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2.
Proc Natl Acad Sci U S A ; 117(4): 2099-2107, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31953264

RESUMO

Nonsegmented negative-stranded (NNS) RNA viruses, among them the virus that causes rabies (RABV), include many deadly human pathogens. The large polymerase (L) proteins of NNS RNA viruses carry all of the enzymatic functions required for viral messenger RNA (mRNA) transcription and replication: RNA polymerization, mRNA capping, and cap methylation. We describe here a complete structure of RABV L bound with its phosphoprotein cofactor (P), determined by electron cryo-microscopy at 3.3 Å resolution. The complex closely resembles the vesicular stomatitis virus (VSV) L-P, the one other known full-length NNS-RNA L-protein structure, with key local differences (e.g., in L-P interactions). Like the VSV L-P structure, the RABV complex analyzed here represents a preinitiation conformation. Comparison with the likely elongation state, seen in two structures of pneumovirus L-P complexes, suggests differences between priming/initiation and elongation complexes. Analysis of internal cavities within RABV L suggests distinct template and product entry and exit pathways during transcription and replication.


Assuntos
RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Chaperonas Moleculares/química , Vírus da Raiva/enzimologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Proteínas Estruturais Virais/química , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/genética , Regulação Viral da Expressão Gênica , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Vírus da Raiva/química , Vírus da Raiva/genética , Vírus da Raiva/metabolismo , Transcrição Genética , Proteínas Virais/genética , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo
3.
Virology ; 539: 80-91, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31706163

RESUMO

To identify potential pathogens responsible for a disease outbreak of cultured peafowls in China in 2013, metagenomic sequencing was conducted. The genomes of two closely related parvoviruses, namely peafowl parvovirus 1 (PePV1) and PePV2, were identified with size of 4428 bp and 4348 bp, respectively. Phylogenetic analysis revealed that both viruses are novel parvoviruses, belonging to the proposed genus Chapparvovirus of Parvoviridae. The transcriptional profile of PePV1 was analyzed by transfecting a nearly complete PePV1 genome into HEK-293T cells. Results revealed that PePV1 employs one promoter and two polyadenylation sites to start and terminate its transcriptions, with one donor site and two acceptor sites for pre-mRNA splicing. PePV1 DNA and structural protein were detected in several tissues of a dead peafowl, which appeared to have suffered enteritis, pneumonia and viremia. These results provide novel information of chapparvoviruses, and call for attention to the potential pathogens.


Assuntos
Doenças das Aves/virologia , Galliformes/virologia , Perfilação da Expressão Gênica , Genoma Viral/genética , Infecções por Parvoviridae/veterinária , Parvovirinae/genética , Processamento Alternativo/genética , Animais , Doenças das Aves/epidemiologia , China/epidemiologia , DNA Viral/genética , DNA Viral/metabolismo , Células HEK293 , Humanos , Metagenômica , Infecções por Parvoviridae/epidemiologia , Infecções por Parvoviridae/virologia , Parvovirinae/classificação , Filogenia , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo
4.
Nat Commun ; 10(1): 4840, 2019 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-31649265

RESUMO

Assembly of tailed bacteriophages and herpesviruses starts with formation of procapsids (virion precursors without DNA). Scaffolding proteins (SP) drive assembly by chaperoning the major capsid protein (MCP) to build an icosahedral lattice. Here we report near-atomic resolution cryo-EM structures of the bacteriophage SPP1 procapsid, the intermediate expanded procapsid with partially released SPs, and the mature capsid with DNA. In the intermediate state, SPs are bound only to MCP pentons and to adjacent subunits from hexons. SP departure results in the expanded state associated with unfolding of the MCP N-terminus and straightening of E-loops. The newly formed extensive inter-capsomere bonding appears to compensate for release of SPs that clasp MCP capsomeres together. Subsequent DNA packaging instigates bending of MCP A domain loops outwards, closing the hexons central opening and creating the capsid auxiliary protein binding interface. These findings provide a molecular basis for the sequential structural rearrangements during viral capsid maturation.


Assuntos
Bacteriófagos/ultraestrutura , Proteínas do Capsídeo/ultraestrutura , Capsídeo/ultraestrutura , Montagem de Vírus , Bacteriófagos/metabolismo , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Proteínas Estruturais Virais/metabolismo , Proteínas Estruturais Virais/ultraestrutura
5.
PLoS Pathog ; 15(10): e1007956, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31589653

