan Province, China
- Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affairs of the People’s Republic of China, Zhengzhou, Henan Province, China
- Key Laboratory of Animal Growth and Development, The Education Department of Henan Province, Zhengzhou, Henan Province, China
| | - Congrong Wang
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan Province, China
- Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affairs of the People’s Republic of China, Zhengzhou, Henan Province, China
- Key Laboratory of Animal Growth and Development, The Education Department of Henan Province, Zhengzhou, Henan Province, China
| | - Yiqian Fu
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan Province, China
- Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affairs of the People’s Republic of China, Zhengzhou, Henan Province, China
- Key Laboratory of Animal Growth and Development, The Education Department of Henan Province, Zhengzhou, Henan Province, China
| | - Kun Li
- State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China
| | - Heshui Zhu
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan Province, China
- Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affairs of the People’s Republic of China, Zhengzhou, Henan Province, China
- Key Laboratory of Animal Growth and Development, The Education Department of Henan Province, Zhengzhou, Henan Province, China
| | - Weifei Lu
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan Province, China
- Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affairs of the People’s Republic of China, Zhengzhou, Henan Province, China
- Key Laboratory of Animal Growth and Development, The Education Department of Henan Province, Zhengzhou, Henan Province, China
| | - Jiang Wang
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan Province, China
- Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affairs of the People’s Republic of China, Zhengzhou, Henan Province, China
- Key Laboratory of Animal Growth and Development, The Education Department of Henan Province, Zhengzhou, Henan Province, China
| | - Beibei Chu
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan Province, China
- Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affairs of the People’s Republic of China, Zhengzhou, Henan Province, China
- Key Laboratory of Animal Growth and Development, The Education Department of Henan Province, Zhengzhou, Henan Province, China
- International Joint Research Center of National Animal Immunology, Henan Agricultural University, Zhengzhou, Henan Province, China
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Lakshmanan RV, Hull JA, Berry L, Burg M, Bothner B, McKenna R, Agbandje-McKenna M. Structural Dynamics and Activity of B19V VP1u during the pHs of Cell Entry and Endosomal Trafficking. Viruses 2022; 14:1922. [PMID: 36146728 PMCID: PMC9505059 DOI: 10.3390/v14091922] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2022] [Revised: 08/28/2022] [Accepted: 08/28/2022] [Indexed: 12/28/2022] Open
Abstract
Parvovirus B19 (B19V) is a human pathogen that is the causative agent of fifth disease in children. It is also known to cause hydrops in fetuses, anemia in AIDS patients, and transient aplastic crisis in patients with sickle cell disease. The unique N-terminus of Viral Protein 1 (VP1u) of parvoviruses, including B19V, exhibits phospholipase A2 (PLA2) activity, which is required for endosomal escape. Presented is the structural dynamics of B19V VP1u under conditions that mimic the pHs of cell entry and endosomal trafficking to the nucleus. Using circular dichroism spectroscopy, the receptor-binding domain of B19V VP1u is shown to exhibit an α-helical fold, whereas the PLA2 domain exhibits a probable molten globule state, both of which are pH invariant. Differential scanning calorimetry performed at endosomal pHs shows that the melting temperature (Tm) of VP1u PLA2 domain is tuned to body temperature (37 °C) at pH 7.4. In addition, PLA2 assays performed at temperatures ranging from 25-45 °C show both a temperature and pH-dependent change in activity. We hypothesize that VP1u PLA2 domain differences in Tm at differing pHs have enabled the virus to "switch on/off" the phospholipase activity during capsid trafficking. Furthermore, we propose the environment of the early endosome as the optimal condition for endosomal escape leading to B19V infection.
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Affiliation(s)
- Renuk V. Lakshmanan
- Department of Biochemistry and Molecular Biology, Center for Structural Biology, The McKnight Brain Institute, University of Florida, Gainesville, FL 32610, USA
| | - Joshua A. Hull
- Department of Biochemistry and Molecular Biology, Center for Structural Biology, The McKnight Brain Institute, University of Florida, Gainesville, FL 32610, USA
| | - Luke Berry
- Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717, USA
| | - Matthew Burg
- Department of Biochemistry and Molecular Biology, Center for Structural Biology, The McKnight Brain Institute, University of Florida, Gainesville, FL 32610, USA
| | - Brian Bothner
- Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717, USA
| | - Robert McKenna
- Department of Biochemistry and Molecular Biology, Center for Structural Biology, The McKnight Brain Institute, University of Florida, Gainesville, FL 32610, USA
| | - Mavis Agbandje-McKenna
- Department of Biochemistry and Molecular Biology, Center for Structural Biology, The McKnight Brain Institute, University of Florida, Gainesville, FL 32610, USA
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13
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Wang J, Chen X, Zhou Y, Yue H, Zhou N, Gong H, Tang C. Prevalence and characteristics of a feline parvovirus-like virus in dogs in China. Vet Microbiol 2022; 270:109473. [DOI: 10.1016/j.vetmic.2022.109473] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2022] [Revised: 05/18/2022] [Accepted: 05/19/2022] [Indexed: 12/01/2022]
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Advances in research on genetic relationships of waterfowl parvoviruses. J Vet Res 2021; 65:391-399. [PMID: 35111991 PMCID: PMC8775729 DOI: 10.2478/jvetres-2021-0063] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2021] [Accepted: 11/18/2021] [Indexed: 01/23/2023] Open
Abstract
Abstract
Derzsy’s disease and Muscovy duck parvovirus disease have become common diseases in waterfowl culture in the world and their potential to cause harm has risen. The causative agents are goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV), which can provoke similar clinical symptoms and high mortality and morbidity rates. In recent years, duck short beak and dwarfism syndrome has been prevalent in the Cherry Valley duck population in eastern China. It is characterised by the physical signs for which it is named. Although the mortality rate is low, it causes stunting and weight loss, which have caused serious economic losses to the waterfowl industry. The virus that causes this disease was named novel goose parvovirus (NGPV). This article summarises the latest research on the genetic relationships of the three parvoviruses, and reviews the aetiology, epidemiology, and necropsy characteristics in infected ducks, in order to facilitate further study.
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15
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Mattola S, Hakanen S, Salminen S, Aho V, Mäntylä E, Ihalainen TO, Kann M, Vihinen-Ranta M. Concepts to Reveal Parvovirus-Nucleus Interactions. Viruses 2021; 13:1306. [PMID: 34372512 PMCID: PMC8310053 DOI: 10.3390/v13071306] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2021] [Revised: 06/24/2021] [Accepted: 07/02/2021] [Indexed: 01/23/2023] Open
Abstract
Parvoviruses are small single-stranded (ss) DNA viruses, which replicate in the nucleoplasm and affect both the structure and function of the nucleus. The nuclear stage of the parvovirus life cycle starts at the nuclear entry of incoming capsids and culminates in the successful passage of progeny capsids out of the nucleus. In this review, we will present past, current, and future microscopy and biochemical techniques and demonstrate their potential in revealing the dynamics and molecular interactions in the intranuclear processes of parvovirus infection. In particular, a number of advanced techniques will be presented for the detection of infection-induced changes, such as DNA modification and damage, as well as protein-chromatin interactions.
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Affiliation(s)
- Salla Mattola
- Department of Biological and Environmental Science, University of Jyvaskyla, 40500 Jyvaskyla, Finland; (S.M.); (S.H.); (S.S.); (V.A.)
| | - Satu Hakanen
- Department of Biological and Environmental Science, University of Jyvaskyla, 40500 Jyvaskyla, Finland; (S.M.); (S.H.); (S.S.); (V.A.)
| | - Sami Salminen
- Department of Biological and Environmental Science, University of Jyvaskyla, 40500 Jyvaskyla, Finland; (S.M.); (S.H.); (S.S.); (V.A.)
| | - Vesa Aho
- Department of Biological and Environmental Science, University of Jyvaskyla, 40500 Jyvaskyla, Finland; (S.M.); (S.H.); (S.S.); (V.A.)
| | - Elina Mäntylä
- BioMediTech, Faculty of Medicine and Health Technology, Tampere University, 33520 Tampere, Finland; (E.M.); (T.O.I.)
| | - Teemu O. Ihalainen
- BioMediTech, Faculty of Medicine and Health Technology, Tampere University, 33520 Tampere, Finland; (E.M.); (T.O.I.)
| | - Michael Kann
- Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, 41390 Gothenburg, Sweden;
- Department of Clinical Microbiology, Sahlgrenska University Hospital, 41345 Gothenburg, Sweden
| | - Maija Vihinen-Ranta
- Department of Biological and Environmental Science, University of Jyvaskyla, 40500 Jyvaskyla, Finland; (S.M.); (S.H.); (S.S.); (V.A.)
