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1.
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
2.
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
3.
Sci Rep ; 5: 14794, 2015 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-26440769

RESUMO

Unlike other viral protease, Avibirnavirus infectious bursal disease virus (IBDV)-encoded viral protease VP4 forms unusual intracellular tubule-like structures during viral infection. However, the formation mechanism and potential biological functions of intracellular VP4 tubules remain largely elusive. Here, we show that VP4 can assemble into tubules in diverse IBDV-infected cells. Dynamic analysis show that VP4 initiates the assembly at early stage of IBDV infection, and gradually assembles into larger size of fibrils within the cytoplasm and nucleus. Intracellular assembly of VP4 doesn't involve the host cytoskeleton, other IBDV-encoded viral proteins or vital subcellular organelles. Interestingly, the last C-terminal hydrophobic and amyloidogenic stretch (238)YHLAMA(243) with two "aggregation-prone" alanine residues was found to be essential for its intracellular self-assembly. The assembled VP4 fibrils show significantly low solubility, subsequently, the deposition of highly assembled VP4 structures ultimately deformed the host cytoskeleton and nucleus, which was potentially associated with IBDV lytic infection. Importantly, the assembly of VP4 significantly reduced the cytotoxicity of protease activity in host cells which potentially prevent the premature cell death and facilitate viral replication. This study provides novel insights into the formation mechanism and biological functions of the Avibirnavirus protease-related fibrils.


Assuntos
Avibirnavirus/metabolismo , Interações Hospedeiro-Patógeno , Serina Endopeptidases/metabolismo , Proteínas Virais/metabolismo , Proteínas Estruturais Virais/metabolismo , Proteínas Amiloidogênicas/química , Proteínas Amiloidogênicas/metabolismo , Animais , Avibirnavirus/patogenicidade , Embrião de Galinha , Chlorocebus aethiops , Citoesqueleto/metabolismo , Células HEK293/virologia , Humanos , Vírus da Doença Infecciosa da Bursa/metabolismo , Vírus da Doença Infecciosa da Bursa/patogenicidade , Peptídeos/química , Peptídeos/metabolismo , Serina Endopeptidases/química , Solubilidade , Células Vero/virologia , Proteínas Virais/química , Proteínas Estruturais Virais/química
4.
Autophagy ; 11(3): 503-15, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25714412

RESUMO

Autophagy is an essential component of host innate and adaptive immunity. Viruses have developed diverse strategies for evading or utilizing autophagy for survival. The response of the autophagy pathways to virus invasion is poorly documented. Here, we report on the induction of autophagy initiated by the pathogen receptor HSP90AA1 (heat shock protein 90 kDa α [cytosolic], class A member 1) via the AKT-MTOR (mechanistic target of rapamycin)-dependent pathway. Transmission electron microscopy and confocal microscopy revealed that intracellular autolysosomes packaged avibirnavirus particles. Autophagy detection showed that early avibirnavirus infection not only increased the amount of light chain 3 (LC3)-II, but also upregulated AKT-MTOR dephosphorylation. HSP90AA1-AKT-MTOR knockdown by RNA interference resulted in inhibition of autophagy during avibirnavirus infection. Virus titer assays further verified that autophagy inhibition, but not induction, enhanced avibirnavirus replication. Subsequently, we found that HSP90AA1 binding to the viral protein VP2 resulted in induction of autophagy and AKT-MTOR pathway inactivation. Collectively, our findings suggest that the cell surface protein HSP90AA1, an avibirnavirus-binding receptor, induces autophagy through the HSP90AA1-AKT-MTOR pathway in early infection. We reveal that upon viral recognition, a direct connection between HSP90AA1 and the AKT-MTOR pathway trigger autophagy, a critical step for controlling infection.


Assuntos
Autofagia , Avibirnavirus/metabolismo , Proteínas do Capsídeo/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Membrana Celular/metabolismo , Galinhas , Citosol/metabolismo , Células HEK293 , Humanos , Lisossomos/metabolismo , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/metabolismo , Fagossomos/metabolismo , Fosforilação , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo
5.
J Virol ; 77(1): 719-25, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12477876

RESUMO

By different approaches, we characterized the birnavirus blotched snakehead virus (BSNV). The sequence of genomic segment A revealed the presence of two open reading frames (ORFs): a large ORF with a 3,207-bp-long nucleotide sequence and a 417-nucleotide-long small ORF located within the N-terminal half of the large ORF, but in a different reading frame. The large ORF was found to encode a polyprotein cotranslationally processed by the viral protease VP4 to generate pVP2 (the VP2 precursor), a 71-amino-acid-long peptide ([X]), VP4, and VP3. The two cleavage sites at the [X]-VP4 and VP4-VP3 junctions were identified by N-terminal sequencing. We showed that the processing of pVP2 generated VP2 and several small peptides (amino acids [aa] 418 to 460, 461 to 467, 468 to 474, and 475 to 486). Two of these peptides (aa 418 to 460 and 475 to 486) were positively identified in the viral particles with 10 additional peptides derived from further processing of the peptide aa 418 to 460. The results suggest that VP4 cleaves multiple Pro-X-Ala downward arrow Ala motifs, with the notable exception of the VP4-VP3 junction. Replacement of the members of the predicted VP4 catalytic dyad (Ser-692 and Lys-729) confirmed their indispensability in the polyprotein processing. The genomic segment B sequence revealed a single large ORF encoding a putative polymerase, VP1. Our results demonstrate that BSNV should be considered a new aquatic birnavirus species, slightly more related to IBDV than to IPNV.


Assuntos
Aquabirnavirus/classificação , Avibirnavirus/classificação , Birnaviridae/classificação , Sequência de Aminoácidos , Birnaviridae/química , Birnaviridae/genética , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Clonagem Molecular , Genoma Viral , Dados de Sequência Molecular , Filogenia , Vírion/química
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