RESUMO

We report the analysis of a complex enveloped human virus, herpes simplex virus (HSV), assembled after in vivo incorporation of bio-orthogonal methionine analogues homopropargylglycine (HPG) or azidohomoalanine (AHA). We optimised protocols for the production of virions incorporating AHA (termed HSVAHA), identifying conditions which resulted in normal yields of HSV and normal particle/pfu ratios. Moreover we show that essentially every single HSVAHA capsid-containing particle was detectable at the individual particle level by chemical ligation of azide-linked fluorochromes to AHA-containing structural proteins. This was a completely specific chemical ligation, with no capsids assembled under normal methionine-containing conditions detected in parallel. We demonstrate by quantitative mass spectrometric analysis that HSVAHA virions exhibit no qualitative or quantitative differences in the repertoires of structural proteins compared to virions assembled under normal conditions. Individual proteins and AHA incorporation sites were identified in capsid, tegument and envelope compartments, including major essential structural proteins. Finally we reveal novel aspects of entry pathways using HSVAHA and chemical fluorochrome ligation that were not apparent from conventional immunofluorescence. Since ligation targets total AHA-containing protein and peptides, our results demonstrate the presence of abundant AHA-labelled products in cytoplasmic macrodomains and tubules which no longer contain intact particles detectable by immunofluorescence. Although these do not co-localise with lysosomal markers, we propose they may represent sites of proteolytic virion processing. Analysis of HSVAHA also enabled the discrimination from primary entering from secondary assembling virions, demonstrating assembly and second round infection within 6 hrs of initial infection and dual infections of primary and secondary virus in spatially restricted cytoplasmic areas of the same cell. Together with other demonstrated applications e.g., in genome biology, lipid and protein trafficking, this work further exemplifies the utility and potential of bio-orthogonal chemistry for studies in many aspects of virus-host interactions.


Assuntos
Aminoácidos/metabolismo , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Epitélio Pigmentado da Retina/virologia , Proteínas Estruturais Virais/metabolismo , Montagem de Vírus , Internalização do Vírus , Proliferação de Células , Células Cultivadas , Herpes Simples/metabolismo , Humanos , Epitélio Pigmentado da Retina/metabolismo
6.
Mol Biotechnol ; 61(11): 852-859, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31473916

RESUMO

To explore virus-like particles formation of dengue virus serotype type 2 (DENV-2) structural proteins of, C, prM, E were expressed in silkworm larvae using recombinant Bombyx mori nucleopolyhedroviruses (BmNPV). Each recombinant BmNPV bacmid coding the 2C-prM-E polypeptide and E protein fused with the signal peptide of bombyxin from B. mori was injected into silkworm larvae. The expressed proteins were collected from hemolymph and fat body, and purified using affinity chromatography. E protein was observed at 55 kDa. The DENV virus-like particles (DENV-LPs) with a diameter approximately 35 nm was observed using transmission electron microscopy (TEM) and immunogold-labelling TEM analysis. The binding of each partially purified proteins to heparin, one of receptors for DENV was confirmed. DENV-LPs were secreted in silkworm larval hemolymph even still low amount, but the E protein and heparin binding function were confirmed.


Assuntos
Proteínas do Capsídeo/metabolismo , Vírus da Dengue/genética , Nucleopoliedrovírus/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Estruturais Virais/metabolismo , Vírion/genética , Animais , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/isolamento & purificação , Vírus da Dengue/metabolismo , Corpo Adiposo/metabolismo , Expressão Gênica , Vetores Genéticos , Hemolinfa/metabolismo , Heparina/metabolismo , Larva/metabolismo , Nucleopoliedrovírus/metabolismo , Sinais Direcionadores de Proteínas/genética , Sorogrupo , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/isolamento & purificação , Proteínas Estruturais Virais/biossíntese , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/isolamento & purificação , Vírion/ultraestrutura
7.
Future Microbiol ; 14: 1147-1157, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31368347

RESUMO

Members of the genus Enterovirus have a significant effect on human health, especially in infants and children. Since the viral genome has limited coding capacity, Enteroviruses subvert a range of cellular processes for viral infection via the interaction of viral proteins and numerous cellular factors. Intriguingly, the capsid-receptor interaction plays a crucial role in viral entry and has significant implications in viral pathogenesis. Moreover, interactions between structural proteins and host factors occur directly or indirectly in multiple steps of viral replication. In this review, we focus on the current understanding of the multifunctionality of structural proteins in the viral life cycle, which may constitute valuable targets for antiviral and therapeutic interventions.