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16
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Molecular Epidemiological Survey of Canine Parvovirus Circulating in China from 2014 to 2019. PATHOGENS (BASEL, SWITZERLAND) 2021; 10:pathogens10050588. [PMID: 34064982 PMCID: PMC8150379 DOI: 10.3390/pathogens10050588] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/24/2021] [Revised: 05/01/2021] [Accepted: 05/04/2021] [Indexed: 11/17/2022]
Abstract
The global distribution of canine parvovirus (CPV-2) derived from a closely related carnivore parvovirus poses a considerable threat to the dog population. The virus is continuously undergoing genetic evolution, giving rise to several variants. To investigate the prevalence of Chinese CPV-2 strains in recent years, a total of 30 CPV-2 strains were collected from 2018 to 2021 and the VP2 gene was sequenced and analyzed. Two variants, new CPV-2a (297Ala, 426Asn) and CPV-2c (426Glu), were identified. In contrast to previous reports, the CPV-2c variant has gained an epidemiological advantage over the new CPV-2a variant in China. To compensate for the relatively small sample size, 683 Chinese CPV-2 strains identified between 2014 and 2019 were retrieved from the GenBank database and previous publications, and analyses of these strains further supported our findings, which should be considered since the CPV-2c variant has been frequently associated with immune failure in adult dogs. VP2 protein sequence analysis revealed several amino acid substitutions, including Ala5Gly, Pro13Ser, Phe267Tyr, Tyr324Ile, Gln370Arg, Thr440Ala, and Lys570Arg. Phylogenetic analysis of full-length VP2 gene indicated a close relationship between Chinese strains and other Asian strains, suggesting mutual transmission between Asian countries. Furthermore, intercontinental transmission is a cause for concern. Surprisingly, two feline panleukopenia virus (FPV) strains with the Ile101Thr mutation in the VP2 protein were identified in canine fecal samples; FPV has been considered incapable of infecting dogs. This study clarified the epidemic characteristics of Chinese CPV-2 strains detected between 2014 and 2019, offering a reference for epidemic control. In addition, the detection of FPV in canine samples may provide information for future studies on the evolution of carnivore parvoviruses.
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17
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The VP1u of Human Parvovirus B19: A Multifunctional Capsid Protein with Biotechnological Applications. Viruses 2020; 12:v12121463. [PMID: 33352888 PMCID: PMC7765992 DOI: 10.3390/v12121463] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2020] [Revised: 12/15/2020] [Accepted: 12/16/2020] [Indexed: 02/06/2023] Open
Abstract
The viral protein 1 unique region (VP1u) of human parvovirus B19 (B19V) is a multifunctional capsid protein with essential roles in virus tropism, uptake, and subcellular trafficking. These functions reside on hidden protein domains, which become accessible upon interaction with cell membrane receptors. A receptor-binding domain (RBD) in VP1u is responsible for the specific targeting and uptake of the virus exclusively into cells of the erythroid lineage in the bone marrow. A phospholipase A2 domain promotes the endosomal escape of the incoming virus. The VP1u is also the immunodominant region of the capsid as it is the target of neutralizing antibodies. For all these reasons, the VP1u has raised great interest in antiviral research and vaccinology. Besides the essential functions in B19V infection, the remarkable erythroid specificity of the VP1u makes it a unique erythroid cell surface biomarker. Moreover, the demonstrated capacity of the VP1u to deliver diverse cargo specifically to cells around the proerythroblast differentiation stage, including erythroleukemic cells, offers novel therapeutic opportunities for erythroid-specific drug delivery. In this review, we focus on the multifunctional role of the VP1u in B19V infection and explore its potential in diagnostics and erythroid-specific therapeutics.
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18
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Ferreira T, Kulkarni A, Bretscher C, Richter K, Ehrlich M, Marchini A. Oncolytic H-1 Parvovirus Enters Cancer Cells through Clathrin-Mediated Endocytosis. Viruses 2020; 12:v12101199. [PMID: 33096814 PMCID: PMC7594094 DOI: 10.3390/v12101199] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2020] [Revised: 10/16/2020] [Accepted: 10/19/2020] [Indexed: 12/11/2022] Open
Abstract
H-1 protoparvovirus (H-1PV) is a self-propagating virus that is non-pathogenic in humans and has oncolytic and oncosuppressive activities. H-1PV is the first member of the Parvoviridae family to undergo clinical testing as an anticancer agent. Results from clinical trials in patients with glioblastoma or pancreatic carcinoma show that virus treatment is safe, well-tolerated and associated with first signs of efficacy. Characterisation of the H-1PV life cycle may help to improve its efficacy and clinical outcome. In this study, we investigated the entry route of H-1PV in cervical carcinoma HeLa and glioma NCH125 cell lines. Using electron and confocal microscopy, we detected H-1PV particles within clathrin-coated pits and vesicles, providing evidence that the virus uses clathrin-mediated endocytosis for cell entry. In agreement with these results, we found that blocking clathrin-mediated endocytosis using specific inhibitors or small interfering RNA-mediated knockdown of its key regulator, AP2M1, markedly reduced H-1PV entry. By contrast, we found no evidence of viral entry through caveolae-mediated endocytosis. We also show that H-1PV entry is dependent on dynamin, while viral trafficking occurs from early to late endosomes, with acidic pH necessary for a productive infection. This is the first study that characterises the cell entry pathways of oncolytic H-1PV.
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Affiliation(s)
- Tiago Ferreira
- Laboratory of Oncolytic Virus Immuno-Therapeutics, German Cancer Research Centre, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany; (T.F.); (C.B.)
| | - Amit Kulkarni
- Laboratory of Oncolytic Virus Immuno-Therapeutics, Luxembourg Institute of Health, 84 Val Fleuri, L-1526 Luxembourg, Luxembourg;
| | - Clemens Bretscher
- Laboratory of Oncolytic Virus Immuno-Therapeutics, German Cancer Research Centre, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany; (T.F.); (C.B.)
| | - Karsten Richter
- Core Facility Electron Microscopy, German Cancer Research Centre, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany;
| | - Marcelo Ehrlich
- Laboratory of Signal Transduction and Membrane Biology, The Shumins School for Biomedicine and Cancer Research, George S. Wise Faculty of Life Sciences, Tel Aviv University, 69978 Tel Aviv, Israel;
| | - Antonio Marchini
- Laboratory of Oncolytic Virus Immuno-Therapeutics, German Cancer Research Centre, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany; (T.F.); (C.B.)
- Laboratory of Oncolytic Virus Immuno-Therapeutics, Luxembourg Institute of Health, 84 Val Fleuri, L-1526 Luxembourg, Luxembourg;
- Correspondence: or ; Tel.: +49-6221-424969 or +352-26-970-856
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Liu Y, Wang J, Chen Y, Wang A, Wei Q, Yang S, Feng H, Chai S, Liu D, Zhang G. Identification of a dominant linear epitope on the VP2 capsid protein of porcine parvovirus and characterization of two monoclonal antibodies with neutralizing abilities. Int J Biol Macromol 2020; 163:2013-2022. [PMID: 32931829 DOI: 10.1016/j.ijbiomac.2020.09.055] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2020] [Revised: 09/06/2020] [Accepted: 09/08/2020] [Indexed: 10/23/2022]
Abstract
Porcine parvovirus (PPV) is a major cause of reproductive failure in swine, and has caused huge losses throughout the world. The structural viral protein VP2, which is able to self-assemble into empty capsids, known as virus-like particles (VLPs), is crucial to induce PPV-specific neutralizing antibodies and protective immunity. In this study, twelve monoclonal antibodies (mAbs) against PPV were generated. The mAbs were characterized by indirect enzyme-linked immunosorbent assay (ELISA), western blotting (WB) and virus neutralization (VN) assay. Two mAbs were defined to be able to neutralize the standard PPV 7909 strain. Subsequently, peptide scanning was applied to identify linear epitopes. The peptide, 89ESGVAGQMV97 was defined as a precise linear epitope. Results from structural analysis showed that the epitope was exposed on the virion surface. Multiple sequence alignment analysis indicated that peptide 89ESGVAGQMV97 was not completely conserved, with a higher amino acid mutation rate at 91G, 92V and 93A position. Alanine-scanning mutagenesis further revealed that residues 89E, 90S, 91G, 92V and 94G were the core sites involved in antibody recognition. These findings may facilitate further understanding the function of the VP2 protein and development of diagnostic tools.
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Affiliation(s)
- Yunchao Liu
- Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
| | - Jucai Wang
- Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China; College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China
| | - Yumei Chen
- School of Life Sciences, Zhengzhou University, Zhengzhou 450001, China
| | - Aiping Wang
- School of Life Sciences, Zhengzhou University, Zhengzhou 450001, China
| | - Qiang Wei
- Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
| | - Suzhen Yang
- Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
| | - Hua Feng
- Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
| | - Shujun Chai
- Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
| | - Dongmin Liu
- Henan Zhongze Biological Engineering Co., Ltd, Zhengzhou, China
| | - Gaiping Zhang
- Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China; College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China; School of Life Sciences, Zhengzhou University, Zhengzhou 450001, China; Henan Zhongze Biological Engineering Co., Ltd, Zhengzhou, China; Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonose, Yangzhou University, Yangzhou, China.