Assuntos
Enterovirus/crescimento & desenvolvimento , Interações Hospedeiro-Patógeno , Proteínas Estruturais Virais/metabolismo , Internalização do Vírus , Replicação Viral , Humanos
8.
Virology ; 535: 241-254, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31344549

RESUMO

A Japanese isolate of Magnaporthe oryzae is infected by Magnaporthe oryzae chrysovirus 1-D (MoCV1-D), which is classified in cluster II of the family Chrysoviridae. The genome of MoCV1-D consists of five dsRNAs. dsRNAs 1-4 show high identity with those of related MoCV1 viruses, whereas dsRNA5 shows relatively low identity and is sometimes deleted during virus propagation. MoCV1-D causes growth inhibition of its host fungus, and the protein encoded by its dsRNA4 impairs cell growth when expressed in yeast cells. It also causes abnormal pigmentation and colony albinization, and we showed that these phenotypes are associated with reduced accumulation of the melanin biosynthesis intermediate scylatone. MoCV1-D exhibits multiform viral structural proteins during prolonged culture. The original host isolate is co-infected with MoCV1-D, a victorivirus, and a partitivirus, and these mycoviruses are detected in cell-free supernatant fractions after prolonged liquid culturing. Hyphal fusion experiments demonstrated that MoCV1-D is transmissible via anastomosis.


Assuntos
Ascomicetos/crescimento & desenvolvimento , Ascomicetos/virologia , Micovírus/crescimento & desenvolvimento , Vírus de RNA/crescimento & desenvolvimento , RNA Viral/genética , Proteínas Estruturais Virais/metabolismo , Ascomicetos/metabolismo , Micovírus/genética , Melaninas/biossíntese , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Vírus de RNA/genética , RNA de Cadeia Dupla/genética , Proteínas Estruturais Virais/genética
9.
Microb Pathog ; 135: 103632, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31325569

RESUMO

Infectious bursal disease virus (IBDV) is the etiological agent of a highly contagious and immunosuppressive disease that affects domestic chickens. Toll-like receptors (TLRs), a kind of pattern recognition receptors, help the host to detect invading pathogens. To date, few systematic studies have been reported about the expression changes of TLR in chickens infected with pathogens. In the present study, layer chickens were infected with IBDV and the expression of chicken TLRs (chTLRs) was assayed by quantitative real-time PCR. The results showed that the expression of chTLR1a, 1b, 2a, 3, 4 and 15 was upregulated in the bursa of chickens infected with IBDV compared with noninfected chickens, while chTLR2b, 5, 7 and 21 expression was downregulated. Correlation analysis showed that chTLR3 expressions was directly associated with IBDV VP2 mRNA expression in bursa. These results suggested that different TLRs have different responses to the same viral infection. Some TLRs were activated early on, some later, and some were suppressed. This is the first study to report on the response of all chTLRs to one virus. This provids a valuable overview of the expression pattern of chTLRs when chickens are challenged by pathogens.


Assuntos
Infecções por Birnaviridae/imunologia , Galinhas/virologia , Vírus da Doença Infecciosa da Bursa/metabolismo , Doenças das Aves Domésticas/imunologia , Receptores Toll-Like/metabolismo , Animais , Infecções por Birnaviridae/genética , Infecções por Birnaviridae/virologia , Regulação Viral da Expressão Gênica , Imunidade Inata , Vírus da Doença Infecciosa da Bursa/genética , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/virologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo , Receptores Toll-Like/genética , Proteínas Estruturais Virais/metabolismo
10.
PLoS Pathog ; 15(7): e1007914, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31356650

RESUMO

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that can cause disability in newborns and serious clinical diseases in immunocompromised patients. HCMV has a large genome with enormous coding potential; its viral particles are equipped with complicated glycoprotein complexes and can infect a wide range of human cells. Although multiple host cellular receptors interacting with viral glycoproteins have been reported, the mechanism of HCMV infection remains a mystery. Here we report identification of adipocyte plasma membrane-associated protein (APMAP) as a novel modulator active in the early stage of HCMV infection. APMAP is necessary for HCMV infection in both epithelial cells and fibroblasts; knockdown of APMAP expression significantly reduced HCMV infection of these cells. Interestingly, ectopic expression of human APMAP in cells refractory to HCMV infection, such as canine MDCK and murine NIH/3T3 cells, promoted HCMV infection. Furthermore, reduction in viral immediate early (IE) gene transcription at 6 h post infection and delayed nucleus translocation of tegument delivered pp65 at 4 h post infection were detected in APMAP-deficient cells but not in the wildtype cells. These results suggest that APMAP plays a role in the early stage of HCMV infection. Results from biochemical studies of APMAP and HCMV proteins suggest that APMAP could participate in HCMV infection through interaction with gH/gL containing glycoprotein complexes at low pH and mediate nucleus translocation of tegument pp65. Taken together, our results suggest that APMAP functions as a modulator promoting HCMV infection in multiple cell types and is an important player in the complex HCMV infection mechanism.


Assuntos
Infecções por Citomegalovirus/metabolismo , Infecções por Citomegalovirus/virologia , Citomegalovirus/patogenicidade , Glicoproteínas de Membrana/metabolismo , Adipócitos/metabolismo , Adipócitos/virologia , Animais , Membrana Celular/metabolismo , Membrana Celular/virologia , Citomegalovirus/genética , Citomegalovirus/metabolismo , Infecções por Citomegalovirus/etiologia , Cães , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Técnicas de Inativação de Genes , Interações entre Hospedeiro e Microrganismos , Humanos , Células Madin Darby de Rim Canino , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Camundongos , Células NIH 3T3 , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Estruturais Virais/metabolismo , Virulência , Internalização do Vírus
11.
Virol J ; 16(1): 81, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221185

RESUMO

BACKGROUND: Pseudorabies virus (PRV) protein UL56 (pUL56) has been implicated in viral dissemination and virulence in vivo. However, the properties of PRV pUL56 remain largely unknown. In the present study, we aim to investigate the subcellular localization of pUL56 and the underlying molecular basis in transfected cells. METHODS: Constructs of N-terminal green fluorescent protein (GFP) fused pUL56 and its truncations were employed for investigating subcellular localization and further identifying amino acids crucial for pUL56 localization in transfected Vero cells. Finally, the identified amino acids were replaced with alanine for confirming if these mutations could impair the specific localization of pUL56. RESULTS: The pUL56 predominantly localized at the Golgi and trans-Golgi network (TGN) through its predicted C-terminal transmembrane helix in transfected Vero cells. A Golgi-associated protein Rab6a, independent of interaction with pUL56, was significantly downregulated by pUL56. Further, we found three truncated pUL56 C-terminal fragments (174-184, 175-185 and 191-195) could restrict GFP in the perinuclear region, respectively. Within these truncations, the 174proline (P), 181leucine (L), 185L and 191L were essential for maintaining perinuclear accumulation, thus suggesting an important role of leucine. Alanine (A) mutagenesis assays were employed to generate a series of pUL56 C-terminal mutants on the basis of leucine. Finally, a pUL56 mutant M10 (174P/A-177L/A-181L/A-185L/A-191L/A-194L/A-195I/A-196-197L/A-200L/A) lost Golgi-TGN localization. Thus, our data revealed that the leucine-rich transmembrane helix was responsible for pUL56 Golgi-TGN localization and retention, probably through specific intracellular membrane insertion. CONCLUSION: Our data indicated that the C-terminal transmembrane helix was responsible for the Golgi-TGN localization of pUL56. In addition, the leucines within C-terminal transmembrane helix were essential for maintaining pUL56 Golgi-TGN retention in cells. Further, the pUL56 can induce downregulation of Golgi-associated protein Rab6a.


Assuntos
Complexo de Golgi/fisiologia , Leucina/química , Pseudorraiva , Proteínas Estruturais Virais/metabolismo , Rede trans-Golgi/fisiologia , Animais , Chlorocebus aethiops , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Transporte Proteico , Transfecção , Células Vero , Proteínas Estruturais Virais/genética
12.
Int J Mol Med ; 44(2): 759-767, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31173167

RESUMO

Enterovirus 71 (EV71) accounts for the majority of hand, foot and mouth disease­related deaths due to fatal neurological complications. EV71 structural viral protein 1 (VP1) promotes viral replication by inducing autophagy in neuron cells, but the effect of VP1 on myelin cells is unclear. The present study aimed to investigate the role and mechanism of VP1 in autophagy of mouse Schwann cells. An EV71 VP1­expressing vector (pEGFP­C3­VP1) was generated and transfected into mouse Schwann cells. Transmission electron microscopy and western blot analysis for microtubule­associated protein 1 light chain 3 α (LC3) II (an autophagy marker) were used to assess autophagy. Reverse transcription­quantitative PCR and immunofluorescence were performed to determine the expression of peripheral myelin protein 22 (PMP22). Small interfering RNA against PMP22 was used to investigate the role of PMP22 in mouse Schwann cell autophagy. Salubrinal [a selective endoplasmic reticulum (ER) stress inhibitor] was used to determine whether PMP22 expression was affected by ER stress. The present results indicated that VP1 promoted mouse Schwann cell autophagy. Overexpression of VP1 upregulated PMP22. PMP22 deficiency downregulated LC3II and thus inhibited autophagy. Furthermore, PMP22 expression was significantly suppressed by salubrinal. In conclusion, VP1 promoted mouse Schwann cell autophagy through upregulation of ER stress­mediated PMP22 expression. Therefore, the VP1/ER stress/PMP22 autophagy axis may be a potential therapeutic target for EV71 infection­induced fatal neuronal damage.