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20
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Moon BY, Jang J, Kim SH, Kim YH, Lee HK, So B, Park CK, Lee KK. Genetic characterization of canine parvovirus type 2c from domestic dogs in Korea. Transbound Emerg Dis 2020; 67:1645-1653. [PMID: 32009300 PMCID: PMC7228216 DOI: 10.1111/tbed.13501] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2019] [Revised: 01/27/2020] [Accepted: 01/28/2020] [Indexed: 01/01/2023]
Abstract
Canine parvovirus type 2 (CPV‐2) is an aetiological agent that causes acute haemorrhagic enteritis and fatal myocarditis in dogs. Since CPV‐2 first emerged in the late 1970s, its rapid evolution has resulted in three antigenic variants: CPV‐2a, CPV‐2b and CPV‐2c. Here, we report, for the first time in Korea, two cases of CPV‐2c infection in two dogs with severe diarrhoea. The complete open reading frame (4,269nt) of CPV‐2, encoding both non‐structural (NS) and structural (VP) proteins, was sequenced. Based on the amino acid Gln present at residue 426 of the VP2 gene, these strains were typed as CPV‐2c, and were named Korea CPV‐2c_1 and Korea CPV‐2c_2. These strains shared 99.48% reciprocal nucleotide sequence identity and had the highest nucleotide identity (99.77%–99.34%) with Asian CPV strains isolated in China, Italy (found in a dog imported from Thailand), and Vietnam from 2013 to 2017. Phylogenetic analysis based on the non‐structural (NS1) and capsid (VP2) genes revealed that Korean CPV‐2c strains clustered closely to Asian CPV strains, and separately from strains isolated in Europe, South America and North America. Amino acid changes never reported before were observed in NS1 (Thr70Pro, Cys287Tyr), VP1 (Lys17Arg, Phe33Leu) and VP2 (Gln365His, Ala516Val). Additional observed mutations, including Phe267Tyr, Tyr324Ile and Gln370Arg, have been previously reported in the recent CPV‐2c strains with Asian origins. These results suggest that the Korean CPV‐2c strains were potentially introduced via neighbouring Asian countries.
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Affiliation(s)
- Bo-Youn Moon
- Animal Disease Diagnosis Division, Animal and Plant Quarantine Agency, Gimcheon, Republic of Korea
| | - Jiung Jang
- Animal Disease Diagnosis Division, Animal and Plant Quarantine Agency, Gimcheon, Republic of Korea.,College of Veterinary Medicine & Animal Disease Intervention Center, Kyungpook National University, Daegu, Republic of Korea
| | - Seong-Hee Kim
- Animal Disease Diagnosis Division, Animal and Plant Quarantine Agency, Gimcheon, Republic of Korea
| | - Yeon-Hee Kim
- Animal Disease Diagnosis Division, Animal and Plant Quarantine Agency, Gimcheon, Republic of Korea
| | | | - ByungJae So
- Animal Disease Diagnosis Division, Animal and Plant Quarantine Agency, Gimcheon, Republic of Korea
| | - Choi-Kyu Park
- College of Veterinary Medicine & Animal Disease Intervention Center, Kyungpook National University, Daegu, Republic of Korea
| | - Kyoung-Ki Lee
- Animal Disease Diagnosis Division, Animal and Plant Quarantine Agency, Gimcheon, Republic of Korea
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21
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Cytoplasmic Parvovirus Capsids Recruit Importin Beta for Nuclear Delivery. J Virol 2020; 94:JVI.01532-19. [PMID: 31748386 DOI: 10.1128/jvi.01532-19] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2019] [Accepted: 11/16/2019] [Indexed: 02/06/2023] Open
Abstract
Parvoviruses are an important platform for gene and cancer therapy. Their cell entry and the following steps, including nuclear import, are inefficient, limiting their use in therapeutic applications. Two models exist on parvoviral nuclear entry: the classical import of the viral capsid using nuclear transport receptors of the importin (karyopherin) family or the direct attachment of the capsid to the nuclear pore complex leading to the local disintegration of the nuclear envelope. Here, by laser scanning confocal microscopy and in situ proximity ligation analyses combined with coimmunoprecipitation, we show that infection requires importin β-mediated access to the nuclear pore complex and nucleoporin 153-mediated interactions on the nuclear side. The importin β-capsid interaction continued within the nucleoplasm, which suggests a mixed model of nuclear entry in which the classical nuclear import across the nuclear pore complex is accompanied by transient ruptures of the nuclear envelope, also allowing the passive entry of importin β-capsid complexes into the nucleus.IMPORTANCE Parvoviruses are small DNA viruses that deliver their DNA into the postmitotic nuclei, which is an important step for parvoviral gene and cancer therapies. Limitations in virus-receptor interactions or endocytic entry do not fully explain the low transduction/infection efficiency, indicating a bottleneck after virus entry into the cytoplasm. We thus investigated the transfer of parvovirus capsids from the cytoplasm to the nucleus, showing that the nuclear import of the parvovirus capsid follows a unique strategy, which differs from classical nuclear import and those of other viruses.
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Dudek AM, Zabaleta N, Zinn E, Pillay S, Zengel J, Porter C, Franceschini JS, Estelien R, Carette JE, Zhou GL, Vandenberghe LH. GPR108 Is a Highly Conserved AAV Entry Factor. Mol Ther 2019; 28:367-381. [PMID: 31784416 DOI: 10.1016/j.ymthe.2019.11.005] [Citation(s) in RCA: 89] [Impact Index Per Article: 14.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2019] [Revised: 10/26/2019] [Accepted: 11/05/2019] [Indexed: 12/19/2022] Open
Abstract
Adeno-associated virus (AAV) is a highly promising gene transfer vector, yet major cellular requirements for AAV entry are poorly understood. Using a genome-wide CRISPR screen for entry of evolutionarily divergent serotype AAVrh32.33, we identified GPR108, a member of the G protein-coupled receptor superfamily, as an AAV entry factor. Of greater than 20 divergent AAVs across all AAV clades tested in human cell lines, only AAV5 transduction was unaffected in the GPR108 knockout (KO). GPR108 dependency was further shown in murine and primary cells in vitro. These findings are further validated in vivo, as the Gpr108 KO mouse demonstrates 10- to 100-fold reduced expression for AAV8 and rh32.33 but not AAV5. Mechanistically, both GPR108 N- and C-terminal domains are required for transduction, and on the capsid, a VP1 unique domain that is not conserved on AAV5 can be transferred to confer GPR108 independence onto AAV2 chimeras. In vitro binding and fractionation studies indicate reduced nuclear import and cytosolic accumulation in the absence of GPR108. We thus have identified the second of two AAV entry factors that is conserved between mice and humans relevant both in vitro and in vivo, further providing a mechanistic understanding to the tropism of AAV gene therapy vectors.
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Affiliation(s)
- Amanda M Dudek
- Grousbeck Gene Therapy Center, Schepens Eye Research Institute, Mass Eye and Ear, Boston, MA, USA; Ocular Genomics Institute, Mass Eye and Ear, Boston, MA, USA; Department of Ophthalmology, Harvard Medical School, Boston, MA, USA
| | - Nerea Zabaleta
- Grousbeck Gene Therapy Center, Schepens Eye Research Institute, Mass Eye and Ear, Boston, MA, USA; Ocular Genomics Institute, Mass Eye and Ear, Boston, MA, USA; Department of Ophthalmology, Harvard Medical School, Boston, MA, USA
| | - Eric Zinn
- Grousbeck Gene Therapy Center, Schepens Eye Research Institute, Mass Eye and Ear, Boston, MA, USA; Ocular Genomics Institute, Mass Eye and Ear, Boston, MA, USA; Department of Ophthalmology, Harvard Medical School, Boston, MA, USA
| | - Sirika Pillay
- Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA
| | - James Zengel
- Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA
| | - Caryn Porter
- Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA, USA; Department of Genetics, Harvard Medical School, Boston, MA, USA
| | - Jennifer Santos Franceschini
- Grousbeck Gene Therapy Center, Schepens Eye Research Institute, Mass Eye and Ear, Boston, MA, USA; Ocular Genomics Institute, Mass Eye and Ear, Boston, MA, USA; Department of Ophthalmology, Harvard Medical School, Boston, MA, USA
| | - Reynette Estelien
- Grousbeck Gene Therapy Center, Schepens Eye Research Institute, Mass Eye and Ear, Boston, MA, USA; Ocular Genomics Institute, Mass Eye and Ear, Boston, MA, USA; Department of Ophthalmology, Harvard Medical School, Boston, MA, USA
| | - Jan E Carette
- Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA
| | - Guo Ling Zhou
- Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA, USA; Department of Genetics, Harvard Medical School, Boston, MA, USA
| | - Luk H Vandenberghe
- Grousbeck Gene Therapy Center, Schepens Eye Research Institute, Mass Eye and Ear, Boston, MA, USA; Ocular Genomics Institute, Mass Eye and Ear, Boston, MA, USA; Department of Ophthalmology, Harvard Medical School, Boston, MA, USA; The Broad Institute of Harvard and MIT, Cambridge, MA, USA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA.
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Parvovirus B19 Uncoating Occurs in the Cytoplasm without Capsid Disassembly and It Is Facilitated by Depletion of Capsid-Associated Divalent Cations. Viruses 2019; 11:v11050430. [PMID: 31083301 PMCID: PMC6563316 DOI: 10.3390/v11050430] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2019] [Revised: 05/07/2019] [Accepted: 05/09/2019] [Indexed: 12/19/2022] Open
Abstract
Human parvovirus B19 (B19V) traffics to the cell nucleus where it delivers the genome for replication. The intracellular compartment where uncoating takes place, the required capsid structural rearrangements and the cellular factors involved remain unknown. We explored conditions that trigger uncoating in vitro and found that prolonged exposure of capsids to chelating agents or to buffers with chelating properties induced a structural rearrangement at 4 °C resulting in capsids with lower density. These lighter particles remained intact but were unstable and short exposure to 37 °C or to a freeze-thaw cycle was sufficient to trigger DNA externalization without capsid disassembly. The rearrangement was not observed in the absence of chelating activity or in the presence of MgCl2 or CaCl2, suggesting that depletion of capsid-associated divalent cations facilitates uncoating. The presence of assembled capsids with externalized DNA was also detected during B19V entry in UT7/Epo cells. Following endosomal escape and prior to nuclear entry, a significant proportion of the incoming capsids rearranged and externalized the viral genome without capsid disassembly. The incoming capsids with accessible genomes accumulated in the nuclear fraction, a process that was prevented when endosomal escape or dynein function was disrupted. In their uncoated conformation, capsids immunoprecipitated from cytoplasmic or from nuclear fractions supported in vitro complementary-strand synthesis at 37 °C. This study reveals an uncoating strategy of B19V based on a limited capsid rearrangement prior to nuclear entry, a process that can be mimicked in vitro by depletion of divalent cations.