Assuntos
Enterovirus Humano A/fisiologia , Infecções por Enterovirus/metabolismo , Proteínas da Mielina/metabolismo , Células de Schwann/virologia , Proteínas Estruturais Virais/metabolismo , Animais , Autofagia , Linhagem Celular , Estresse do Retículo Endoplasmático , Infecções por Enterovirus/virologia , Humanos , Camundongos , Células de Schwann/metabolismo , Células de Schwann/patologia
13.
PLoS Biol ; 17(6): e3000316, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31199794

RESUMO

Infections with human herpesviruses are ubiquitous and a public health concern worldwide. Current treatments reduce the severity of some symptoms associated to herpetic infections but neither remove the viral reservoir from the infected host nor protect from the recurrent symptom outbreaks that characterise herpetic infections. The difficulty in therapeutically tackling these viral systems stems in part from their remarkably large proteomes and the complex networks of physical and functional associations that they tailor. This study presents our efforts to unravel the complexity of the interactome of herpes simplex virus type 1 (HSV1), the prototypical herpesvirus species. Inspired by our previous work, we present an improved and more integrative computational pipeline for the protein-protein interaction (PPI) network reconstruction in HSV1, together with a newly developed consensus clustering framework, which allowed us to extend the analysis beyond binary physical interactions and revealed a system-level layout of higher-order functional associations in the virion proteome. Additionally, the analysis provided new functional annotation for the currently undercharacterised protein pUS10. In-depth bioinformatics sequence analysis unravelled structural features in pUS10 reminiscent of those observed in some capsid-associated proteins in tailed bacteriophages, with which herpesviruses are believed to share a common ancestry. Using immunoaffinity purification (IP)-mass spectrometry (MS), we obtained additional support for our bioinformatically predicted interaction between pUS10 and the inner tegument protein pUL37, which binds cytosolic capsids, contributing to initial tegumentation and eventually virion maturation. In summary, this study unveils new, to our knowledge, insights at both the system and molecular levels that can help us better understand the complexity behind herpesvirus infections.


Assuntos
Biologia Computacional/métodos , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 1/ultraestrutura , Animais , Capsídeo/química , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Bases de Dados Factuais , Herpes Simples/metabolismo , Humanos , Hidroliases/metabolismo , Ligação Proteica , Mapas de Interação de Proteínas , Relação Estrutura-Atividade , Proteínas Virais/metabolismo , Proteínas Estruturais Virais/metabolismo , Vírion/metabolismo , Montagem de Vírus
14.
J Virol ; 93(10)2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30842328

RESUMO

SUMOylation is a posttranslational modification that has crucial roles in diverse cellular biological pathways and in various viral life cycles. In this study, we found that the VP1 protein, the RNA-dependent RNA polymerase of avibirnavirus infectious bursal disease virus (IBDV), regulates virus replication by SUMOylation during infection. Our data demonstrated that the polymerase VP1 is efficiently modified by small ubiquitin-like modifier 1 (SUMO1) in avibirnavirus-infected cell lines. Mutation analysis showed that residues 404I and 406I within SUMO interaction motif 3 of VP1 constitute the critical site for SUMO1 modification. Protein stability assays showed that SUMO1 modification enhanced significantly the stability of polymerase VP1 by inhibiting K48-linked ubiquitination. A reverse genetic approach showed that only IBDV with I404C/T and I406C/F mutations of VP1 could be rescued successfully with decreased replication ability. Our data demonstrated that SUMO1 modification is essential to sustain the stability of polymerase VP1 during IBDV replication and provides a potential target for designing antiviral drugs targeting IBDV.IMPORTANCE SUMOylation is an extensively discussed posttranslational modification in diverse cellular biological pathways. However, there is limited understanding about SUMOylation of viral proteins of IBDV during infection. In the present study, we revealed a SUMO1 modification of VP1 protein, the RNA-dependent RNA polymerase of avibirnavirus infectious bursal disease virus (IBDV). The required site of VP1 SUMOylation comprised residues 404I and 406I of SUMO interaction motif 3, which was essential for maintaining its stability by inhibiting K48-linked ubiquitination. We also showed that IBDV with SUMOylation-deficient VP1 had decreased replication ability. These data demonstrated that the SUMOylation of IBDV VP1 played an important role in maintaining IBDV replication.


Assuntos
Vírus da Doença Infecciosa da Bursa/metabolismo , Proteína SUMO-1/metabolismo , Proteínas Estruturais Virais/metabolismo , Avibirnavirus/metabolismo , Avibirnavirus/patogenicidade , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Vírus da Doença Infecciosa da Bursa/patogenicidade , Vírus da Doença Infecciosa da Bursa/fisiologia , Processamento de Proteína Pós-Traducional , RNA Replicase/genética , Proteína SUMO-1/fisiologia , Sumoilação , Ubiquitinação , Proteínas Virais/metabolismo , Proteínas Estruturais Virais/genética , Replicação Viral/fisiologia
15.
J Virol ; 93(7)2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30651355

RESUMO

Viruses associated with sleeping disease (SD) in crabs cause great economic losses to aquaculture, and no effective measures are available for their prevention. In this study, to help develop novel antiviral strategies, single-particle cryo-electron microscopy was applied to investigate viruses associated with SD. The results not only revealed the structure of mud crab dicistrovirus (MCDV) but also identified a novel mud crab tombus-like virus (MCTV) not previously detected using molecular biology methods. The structure of MCDV at a 3.5-Å resolution reveals three major capsid proteins (VP1 to VP3) organized into a pseudo-T=3 icosahedral capsid, and affirms the existence of VP4. Unusually, MCDV VP3 contains a long C-terminal region and forms a novel protrusion that has not been observed in other dicistrovirus. Our results also reveal that MCDV can release its genome via conformation changes of the protrusions when viral mixtures are heated. The structure of MCTV at a 3.3-Å resolution reveals a T= 3 icosahedral capsid with common features of both tombusviruses and nodaviruses. Furthermore, MCTV has a novel hydrophobic tunnel beneath the 5-fold vertex and 30 dimeric protrusions composed of the P-domains of the capsid protein at the 2-fold axes that are exposed on the virion surface. The structural features of MCTV are consistent with a novel type of virus.IMPORTANCE Pathogen identification is vital for unknown infectious outbreaks, especially for dual or multiple infections. Sleeping disease (SD) in crabs causes great economic losses to aquaculture worldwide. Here we report the discovery and identification of a novel virus in mud crabs with multiple infections that was not previously detected by molecular, immune, or traditional electron microscopy (EM) methods. High-resolution structures of pathogenic viruses are essential for a molecular understanding and developing new disease prevention methods. The three-dimensional (3D) structure of the mud crab tombus-like virus (MCTV) and mud crab dicistrovirus (MCDV) determined in this study could assist the development of antiviral inhibitors. The identification of a novel virus in multiple infections previously missed using other methods demonstrates the usefulness of this strategy for investigating multiple infectious outbreaks, even in humans and other animals.


Assuntos
Braquiúros/virologia , Dicistroviridae/fisiologia , Animais , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Microscopia Crioeletrônica/métodos , Dicistroviridae/metabolismo , Proteínas Estruturais Virais/metabolismo , Vírion/metabolismo , Vírion/fisiologia , Viroses/virologia
16.
PLoS Pathog ; 15(1): e1007509, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30657784

RESUMO

Many picornaviruses cause important diseases in humans and other animals including poliovirus, rhinoviruses (causing the common cold) and foot-and-mouth disease virus (FMDV). These small, non-enveloped viruses comprise a positive-stranded RNA genome (ca. 7-9 kb) enclosed within a protein shell composed of 60 copies of three or four different capsid proteins. For the aphthoviruses (e.g. FMDV) and cardioviruses, the capsid precursor, P1-2A, is cleaved by the 3C protease (3Cpro) to generate VP0, VP3 and VP1 plus 2A. For enteroviruses, e.g. poliovirus, the capsid precursor is P1 alone, which is cleaved by the 3CD protease to generate just VP0, VP3 and VP1. The sequences required for correct processing of the FMDV capsid protein precursor in mammalian cells were analyzed. Truncation of the P1-2A precursor from its C-terminus showed that loss of the 2A peptide (18 residues long) and 27 residues from the C-terminus of VP1 (211 residues long) resulted in a precursor that cannot be processed by 3Cpro although it still contained two unmodified internal cleavage sites (VP0/VP3 and VP3/VP1 junctions). Furthermore, introduction of small deletions within P1-2A identified residues 185-190 within VP1 as being required for 3Cpro-mediated processing and for optimal accumulation of the precursor. Within this C-terminal region of VP1, five of these residues (YCPRP), are very highly conserved in all FMDVs and are also conserved amongst other picornaviruses. Mutant FMDV P1-2A precursors with single amino acid substitutions within this motif were highly resistant to cleavage at internal junctions. Such substitutions also abrogated virus infectivity. These results can explain earlier observations that loss of the C-terminus (including the conserved motif) from the poliovirus capsid precursor conferred resistance to processing. Thus, this motif seems essential for maintaining the correct structure of picornavirus capsid precursors prior to processing and subsequent capsid assembly; it may represent a site that interacts with cellular chaperones.