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Callaway HM, Subramanian S, Urbina CA, Barnard KN, Dick RA, Bator CM, Hafenstein SL, Gifford RJ, Parrish CR. Examination and Reconstruction of Three Ancient Endogenous Parvovirus Capsid Protein Gene Remnants Found in Rodent Genomes. J Virol 2019; 93:e01542-18. [PMID: 30626673 PMCID: PMC6401472 DOI: 10.1128/jvi.01542-18] [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: 09/17/2018] [Accepted: 12/24/2018] [Indexed: 11/20/2022] Open
Abstract
Parvovirus-derived endogenous viral elements (EVEs) have been found in the genomes of many different animal species, resulting from integration events that may have occurred from more than 50 million years ago to much more recently. Here, we further investigate the properties of autonomous parvovirus EVEs and describe their relationships to contemporary viruses. While we did not find any intact capsid protein open reading frames in the integrated viral sequences, we examined three EVEs that were repaired to form full-length sequences with relatively few changes. These sequences were found in the genomes of Rattus norvegicus (brown rat), Mus spretus (Algerian mouse), and Apodemus sylvaticus (wood mouse). The R. norvegicus sequence was not present in the genomes of the closely related species R. rattus, R. tanezumi, R. exulans, and R. everetti, indicating that it was less than 2 million years old, and the M. spretus and A. sylvaticus sequences were not found in the published genomes of other mouse species, also indicating relatively recent insertions. The M. spretus VP2 sequence assembled into capsids, which had high thermal stability, bound the sialic acid N-acetylneuraminic acid, and entered murine L cells. The 3.89-Å structure of the M. spretus virus-like particles (VLPs), determined using cryo-electron microscopy, showed similarities to rodent and porcine parvovirus capsids. The repaired VP2 sequences from R. norvegicus and A. sylvaticus did not assemble as first prepared, but chimeras combining capsid surface loops from R. norvegicus with canine parvovirus assembled, allowing some of that capsid's structures and functions to be examined.IMPORTANCE Parvovirus endogenous viral elements (EVEs) that have been incorporated into the genomes of different animals represent remnants of the DNA sequences of ancient viruses that infected the ancestors of those animals millions of years ago, but we know little about their properties or how they differ from currently circulating parvoviruses. By expressing the capsid proteins of different parvovirus EVEs that were found integrated into the genomes of three different rodents, we can examine their structures and functions. A VP2 (major capsid protein) EVE sequence from a mouse genome assembled into capsids that had a similar structure and biophysical properties to extant parvoviruses and also bound sialic acids and entered rodent cells. Chimeras formed from combinations of canine parvovirus and portions of the parvovirus sequences from the brown rat genome allowed us to examine the structures and functions of the surface loops of that EVE capsid.
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Affiliation(s)
- Heather M Callaway
- Baker Institute for Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA
| | - Suriyasri Subramanian
- Department of Medicine, Penn State University College of Medicine, Hershey, Pennsylvania, USA
- Department of Biochemistry and Molecular Biology, Penn State University, University Park, Pennsylvania, USA
| | - Christian A Urbina
- Baker Institute for Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA
| | - Karen N Barnard
- Baker Institute for Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA
| | - Robert A Dick
- Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA
| | - Carol M Bator
- Department of Medicine, Penn State University College of Medicine, Hershey, Pennsylvania, USA
- Department of Biochemistry and Molecular Biology, Penn State University, University Park, Pennsylvania, USA
| | - Susan L Hafenstein
- Department of Medicine, Penn State University College of Medicine, Hershey, Pennsylvania, USA
- Department of Biochemistry and Molecular Biology, Penn State University, University Park, Pennsylvania, USA
| | - Robert J Gifford
- MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom
| | - Colin R Parrish
- Baker Institute for Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA
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25
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Joshi PR, Cervera L, Ahmed I, Kondratov O, Zolotukhin S, Schrag J, Chahal PS, Kamen AA. Achieving High-Yield Production of Functional AAV5 Gene Delivery Vectors via Fedbatch in an Insect Cell-One Baculovirus System. MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT 2019; 13:279-289. [PMID: 30886878 PMCID: PMC6404649 DOI: 10.1016/j.omtm.2019.02.003] [Citation(s) in RCA: 32] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/21/2018] [Accepted: 02/07/2019] [Indexed: 11/19/2022]
Abstract
Despite numerous advancements in production protocols, manufacturing AAV to meet exceptionally high demand (1016–1017 viral genomes [VGs]) in late clinical stages and for eventual systemic delivery poses significant challenges. Here, we report an efficient, simple, scalable, robust AAV5 production process utilizing the most recent modification of the OneBac platform. An increase in volumetric yield of genomic particles by ∼6-fold and functional particles by ∼20-fold was achieved by operating a high-cell-density process in shake flasks and bioreactors that involves an Sf9-based rep/cap stable cell line grown at a density of about 10 million cells/mL infected with a single baculovirus. The overall volumetric yields of genomic (VG) and bioactive particles (enhanced transducing units [ETUs]) in representative fedbatch bioreactor runs ranged from 2.5 to 3.5 × 1014 VG/L and from 1 to 2 × 1011 ETU/L. Analytical ultracentrifugation analyses of affinity-purified AAV vector samples from side-by-side batch and fedbatch production runs showed vector preparations with a full and empty particle distribution of 20%–30% genomic and 70%–80% empty particles. Moreover, the stoichiometric analysis of capsid proteins from fedbatch production in shake flask and bioreactor run samples demonstrated the incorporation of higher VP1 subunits, resulting in better functionality.
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Affiliation(s)
- Pranav R.H. Joshi
- Viral Vectors and Vaccine Bioprocessing Group, Department of Bioengineering, McGill University, Montreal, QC H3A 0E9, Canada
| | - Laura Cervera
- Viral Vectors and Vaccine Bioprocessing Group, Department of Bioengineering, McGill University, Montreal, QC H3A 0E9, Canada
| | - Ibrahim Ahmed
- Viral Vectors and Vaccine Bioprocessing Group, Department of Bioengineering, McGill University, Montreal, QC H3A 0E9, Canada
| | - Oleksandr Kondratov
- Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL 32610, USA
| | - Sergei Zolotukhin
- Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL 32610, USA
| | - Joseph Schrag
- Human Health Therapeutics Portfolio, National Research Council of Canada, Montreal, QC H4P 2R2, Canada
| | - Parminder S. Chahal
- Human Health Therapeutics Portfolio, National Research Council of Canada, Montreal, QC H4P 2R2, Canada
| | - Amine A. Kamen
- Viral Vectors and Vaccine Bioprocessing Group, Department of Bioengineering, McGill University, Montreal, QC H3A 0E9, Canada
- Corresponding author: Amine Kamen, Viral Vectors and Vaccine Bioprocessing Group, Department of Bioengineering, McGill University, Montreal, QC H3A 0E9, Canada.
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26
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Dunbar CA, Callaway HM, Parrish CR, Jarrold MF. Probing Antibody Binding to Canine Parvovirus with Charge Detection Mass Spectrometry. J Am Chem Soc 2018; 140:15701-15711. [PMID: 30398860 DOI: 10.1021/jacs.8b08050] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
There are many techniques for monitoring and measuring the interactions between proteins and ligands. Most of these techniques are ensemble methods that can provide association constants and in some cases stoichiometry. Here we use charge detection mass spectrometry (CDMS), a single particle technique, to probe the interactions of antigen binding fragments (Fabs) from a series of antibodies with the canine parvovirus (CPV) capsid. In addition to providing the average number of bound Fabs as a function of Fab concentration (i.e., the binding curve), CDMS measurements provide information about the distribution of bound Fabs. We show that the distribution of bound ligands is much better at distinguishing between different binding models than the binding curve. The binding of Fab E to CPV is a textbook example. A maximum of 60 Fabs bind and the results are consistent with a model where all sites have the same binding affinity. However, for Fabs B, F, and 14, the distributions can only be fit by a model where there are distinct virus subpopulations with different binding affinities. This behavior can be distinguished from a situation where all CPV particles are identical, and each particle has the same distribution of sites with different binding affinities. The different responses to viral heterogeneity can be traced to the Fab binding sites. A comparison of Fab binding to new and aged CPV capsids reveals that a post-translational modification at the binding site for Fab E (M569) probably reduces the binding affinity.