Assuntos
Infecções por Picornaviridae/metabolismo , Picornaviridae/genética , Proteínas Estruturais Virais/genética , Motivos de Aminoácidos/genética , Sequência de Aminoácidos/genética , Substituição de Aminoácidos , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Sequência Conservada , Picornaviridae/metabolismo , Infecções por Picornaviridae/genética , Precursores de Proteínas/genética , Processamento de Proteína Pós-Traducional , Homologia de Sequência de Aminoácidos , Proteínas Virais/metabolismo , Proteínas Estruturais Virais/metabolismo
17.
Virus Res ; 263: 119-128, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30639468

RESUMO

Mycoviruses ubiquitously infect a wide range of fungal hosts in the world. The current study reports a novel double stranded RNA (dsRNA) virus, termed Alternaria alternata botybirnavirus 1 (AaBbV1), infecting a Pakistani strain, 4a, of a phytopathogenic ascomycetous fungus Alternaria alternata. A combined approach of next generation and conventional terminal end sequencing of the viral genome revealed that the virus is a distinct member of the genus Botybirnavirus. This virus comprised of two segments (dsRNA1 and dsRNA2) of sizes 6127 bp and 5860 bp respectively. The dsRNA1-encoded protein carrying the RNA-dependent RNA polymerase domain showed 61% identity to the counterpart of Botrytis porri botybirnavirus 1 and lower levels of amino acid similarity with those of other putative botybirnaviruses and the fungal dsRNA viruses such as members of the families Totiviridae, Chrysoviridae and Megabirnaviridae. The dsRNA2-encoded protein showed resemblance with corresponding proteins of botybirnaviruses. Electron microscopy showed AaBbV1 to form spherical particles of 40 nm in diameter. Biochemical analyses showed that two structural proteins encoded by dsRNA1 and dsRNA2 underwent processing to some extent during particle purification, resulting in the appearance of multiple smaller products. Phylogenetic analyses of structural proteins suggested that their coding region might have been duplicated once and maintained without recombination. Protoplast fusion technique allowed for the introduction of AaBbV1 into a virus free Japanese strain of A. alternata and demonstrated its symptomless infection by the virus. Interesting similarities and dissimilarities between AaBbV1 and other previously reported botybirnaviruses are also discussed.


Assuntos
Alternaria/virologia , Micovírus/classificação , Micovírus/isolamento & purificação , Vírus de RNA/classificação , Vírus de RNA/isolamento & purificação , Análise por Conglomerados , Micovírus/genética , Micovírus/ultraestrutura , Genoma Viral , Microscopia Eletrônica , Paquistão , Filogenia , Processamento de Proteína Pós-Traducional , RNA Replicase/genética , Vírus de RNA/genética , Vírus de RNA/ultraestrutura , RNA de Cadeia Dupla/genética , RNA Viral/genética , Análise de Sequência de DNA , Homologia de Sequência , Proteínas Estruturais Virais/metabolismo , Vírion/ultraestrutura
18.
J Virol ; 93(3)2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30429342

RESUMO

Ubiquitination is critical for several cellular physical processes. However, ubiquitin modification in virus replication is poorly understood. Therefore, the present study aimed to determine the presence and effect of ubiquitination on polymerase activity of viral protein 1 (VP1) of avibirnavirus. We report that the replication of avibirnavirus is regulated by ubiquitination of its VP1 protein, the RNA-dependent RNA polymerase of infectious bursal disease virus (IBDV). In vivo detection revealed the ubiquitination of VP1 protein in IBDV-infected target organs and different cells but not in purified IBDV particles. Further analysis of ubiquitination confirms that VP1 is modified by K63-linked ubiquitin chain. Point mutation screening showed that the ubiquitination site of VP1 was at the K751 residue in the C terminus. The K751 ubiquitination is independent of VP1's interaction with VP3 and eukaryotic initiation factor 4A II. Polymerase activity assays indicated that the K751 ubiquitination at the C terminus of VP1 enhanced its polymerase activity. The K751-to-R mutation of VP1 protein did not block the rescue of IBDV but decreased the replication ability of IBDV. Our data demonstrate that the ubiquitination of VP1 is crucial to regulate its polymerase activity and IBDV replication.IMPORTANCE Avibirnavirus protein VP1, the RNA-dependent RNA polymerase, is responsible for IBDV genome replication, gene expression, and assembly. However, little is known about its chemical modification relating to its polymerase activity. In this study, we revealed the molecular mechanism of ubiquitin modification of VP1 via a K63-linked ubiquitin chain during infection. Lysine (K) residue 751 at the C terminus of VP1 is the target site for ubiquitin, and its ubiquitination is independent of VP1's interaction with VP3 and eukaryotic initiation factor 4A II. The K751 ubiquitination promotes the polymerase activity of VP1 and unubiquitinated VP1 mutant IBDV significantly impairs virus replication. We conclude that VP1 is the ubiquitin-modified protein and reveal the mechanism by which VP1 promotes avibirnavirus replication.