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Affiliation(s)
- Carmen A Dunbar
- Department of Chemistry , Indiana University , 800 E. Kirkwood Ave. , Bloomington , Indiana 47405 , United States
| | - Heather M Callaway
- Baker Institute for Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine , Cornell University , Ithaca , New York 14850 , United States
| | - Colin R Parrish
- Baker Institute for Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine , Cornell University , Ithaca , New York 14850 , United States
| | - Martin F Jarrold
- Department of Chemistry , Indiana University , 800 E. Kirkwood Ave. , Bloomington , Indiana 47405 , United States
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Intracellular Localization of Blattella germanica Densovirus (BgDV1) Capsid Proteins. Viruses 2018; 10:v10070370. [PMID: 30011943 PMCID: PMC6071259 DOI: 10.3390/v10070370] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2018] [Revised: 07/10/2018] [Accepted: 07/12/2018] [Indexed: 01/16/2023] Open
Abstract
Densovirus genome replication and capsid assembly take place in the nucleus of the infected cells. However, the mechanisms underlying such processes as the delivery of virus proteins to the nucleus and the export of progeny virus from the nucleus remain elusive. It is evident that nuclear transport signals should be involved in these processes. We performed an in silico search for the putative nuclear localization signal (NLS) and nuclear export signal (NES) motifs in the capsid proteins of the Blattella germanica Densovirus 1 (BgDV1) densovirus. A high probability NLS motif was found in the common C-terminal of capsid proteins together with a NES motif in the unique N-terminal of VP2. We also performed a global search for the nuclear traffic signals in the densoviruses belonging to five Densovirinae genera, which revealed high diversity in the patterns of NLSs and NESs. Using a heterologous system, the HeLa mammalian cell line expressing GFP-fused BgDV1 capsid proteins, we demonstrated that both signals are functionally active. We suggest that the NLS shared by all three BgDV1 capsid proteins drives the trafficking of the newly-synthesized proteins into the nucleus, while the NES may play a role in the export of the newly-assembled BgDV1 particles into the cytoplasm through nuclear pore complexes.
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Pénzes JJ, Marsile-Medun S, Agbandje-McKenna M, Gifford RJ. Endogenous amdoparvovirus-related elements reveal insights into the biology and evolution of vertebrate parvoviruses. Virus Evol 2018; 4:vey026. [PMID: 30443409 PMCID: PMC6232428 DOI: 10.1093/ve/vey026] [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] [Indexed: 02/06/2023] Open
Abstract
Amdoparvoviruses (family Parvoviridae: genus Amdoparvovirus) infect carnivores, and are a major cause of morbidity and mortality in farmed animals. In this study, we systematically screened animal genomes to identify endogenous parvoviral elements (EPVs) disclosing a high degree of similarity to amdoparvoviruses, and investigated their genomic, phylogenetic and protein structural features. We report the first examples of full-length, amdoparvovirus-derived EPVs in the genome of the Transcaucasian mole vole (Ellobius lutescens). We also identify four EPVs in mammal and reptile genomes that are intermediate between amdoparvoviruses and their sister genus (Protoparvovirus) in terms of their phylogenetic placement and genomic features. In particular, we identify a genome-length EPV in the genome of a pit viper (Protobothrops mucrosquamatus) that is more similar to a protoparvovirus than an amdoparvovirus in terms of its phylogenetic placement and the structural features of its capsid protein (as revealed by homology modeling), yet exhibits characteristically amdoparvovirus-like genome features including: (1) a putative middle ORF gene; (2) a capsid gene that lacks a phospholipase A2 domain; (3) a genome structure consistent with an amdoparvovirus-like mechanism of capsid gene expression. Our findings indicate that amdoparvovirus host range extends to rodents, and that parvovirus lineages possessing a mixture of proto- and amdoparvovirus-like characteristics have circulated in the past. In addition, we show that EPV sequences in the mole vole and pit viper encode intact, expressible replicase genes that have potentially been co-opted or exapted in these host species.
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Affiliation(s)
- Judit J Pénzes
- University of Florida McKnight Brain Institute, 1149 Newell Dr, Gainesville, USA
| | - Soledad Marsile-Medun
- Agrocampus Ouest, 65 Rue de Saint-Brieuc, Rennes, France
- MRC-University of Glasgow Centre for Virus Research, 464 Bearsden Road, Glasgow, UK
| | | | - Robert James Gifford
- MRC-University of Glasgow Centre for Virus Research, 464 Bearsden Road, Glasgow, UK
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29
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The 164 K, 165 K and 167 K residues in 160YPVVKKPKLTEE171 are required for the nuclear import of goose parvovirus VP1. Virology 2018; 519:17-22. [DOI: 10.1016/j.virol.2018.03.020] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2017] [Revised: 03/12/2018] [Accepted: 03/22/2018] [Indexed: 11/23/2022]
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30
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Karyopherin Alpha 6 Is Required for Replication of Porcine Reproductive and Respiratory Syndrome Virus and Zika Virus. J Virol 2018; 92:JVI.00072-18. [PMID: 29444946 DOI: 10.1128/jvi.00072-18] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2018] [Accepted: 02/09/2018] [Indexed: 12/16/2022] Open
Abstract
Movement of macromolecules between the cytoplasm and the nucleus occurs through the nuclear pore complex (NPC). Karyopherins comprise a family of soluble transport factors facilitating the nucleocytoplasmic translocation of proteins through the NPC. In this study, we found that karyopherin α6 (KPNA6; also known as importin α7) was required for the optimal replication of porcine reproductive and respiratory syndrome virus (PRRSV) and Zika virus (ZIKV), which are positive-sense, single-stranded RNA viruses replicating in the cytoplasm. The KPNA6 protein level in virus-infected cells was much higher than that in mock-infected controls, whereas the KPNA6 transcript remains stable. Viral infection blocked the ubiquitin-proteasomal degradation of KPNA6, which led to an extension of the KPNA6 half-life and the elevation of the KPNA6 level in comparison to mock-infected cells. PRRSV nsp12 protein induced KPNA6 stabilization. KPNA6 silencing was detrimental to the replication of PRRSV, and KPNA6 knockout impaired ZIKV replication. Moreover, KPNA6 knockout blocked the nuclear translocation of PRRSV nsp1β but had a minimal effect on two other PRRSV proteins with nuclear localization. Exogenous restitution of KPNA6 expression in the KPNA6-knockout cells results in restoration of the nuclear translocation of PRRSV nsp1β and the replication of ZIKV. These results indicate that KPNA6 is an important cellular factor for the replication of PRRSV and ZIKV.IMPORTANCE Positive-sense, single-stranded RNA (+ssRNA) viruses replicate in the cytoplasm of infected cells. The roles of transport factors in the nucleocytoplasmic trafficking system for the replication of +ssRNA viruses are not known. In this study, we discovered that PRRSV and ZIKV viruses needed karyopherin α6 (KPNA6), one of the transport factors, to enhance the virus replication. Our data showed that viral infection induced an elevation of the KPNA6 protein level due to an extension of the KPNA6 half-life via viral interference of the ubiquitin-proteasomal degradation of KPNA6. Notably, KPNA6 silencing or knockout dramatically reduced the replication of PRRSV and ZIKV. PRRSV nsp1β depended on KPNA6 to translocate into the nucleus. In addition, exogenous restitution of KPNA6 expression in KPNA6-knockout cells led to the restoration of nsp1β nuclear translocation and ZIKV replication. These results reveal a new aspect in the virus-cell interaction and may facilitate the development of novel antiviral therapeutics.
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Gil-Ranedo J, Hernando E, Valle N, Riolobos L, Maroto B, Almendral JM. Differential phosphorylation and n-terminal configuration of capsid subunits in parvovirus assembly and viral trafficking. Virology 2018. [PMID: 29524834 DOI: 10.1016/j.virol.2018.02.018] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/17/2022]
Abstract
The T1 parvovirus Minute Virus of Mice (MVM) was used to study the roles that phosphorylation and N-terminal domains (Nt) configuration of capsid subunits may play in icosahedral nuclear viruses assembly. In synchronous MVM infection, capsid subunits newly assembled as two types of cytoplasmic trimeric intermediates (3VP2, and 1VP1:2VP2) harbored a VP1 phosphorylation level fivefold higher than that of VP2, and hidden Nt. Upon nuclear translocation at S phase, VP1-Nt became exposed in the heterotrimer and subsequent subviral assembly intermediates. Empty capsid subunits showed a phosphorylation level restored to VP1:VP2 stoichiometry, and the Nt concealed in their interior. However ssDNA-filled virus maturing at S/G2 lacked VP1 phosphorylation and one major VP2 phosphopeptide, and exposed VP2-Nt. Endosomal VP2-Nt cleavage resulted in VP3 subunits devoid of any phospholabel, implying that incoming viral particles specifically harbor a low phosphorylation status. Phosphorylation provides a mechanistic coupling of parvovirus nuclear assembly to the cell cycle.
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Affiliation(s)
- Jon Gil-Ranedo
- Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Cantoblanco, 28049 Madrid, Spain
| | - Eva Hernando
- Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Cantoblanco, 28049 Madrid, Spain
| | - Noelia Valle
- Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Cantoblanco, 28049 Madrid, Spain
| | - Laura Riolobos
- Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Cantoblanco, 28049 Madrid, Spain
| | - Beatriz Maroto
- Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Cantoblanco, 28049 Madrid, Spain
| | - José M Almendral
- Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Cantoblanco, 28049 Madrid, Spain.
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32
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Viral highway to nucleus exposed by image correlation analyses. Sci Rep 2018; 8:1152. [PMID: 29348472 PMCID: PMC5773500 DOI: 10.1038/s41598-018-19582-w] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2017] [Accepted: 01/04/2018] [Indexed: 01/26/2023] Open
Abstract
Parvoviral genome translocation from the plasma membrane into the nucleus is a coordinated multistep process mediated by capsid proteins. We used fast confocal microscopy line scan imaging combined with image correlation methods including auto-, pair- and cross-correlation, and number and brightness analysis, to study the parvovirus entry pathway at the single-particle level in living cells. Our results show that the endosome-associated movement of virus particles fluctuates from fast to slow. Fast transit of single cytoplasmic capsids to the nuclear envelope is followed by slow movement of capsids and fast diffusion of capsid fragments in the nucleoplasm. The unique combination of image analyses allowed us to follow the fate of intracellular single virus particles and their interactions with importin β revealing previously unknown dynamics of the entry pathway.