Assuntos
Avibirnavirus/fisiologia , Infecções por Birnaviridae/virologia , Vírus da Doença Infecciosa da Bursa/fisiologia , RNA Replicase/metabolismo , Ubiquitinação , Proteínas Estruturais Virais/metabolismo , Replicação Viral , Animais , Avibirnavirus/classificação , Infecções por Birnaviridae/enzimologia , Células Cultivadas , Galinhas/virologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Células HEK293 , Humanos , RNA Replicase/química , Ubiquitina/metabolismo , Proteínas Estruturais Virais/química
19.
PLoS Pathog ; 14(10): e1007383, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30339712

RESUMO

Rabbit hemorrhagic disease virus (RHDV) is an important member of the Caliciviridae family and a highly lethal pathogen in rabbits. Although the cell receptor of RHDV has been identified, the mechanism underlying RHDV internalization remains unknown. In this study, the entry and post-internalization of RHDV into host cells were investigated using several biochemical inhibitors and RNA interference. Our data demonstrate that rabbit nucleolin (NCL) plays a key role in RHDV internalization. Further study revealed that NCL specifically interacts with the RHDV capsid protein (VP60) through its N-terminal residues (aa 285-318), and the exact position of the VP60 protein for the interaction with NCL is located in a highly conserved region (472Asp-Val-Asn474; DVN motif). Following competitive blocking of the interaction between NCL and VP60 with an artificial DVN peptide (RRTGDVNAAAGSTNGTQ), the internalization efficiency of the virus was markedly reduced. Moreover, NCL also interacts with the C-terminal residues of clathrin light chain A, which is an important component in clathrin-dependent endocytosis. In addition, the results of animal experiments also demonstrated that artificial DVN peptides protected most rabbits from RHDV infection. These findings demonstrate that NCL is involved in RHDV internalization through clathrin-dependent endocytosis.


Assuntos
Infecções por Caliciviridae/virologia , Clatrina/metabolismo , Endocitose , Vírus da Doença Hemorrágica de Coelhos/fisiologia , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Estruturais Virais/metabolismo , Montagem de Vírus , Animais , Masculino , Camundongos , Fosfoproteínas/química , Fosfoproteínas/genética , Conformação Proteica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Coelhos , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/genética , Internalização do Vírus
20.
J Virol ; 92(22)2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30185590

RESUMO

Despite differences in the pathogenesis and host range of alphaherpesviruses, many stages of their morphogenesis are thought to be conserved. Here, an ultrastructural study of bovine herpesvirus 1 (BoHV-1) envelopment revealed profiles similar to those previously found for herpes simplex virus 1 (HSV-1), with BoHV-1 capsids associating with endocytic tubules. Consistent with the similarity of their genomes and envelopment strategies, the proteomic compositions of BoHV-1 and HSV-1 virions were also comparable. However, BoHV-1 morphogenesis exhibited a diversity in envelopment events. First, heterogeneous primary envelopment profiles were readily detectable at the inner nuclear membrane of BoHV-1-infected cells. Second, the BoHV-1 progeny comprised not just full virions but also an abundance of capsidless, noninfectious light particles (L-particles) that were released from the infected cells in numbers similar to those of virions and in the absence of DNA replication. Proteomic analysis of BoHV-1 L-particles and the much less abundant HSV-1 L-particles revealed that they contained the same complement of envelope proteins as virions but showed variations in tegument content. In the case of HSV-1, the UL46 tegument protein was reproducibly found to be >6-fold enriched in HSV-1 L-particles. More strikingly, the tegument proteins UL36, UL37, UL21, and UL16 were depleted in BoHV-1 but not HSV-1 L-particles. We propose that these combined differences reflect the presence of truly segregated "inner" and "outer" teguments in BoHV-1, making it a critical system for studying the structure and process of tegumentation and envelopment.IMPORTANCE The alphaherpesvirus family includes viruses that infect humans and animals. Hence, not only do they have a significant impact on human health, but they also have a substantial economic impact on the farming industry. While the pathogenic manifestations of the individual viruses differ from host to host, their relative genetic compositions suggest similarity at the molecular level. This study provides a side-by-side comparison of the particle outputs from the major human pathogen HSV-1 and the veterinary pathogen BoHV-1. Ultrastructural and proteomic analyses have revealed that both viruses have broadly similar morphogenesis profiles and infectious virus compositions. However, the demonstration that BoHV-1 has the capacity to generate vast numbers of capsidless enveloped particles that differ from those produced by HSV-1 in composition implies a divergence in the cell biology of these viruses that impacts our general understanding of alphaherpesvirus morphogenesis.


Assuntos
Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , Herpesvirus Bovino 1/metabolismo , Herpesvirus Humano 1/metabolismo , Proteínas Estruturais Virais/metabolismo , Animais , Bovinos , Linhagem Celular , Chlorocebus aethiops , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/virologia , Humanos , Células Vero , Vírion/metabolismo , Montagem de Vírus/fisiologia
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