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Ros C, Bayat N, Wolfisberg R, Almendral JM. Protoparvovirus Cell Entry. Viruses 2017; 9:v9110313. [PMID: 29072600 PMCID: PMC5707520 DOI: 10.3390/v9110313] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2017] [Revised: 10/21/2017] [Accepted: 10/23/2017] [Indexed: 01/25/2023] Open
Abstract
The Protoparvovirus (PtPV) genus of the Parvoviridae family of viruses includes important animal pathogens and reference molecular models for the entire family. Some virus members of the PtPV genus have arisen as promising tools to treat tumoral processes, as they exhibit marked oncotropism and oncolytic activities while being nonpathogenic for humans. The PtPVs invade and replicate within the nucleus making extensive use of the transport, transcription and replication machineries of the host cells. In order to reach the nucleus, PtPVs need to cross over several intracellular barriers and traffic through different cell compartments, which limit their infection efficiency. In this review we summarize molecular interactions, capsid structural transitions and hijacking of cellular processes, by which the PtPVs enter and deliver their single-stranded DNA genome into the host cell nucleus. Understanding mechanisms that govern the complex PtPV entry will be instrumental in developing approaches to boost their anticancer therapeutic potential and improving their safety profile.
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Affiliation(s)
- Carlos Ros
- Department of Chemistry and Biochemistry, University of Bern, 3012 Bern, Switzerland.
| | - Nooshin Bayat
- Centro de Biología Molecular "Severo Ochoa", Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
| | - Raphael Wolfisberg
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, 1165 Copenhagen, Denmark.
| | - José M Almendral
- Centro de Biología Molecular "Severo Ochoa", Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
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Protoparvovirus Knocking at the Nuclear Door. Viruses 2017; 9:v9100286. [PMID: 28974036 PMCID: PMC5691637 DOI: 10.3390/v9100286] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2017] [Revised: 09/28/2017] [Accepted: 09/29/2017] [Indexed: 12/20/2022] Open
Abstract
Protoparvoviruses target the nucleus due to their dependence on the cellular reproduction machinery during the replication and expression of their single-stranded DNA genome. In recent years, our understanding of the multistep process of the capsid nuclear import has improved, and led to the discovery of unique viral nuclear entry strategies. Preceded by endosomal transport, endosomal escape and microtubule-mediated movement to the vicinity of the nuclear envelope, the protoparvoviruses interact with the nuclear pore complexes. The capsids are transported actively across the nuclear pore complexes using nuclear import receptors. The nuclear import is sometimes accompanied by structural changes in the nuclear envelope, and is completed by intranuclear disassembly of capsids and chromatinization of the viral genome. This review discusses the nuclear import strategies of protoparvoviruses and describes its dynamics comprising active and passive movement, and directed and diffusive motion of capsids in the molecularly crowded environment of the cell.
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Liu P, Chen S, Wang M, Cheng A. The role of nuclear localization signal in parvovirus life cycle. Virol J 2017; 14:80. [PMID: 28410597 PMCID: PMC5391597 DOI: 10.1186/s12985-017-0745-1] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2017] [Accepted: 03/29/2017] [Indexed: 12/30/2022] Open
Abstract
Parvoviruses are small, non-enveloped viruses with an approximately 5.0 kb, single-stranded DNA genome. Usually, the parvovirus capsid gene contains one or more nuclear localization signals (NLSs), which are required for guiding the virus particle into the nucleus through the nuclear pore. However, several classical NLSs (cNLSs) and non-classical NLSs (ncNLSs) have been identified in non-structural genes, and the ncNLSs can also target non-structural proteins into the nucleus. In this review, we have summarized recent research findings on parvovirus NLSs. The capsid protein of the adeno-associated virus has four potential nuclear localization sequences, named basic region 1 (BR), BR2, BR3 and BR4. BR3 was identified as an NLS by fusing it with green fluorescent protein. Moreover, BR3 and BR4 are required for infectivity and virion assembly. In Protoparvovirus, the canine parvovirus has a common cNLS located in the VP1 unique region, similar to parvovirus minute virus of mice (MVM) and porcine parvovirus. Moreover, an ncNLS is found in the C-terminal region of MVM VP1/2. Parvovirus B19 also contains an ncNLS in the C-terminal region of VP1/2, which is essential for the nuclear transport of VP1/VP2. Approximately 1 or 2 cNLSs and 1 ncNLS have been reported in the non-structural protein of bocaviruses. Understanding the role of the NLS in the process of parvovirus infection and its mechanism of nuclear transport will contribute to the development of therapeutic vaccines and novel antiviral medicines.
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Affiliation(s)
- Peng Liu
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, No. 211 Huimin Road, Wenjiang District, Chengdu, Sichuan, 611130, China
| | - Shun Chen
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, No. 211 Huimin Road, Wenjiang District, Chengdu, Sichuan, 611130, China. .,Research Center of Avian Disease, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, 611130, China. .,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu, Sichuan, 611130, China.
| | - Mingshu Wang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, No. 211 Huimin Road, Wenjiang District, Chengdu, Sichuan, 611130, China.,Research Center of Avian Disease, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, 611130, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu, Sichuan, 611130, China
| | - Anchun Cheng
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, No. 211 Huimin Road, Wenjiang District, Chengdu, Sichuan, 611130, China. .,Research Center of Avian Disease, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, 611130, China. .,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu, Sichuan, 611130, China.
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Zhou P, Zeng W, Zhang X, Li S. The genetic evolution of canine parvovirus - A new perspective. PLoS One 2017; 12:e0175035. [PMID: 28362831 PMCID: PMC5376324 DOI: 10.1371/journal.pone.0175035] [Citation(s) in RCA: 80] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2016] [Accepted: 03/20/2017] [Indexed: 02/03/2023] Open
Abstract
To trace the evolution process of CPV-2, all of the VP2 gene sequences of CPV-2 and FPV (from 1978 to 2015) from GenBank were analyzed in this study. Then, several new ideas regarding CPV-2 evolution were presented. First, the VP2 amino acid 555 and 375 positions of CPV-2 were first ruled out as a universal mutation site in CPV-2a and amino acid 101 position of FPV feature I or T instead of only I in existing rule. Second, the recently confusing nomenclature of CPV-2 variants was substituted with a optional nomenclature that would serve future CPV-2 research. Third, After check the global distribution of variants, CPV-2a is the predominant variant in Asia and CPV-2c is the predominant variant in Europe and Latin America. Fourth, a series of CPV-2-like strains were identified and deduced to evolve from modified live vaccine strains. Finally, three single VP2 mutation (F267Y, Y324I, and T440A) strains were caught concern. Furthermore, these three new VP2 mutation strains may be responsible for vaccine failure, and the strains with VP2 440A may become the novel CPV sub-variant. In conclusion, a summary of all VP2 sequences provides a new perspective regarding CPV-2 evolution and the correlative biological studies needs to be further performed.
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Affiliation(s)
- Pei Zhou
- College of Veterinary Medicine, South China Agricultural University, Tianhe District, Guangzhou, Guangdong Province, People’s Republic of China
- Key Laboratory of Comprehensive Prevention and Control for Severe Clinical Animal Diseases of Guangdong Province, Guangzhou, Guangdong Province, People’s Republic of China
- Guangdong Engineering and Technological Research Center for Pets, Guangzhou, Guangdong Province, People’s Republic of China
| | - Weijie Zeng
- College of Veterinary Medicine, South China Agricultural University, Tianhe District, Guangzhou, Guangdong Province, People’s Republic of China
- Key Laboratory of Comprehensive Prevention and Control for Severe Clinical Animal Diseases of Guangdong Province, Guangzhou, Guangdong Province, People’s Republic of China
- Guangdong Engineering and Technological Research Center for Pets, Guangzhou, Guangdong Province, People’s Republic of China
| | - Xin Zhang
- College of Veterinary Medicine, South China Agricultural University, Tianhe District, Guangzhou, Guangdong Province, People’s Republic of China
- Key Laboratory of Comprehensive Prevention and Control for Severe Clinical Animal Diseases of Guangdong Province, Guangzhou, Guangdong Province, People’s Republic of China
- Guangdong Engineering and Technological Research Center for Pets, Guangzhou, Guangdong Province, People’s Republic of China
| | - Shoujun Li
- College of Veterinary Medicine, South China Agricultural University, Tianhe District, Guangzhou, Guangdong Province, People’s Republic of China
- Key Laboratory of Comprehensive Prevention and Control for Severe Clinical Animal Diseases of Guangdong Province, Guangzhou, Guangdong Province, People’s Republic of China
- Guangdong Engineering and Technological Research Center for Pets, Guangzhou, Guangdong Province, People’s Republic of China
- * E-mail:
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Parvovirus Capsid Structures Required for Infection: Mutations Controlling Receptor Recognition and Protease Cleavages. J Virol 2017; 91:JVI.01871-16. [PMID: 27847360 DOI: 10.1128/jvi.01871-16] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2016] [Accepted: 11/01/2016] [Indexed: 01/11/2023] Open
Abstract
Parvovirus capsids are small but complex molecular machines responsible for undertaking many of the steps of cell infection, genome packing, and cell-to-cell as well as host-to-host transfer. The details of parvovirus infection of cells are still not fully understood, but the processes must involve small changes in the capsid structure that allow the endocytosed virus to escape from the endosome, pass through the cell cytoplasm, and deliver the single-stranded DNA (ssDNA) genome to the nucleus, where viral replication occurs. Here, we examine capsid substitutions that eliminate canine parvovirus (CPV) infectivity and identify how those mutations changed the capsid structure or altered interactions with the infectious pathway. Amino acid substitutions on the exterior surface of the capsid (Gly299Lys/Ala300Lys) altered the binding of the capsid to transferrin receptor type 1 (TfR), particularly during virus dissociation from the receptor, but still allowed efficient entry into both feline and canine cells without successful infection. These substitutions likely control specific capsid structural changes resulting from TfR binding required for infection. A second set of changes on the interior surface of the capsid reduced viral infectivity by >100-fold and included two cysteine residues and neighboring residues. One of these substitutions, Cys270Ser, modulates a VP2 cleavage event found in ∼10% of the capsid proteins that also was shown to alter capsid stability. A neighboring substitution, Pro272Lys, significantly reduced capsid assembly, while a Cys273Ser change appeared to alter capsid transport from the nucleus. These mutants reveal additional structural details that explain cell infection processes of parvovirus capsids. IMPORTANCE Parvoviruses are commonly found in both vertebrate and invertebrate animals and cause widespread disease. They are also being developed as oncolytic therapeutics and as gene therapy vectors. Most functions involved in infection or transduction are mediated by the viral capsid, but the structure-function correlates of the capsids and their constituent proteins are still incompletely understood, especially in relation to identifying capsid processes responsible for infection and release from the cell. Here, we characterize the functional effects of capsid protein mutations that result in the loss of virus infectivity, giving a better understanding of the portions of the capsid that mediate essential steps in successful infection pathways and how they contribute to viral infectivity.
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Abstract
Parvovirus B19 (B19V) and human bocavirus 1 (HBoV1), members of the large Parvoviridae family, are human pathogens responsible for a variety of diseases. For B19V in particular, host features determine disease manifestations. These viruses are prevalent worldwide and are culturable in vitro, and serological and molecular assays are available but require careful interpretation of results. Additional human parvoviruses, including HBoV2 to -4, human parvovirus 4 (PARV4), and human bufavirus (BuV) are also reviewed. The full spectrum of parvovirus disease in humans has yet to be established. Candidate recombinant B19V vaccines have been developed but may not be commercially feasible. We review relevant features of the molecular and cellular biology of these viruses, and the human immune response that they elicit, which have allowed a deep understanding of pathophysiology.
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Affiliation(s)
- Jianming Qiu
- Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA
| | | | - Neal S Young
- Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA
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Mao Y, Su J, Wang J, Zhang X, Hou Q, Bian D, Liu W. Roles of three amino acids of capsid proteins in mink enteritis parvovirus replication. Virus Res 2016; 222:24-28. [PMID: 27212684 DOI: 10.1016/j.virusres.2016.05.019] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2016] [Revised: 05/15/2016] [Accepted: 05/18/2016] [Indexed: 10/21/2022]
Abstract
Virulent mink enteritis parvovirus (MEV) strain MEV-LHV replicated to higher titers in feline F81 cells than attenuated strain MEV-L. Phylogenetic and sequence analyses of the VP2 gene of MEV-LHV, MEV-L and other strains in GenBank revealed two evolutionary branches separating virulent and attenuated strains. Three residues, 101, 232 and 411, differed between virulent and attenuated strains but were conserved within the two branches. Site-directed mutagenesis of the VP2 gene of infectious plasmids of attenuated strain MEV-L respectively replacing residues 101 Ile and 411 Ala with Thr and Glu of virulent strains (MEV-L I101T and MEV-L A411E) increased replication efficiency but still to lower levels than MEV-LHV. However, viruses with mutation of residue 232 (MEV-L I232V and MEV-L I101T/I232V/A411E) decreased viral transcription and replication levels. The three VP2 residues 101, 232 and 411, located on or near the capsid surface, played different roles in the infection processes of MEV.
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Affiliation(s)
- Yaping Mao
- State Key Laboratory of Agrobiotechnology, Department of Biochemistry and Molecular Biology, College of Biological Sciences, China Agricultural University, Beijing 100193, PR China
| | - Jun Su
- State Key Laboratory of Agrobiotechnology, Department of Biochemistry and Molecular Biology, College of Biological Sciences, China Agricultural University, Beijing 100193, PR China
| | - Jigui Wang
- State Key Laboratory of Agrobiotechnology, Department of Biochemistry and Molecular Biology, College of Biological Sciences, China Agricultural University, Beijing 100193, PR China
| | - Xiaomei Zhang
- State Key Laboratory of Agrobiotechnology, Department of Biochemistry and Molecular Biology, College of Biological Sciences, China Agricultural University, Beijing 100193, PR China
| | - Qiang Hou
- State Key Laboratory of Agrobiotechnology, Department of Biochemistry and Molecular Biology, College of Biological Sciences, China Agricultural University, Beijing 100193, PR China
| | - Dawei Bian
- State Key Laboratory of Agrobiotechnology, Department of Biochemistry and Molecular Biology, College of Biological Sciences, China Agricultural University, Beijing 100193, PR China
| | - Weiquan Liu
- State Key Laboratory of Agrobiotechnology, Department of Biochemistry and Molecular Biology, College of Biological Sciences, China Agricultural University, Beijing 100193, PR China.
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40
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Wang J, Lin P, Zhao H, Cheng Y, Jiang Z, Zhu H, Wu H, Cheng S. Continuing evolution of canine parvovirus in China: Isolation of novel variants with an Ala5Gly mutation in the VP2 protein. INFECTION GENETICS AND EVOLUTION 2016; 38:73-78. [DOI: 10.1016/j.meegid.2015.12.009] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/26/2015] [Revised: 12/07/2015] [Accepted: 12/11/2015] [Indexed: 12/27/2022]
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Affiliation(s)
- Shweta Kailasan
- Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, Florida 32610
| | - Mavis Agbandje-McKenna
- Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, Florida 32610
| | - Colin R. Parrish
- Baker Institute for Animal Health and Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853;
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Tu M, Liu F, Chen S, Wang M, Cheng A. Role of capsid proteins in parvoviruses infection. Virol J 2015; 12:114. [PMID: 26239432 PMCID: PMC4524367 DOI: 10.1186/s12985-015-0344-y] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2015] [Accepted: 07/15/2015] [Indexed: 11/30/2022] Open
Abstract
The parvoviruses are widely spread in many species and are among the smallest DNA animal viruses. The parvovirus is composed of a single strand molecule of DNA wrapped into an icosahedral capsid. In a viral infection, the massy capsid participates in the entire viral infection process, which is summarized in this review. The capsid protein VP1 is primarily responsible for the infectivity of the virus, and the nuclear localization signal (NLS) of the VP1 serves as a guide to assist the viral genome in locating the nucleus. The dominant protein VP2 provides an “anti-receptor”, which interacts with the cellular receptor and leads to the further internalization of virus, and, the N-terminal of VP2 also cooperates with the VP1 to prompt the process of nucleus translocation. Additionally, a cleavage protein VP3 is a part of the capsid, which exists only in several members of the parvovirus family; however, the function of this cleavage protein remains to be fully determined. Parvoviruses can suffer from the extreme environmental conditions such as low pH, or even escape from the recognition of pattern recognition receptors (PRRs), due to the protection of the stable capsid, which is thought to be an immune escape mechanism. The applications of the capsid proteins to the screening and the treatment of diseases are also discussed. The processes of viral infection should be noted, because understanding the virus-host interactions will contribute to the development of therapeutic vaccines.
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Affiliation(s)
- Mengyu Tu
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang District, Chengdu City, Sichuan Province, 611130, China.
| | - Fei Liu
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang District, Chengdu City, Sichuan Province, 611130, China.
| | - Shun Chen
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang District, Chengdu City, Sichuan Province, 611130, China. .,Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Wenjiang District, Chengdu City, Sichuan Province, 611130, China. .,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang District, Chengdu City, Sichuan Province, 611130, China.
| | - Mingshu Wang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang District, Chengdu City, Sichuan Province, 611130, China. .,Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Wenjiang District, Chengdu City, Sichuan Province, 611130, China. .,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang District, Chengdu City, Sichuan Province, 611130, China.
| | - Anchun Cheng
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang District, Chengdu City, Sichuan Province, 611130, China. .,Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Wenjiang District, Chengdu City, Sichuan Province, 611130, China. .,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang District, Chengdu City, Sichuan Province, 611130, China.
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Marchini A, Bonifati S, Scott EM, Angelova AL, Rommelaere J. Oncolytic parvoviruses: from basic virology to clinical applications. Virol J 2015; 12:6. [PMID: 25630937 PMCID: PMC4323056 DOI: 10.1186/s12985-014-0223-y] [Citation(s) in RCA: 70] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2014] [Accepted: 12/03/2014] [Indexed: 12/28/2022] Open
Abstract
Accumulated evidence gathered over recent decades demonstrated that some members of the Parvoviridae family, in particular the rodent protoparvoviruses H-1PV, the minute virus of mice and LuIII have natural anticancer activity while being nonpathogenic to humans. These studies have laid the foundations for the launch of a first phase I/IIa clinical trial, in which the rat H-1 parvovirus is presently undergoing evaluation for its safety and first signs of efficacy in patients with glioblastoma multiforme. After a brief overview of the biology of parvoviruses, this review focuses on the studies which unraveled the antineoplastic properties of these agents and supported their clinical use as anticancer therapeutics. Furthermore, the development of novel parvovirus-based anticancer strategies with enhanced specificity and efficacy is discussed, in particular the development of second and third generation vectors and the combination of parvoviruses with other anticancer agents. Lastly, we address the key challenges that remain towards a more rational and efficient use of oncolytic parvoviruses in clinical settings, and discuss how a better understanding of the virus life-cycle and of the cellular factors involved in virus infection, replication and cytotoxicity may promote the further development of parvovirus-based anticancer therapies, open new prospects for treatment and hopefully improve clinical outcome.
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Affiliation(s)
- Antonio Marchini
- Infection and Cancer Program, Tumor Virology Division (F010), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 242, 69120, Heidelberg, Germany.
| | - Serena Bonifati
- Infection and Cancer Program, Tumor Virology Division (F010), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 242, 69120, Heidelberg, Germany.
| | - Eleanor M Scott
- Infection and Cancer Program, Tumor Virology Division (F010), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 242, 69120, Heidelberg, Germany.
| | - Assia L Angelova
- Infection and Cancer Program, Tumor Virology Division (F010), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 242, 69120, Heidelberg, Germany.
| | - Jean Rommelaere
- Infection and Cancer Program, Tumor Virology Division (F010), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 242, 69120, Heidelberg, Germany.
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Bilkova E, Forstova J, Abrahamyan L. Coat as a dagger: the use of capsid proteins to perforate membranes during non-enveloped DNA viruses trafficking. Viruses 2014; 6:2899-937. [PMID: 25055856 PMCID: PMC4113798 DOI: 10.3390/v6072899] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2014] [Revised: 07/09/2014] [Accepted: 07/14/2014] [Indexed: 01/24/2023] Open
Abstract
To get access to the replication site, small non-enveloped DNA viruses have to cross the cell membrane using a limited number of capsid proteins, which also protect the viral genome in the extracellular environment. Most of DNA viruses have to reach the nucleus to replicate. The capsid proteins involved in transmembrane penetration are exposed or released during endosomal trafficking of the virus. Subsequently, the conserved domains of capsid proteins interact with cellular membranes and ensure their efficient permeabilization. This review summarizes our current knowledge concerning the role of capsid proteins of small non-enveloped DNA viruses in intracellular membrane perturbation in the early stages of infection.
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Affiliation(s)
- Eva Bilkova
- Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Vinicna 5, 12844, Prague 2, Czech Republic.
| | - Jitka Forstova
- Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Vinicna 5, 12844, Prague 2, Czech Republic.
| | - Levon Abrahamyan
- Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Vinicna 5, 12844, Prague 2, Czech Republic.
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45
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Abstract
Parvoviruses are small, rugged, nonenveloped protein particles containing a linear, nonpermuted, single-stranded DNA genome of ∼5 kb. Their limited coding potential requires optimal adaptation to the environment of particular host cells, where entry is mediated by a variable program of capsid dynamics, ultimately leading to genome ejection from intact particles within the host nucleus. Genomes are amplified by a continuous unidirectional strand-displacement mechanism, a linear adaptation of rolling circle replication that relies on the repeated folding and unfolding of small hairpin telomeres to reorient the advancing fork. Progeny genomes are propelled by the viral helicase into the preformed capsid via a pore at one of its icosahedral fivefold axes. Here we explore how the fine-tuning of this unique replication system and the mechanics that regulate opening and closing of the capsid fivefold portals have evolved in different viral lineages to create a remarkably complex spectrum of phenotypes.
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Affiliation(s)
| | - Peter Tattersall
- Departments of 1Laboratory Medicine and.,Genetics, Yale University Medical School, New Haven, Connecticut 06510;
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46
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Lyi SM, Tan MJA, Parrish CR. Parvovirus particles and movement in the cellular cytoplasm and effects of the cytoskeleton. Virology 2014; 456-457:342-52. [PMID: 24889253 DOI: 10.1016/j.virol.2014.04.003] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2014] [Revised: 04/03/2014] [Accepted: 04/03/2014] [Indexed: 12/20/2022]
Abstract
Cell infection by parvoviruses requires that capsids be delivered from outside the cell to the cytoplasm, followed by genome trafficking to the nucleus. Here we microinject capsids into cells that lack receptors and followed their movements within the cell over time. In general the capsids remained close to the positions where they were injected, and most particles did not move to the vicinity of or enter the nucleus. When 70 kDa-dextran was injected along with the capsids that did not enter the nucleus in significant amounts. Capsids conjugated to peptides containing the SV40 large T-antigen nuclear localization signal remained in the cytoplasm, although bovine serum albumen conjugated to the same peptide entered the nucleus rapidly. No effects of disruption of microfilaments, intermediate filaments, or microtubules on the distribution of the capsids were observed. These results suggest that movement of intact capsids within cells is primarily associated with passive processes.
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Affiliation(s)
- Sangbom Michael Lyi
- Baker Institute for Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, United States
| | - Min Jie Alvin Tan
- Baker Institute for Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, United States.
| | - Colin R Parrish
- Baker Institute for Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, United States.
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47
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A slender tract of glycine residues is required for translocation of the VP2 protein N-terminal domain through the parvovirus MVM capsid channel to initiate infection. Biochem J 2013; 455:87-94. [PMID: 23875612 DOI: 10.1042/bj20130503] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Viruses constitute paradigms to study conformational dynamics in biomacromolecular assemblies. Infection by the parvovirus MVM (minute virus of mice) requires a conformational rearrangement that involves the intracellular externalization through capsid channels of the 2Nt (N-terminal region of VP2). We have investigated the role in this process of conserved glycine residues in an extended glycine-rich tract located immediately after 2Nt. Based on the virus structure, residues with hydrophobic side chains of increasing volume were substituted for glycine residues 31 or 33. Mutations had no effect on capsid assembly or stability, but inhibited virus infectivity. All mutations, except those to alanine residues which had minor effects, impaired 2Nt externalization in nuclear maturing virions and in purified virions, to an extent that correlated with the side chain size. Different biochemical and biophysical analyses were consistent with this result. Importantly, all of the tested glycine residue replacements impaired the capacity of the virion to initiate infection, at ratios correlating with their restrictive effects on 2Nt externalization. Thus small residues within the evolutionarily conserved glycine-rich tract facilitate 2Nt externalization through the capsid channel, as required by this virus to initiate cell entry. The results demonstrate the exquisite dependence on geometric constraints of a biologically relevant translocation event in a biomolecular complex.
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Wolfisberg R, Ruprecht N, Kempf C, Ros C. Impaired genome encapsidation restricts the in vitro propagation of human parvovirus B19. J Virol Methods 2013; 193:215-25. [DOI: 10.1016/j.jviromet.2013.06.003] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2013] [Revised: 05/24/2013] [Accepted: 06/03/2013] [Indexed: 10/26/2022]
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Snoussi K, Kann M. Interaction of parvoviruses with the nuclear envelope. Adv Biol Regul 2013; 54:39-49. [PMID: 24157125 DOI: 10.1016/j.jbior.2013.09.008] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2013] [Accepted: 09/17/2013] [Indexed: 11/29/2022]
Abstract
Parvoviruses are serious pathogens but also serve as platforms for gene therapy or for using their lytic activity in experimental cancer treatment. Despite of their growing importance during the last decade little is known on how the viral genome is transported into the nucleus of the infected cell, which is crucial for replication. As nucleic acids are not karyophilic per se nuclear import must be driven by proteins attached to the viral genome. In turn, presence and conformation of these proteins depend upon the entry pathway of the virus into the cell. This review focuses on the trafficking of the parvoviral genome from the cellular periphery to nucleus. Despite of the uncertainties in knowledge about the entry pathway we show that parvoviruses developed a unique strategy to pass the nuclear envelope by hijacking enzymes involved in mitosis.
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Affiliation(s)
- Kenza Snoussi
- Department of Infection Biology (Molecular Virology), University of Tsukuba, Japan; Human Biology Program, University of Tsukuba, Japan
| | - Michael Kann
- Univ. de Bordeaux, Microbiologie fondamentale et Pathogénicité, UMR 5234, Bordeaux, France; CHU de Bordeaux, Bordeaux, France.
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Parvovirus B19 uptake is a highly selective process controlled by VP1u, a novel determinant of viral tropism. J Virol 2013; 87:13161-7. [PMID: 24067971 DOI: 10.1128/jvi.02548-13] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The VP1 unique region (VP1u) of human parvovirus B19 (B19V) is the immunodominant part of the viral capsid. Originally inaccessible, the VP1u becomes exposed upon primary attachment to the globoside receptor. To study the function of the exposed VP1u in B19V uptake, we expressed this region as a recombinant protein. Here, we report that purified recombinant VP1u binds and is internalized in UT7/Epo cells. By means of truncations and specific antibodies, we identified the most N-terminal amino acid residues of VP1u as the essential region for binding and internalization. Furthermore, the recombinant VP1u was able to block B19V uptake, suggesting that the protein and the virus undertake the same internalization pathway. Assays with different erythroid and nonerythroid cell lines showed that the N-terminal VP1u binding was restricted to a few cell lines of the erythroid lineage, which were also the only cells that allowed B19V internalization and infection. These results together indicate that the N-terminal region of VP1u is responsible for the internalization of the virus and that the interacting receptor is restricted to B19V-susceptible cells. The highly selective uptake mechanism represents a novel determinant of the tropism and pathogenesis of B19V.
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