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J Virol ; 93(3)2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30429342


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.

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
Virus Res ; 191: 184-91, 2014 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-25128465


The avian influenza (AI) H9N2 virus and IBDV are two major problems in the poultry industry. They have been prevalent among domestic poultry in Asia for many years and have caused considerable economic losses. Retinoic-acid-induced gene I (RIG-I) is a cytoplasmic sensor of dsRNA and ssRNA. It can detect Encephalomyocarditis virus (EMCV) and vesicular stomatitis virus (VSV) in human cells, influenza virus in duck leads to production of IFN-ß and IFN-stimulated antiviral genes and reductions in the replication of RNA virus. Chickens, which lack RIG-I, are more sensitive to influenza virus than ducks. However, little is known about the roles of duck RIG-I (dRIG-I) in the detection of IBDV and AI H9N2 in chicken cells DF-1. The purpose of this study was to examine the function of dRIG-I in the recognition of IBDV Ts strain and H9N2 A/Chicken/Shandong/ZB/2007(ZB07) and in the induction of antiviral gene expression to gain an understanding of antiviral ability of dRIG-I in chicken cells against dsRNA virus IBDV and ssRNA virus ZB07. After challenge with the IBDV Ts strain and ZB07 the expression levels of Type I IFN (IFN-ß and IFN-α) and IFN-induced antiviral genes (Mx and PKR) were significantly up-regulated in dRIG-I-transfected DF-1cells compared with the empty-vector-transfected control. dRIG-I knockdown experiments further proved that dRIG-I is essential to sensing IBDV and ZB07 in duck embryo fibroblasts (DEF). Growth curves showed that dRIG-I repressed the replication of IBDV and almost blunted the growth of ZB07 in DF-1. Apoptosis analysis revealed that dRIG-I increase the number of the survival cells after IBDV Ts strain or ZB07 infection relative to the empty-vector-transfected control. These results indicate that dRIG-I can up-regulates type I IFN and reduce viral gene expression and viral replication and protect chicken cells from virus-induced apoptosis during ZB07 and IBDV infection.

Infecções por Birnaviridae/veterinária , RNA Helicases DEAD-box/imunologia , Patos/imunologia , Vírus da Doença Infecciosa da Bursa/fisiologia , Influenza Aviária/imunologia , Doenças das Aves Domésticas/imunologia , Animais , Infecções por Birnaviridae/enzimologia , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/virologia , Galinhas , RNA Helicases DEAD-box/genética , Patos/genética , Vírus da Doença Infecciosa da Bursa/imunologia , Vírus da Influenza A Subtipo H9N2/imunologia , Influenza Aviária/genética , Influenza Aviária/virologia , Interferon-alfa/imunologia , Interferon beta/imunologia , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/virologia , Replicação Viral
PLoS One ; 7(1): e28064, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22253687


BACKGROUND: Infectious bursal disease virus (IBDV) is a pathogen of worldwide significance to the poultry industry. IBDV has a bi-segmented double-stranded RNA genome. Segments A and B encode the capsid, ribonucleoprotein and non-structural proteins, or the virus polymerase (RdRp), respectively. Since the late eighties, very virulent (vv) IBDV strains have emerged in Europe inducing up to 60% mortality. Although some progress has been made in understanding the molecular biology of IBDV, the molecular basis for the pathogenicity of vvIBDV is still not fully understood. METHODOLOGY, PRINCIPAL FINDINGS: Strain 88180 belongs to a lineage of pathogenic IBDV phylogenetically related to vvIBDV. By reverse genetics, we rescued a molecular clone (mc88180), as pathogenic as its parent strain. To study the molecular basis for 88180 pathogenicity, we constructed and characterized in vivo reassortant or mosaic recombinant viruses derived from the 88180 and the attenuated Cu-1 IBDV strains. The reassortant virus rescued from segments A of 88180 (A88) and B of Cu-1 (BCU1) was milder than mc88180 showing that segment B is involved in 88180 pathogenicity. Next, the exchange of different regions of BCU1 with their counterparts in B88 in association with A88 did not fully restore a virulence equivalent to mc88180. This demonstrated that several regions if not the whole B88 are essential for the in vivo pathogenicity of 88180. CONCLUSION, SIGNIFICANCE: The present results show that different domains of the RdRp, are essential for the in vivo pathogenicity of IBDV, independently of the replication efficiency of the mosaic viruses.

Infecções por Birnaviridae/enzimologia , Infecções por Birnaviridae/virologia , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Vírus da Doença Infecciosa da Bursa/enzimologia , Vírus da Doença Infecciosa da Bursa/patogenicidade , Aminoácidos/genética , Animais , Linhagem Celular , Galinhas/virologia , RNA Polimerases Dirigidas por DNA/genética , Genoma Viral/genética , Vírus da Doença Infecciosa da Bursa/genética , Dados de Sequência Molecular , Mosaicismo , Nucleotídeos/genética , Fenótipo , Doenças das Aves Domésticas/enzimologia , Doenças das Aves Domésticas/virologia , Estrutura Terciária de Proteína , Recombinação Genética/genética , Virulência/genética
J Fish Dis ; 31(6): 451-60, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18471101


Our previous studies found that infectious pancreatic necrosis virus (IPNV) induces host apoptotic cell death, possibly through a newly synthesized protein trigger. Here, we examine whether IPNV infection can induce NF-kappaB activation through tyrosine kinase signalling of CHSE-214 cell death (host cell death). Using the electrophoretic mobility shift assay (EMSA) to detect transcription factor activation, we found that NF-kappaB is apparently activated 6-8 h post-IPNV infection. Using genistein (100 microg mL(-1); a tyrosine kinase inhibitor) to determine whether NF-kappaB activation requires tyrosine kinase activation, we found genistein blocks NF-kappaB activation at 8 h post-infection (p.i), and either enhances cell viability up to 50% at 12 h p.i. or blocks DNA fragmentation at 24 h p.i. Furthermore, the proteasome inhibitors PSI-I and PSI-II (both at 40 microm) also effectively blocked the NF-kappaB activation as well as stimulating a 30% increase in cell viability (30% decrease in apoptosis) at 8 and 12 h p.i. Taken together our data suggest that IPNV may induce NF-kappaB activation through tyrosine kinase signalling, which may be associated with induction of apoptosis.

Infecções por Birnaviridae/veterinária , Birnaviridae/fisiologia , Doenças dos Peixes/patologia , NF-kappa B/metabolismo , Proteínas Tirosina Quinases/metabolismo , Salmão/virologia , Transdução de Sinais , Animais , Apoptose/efeitos dos fármacos , Infecções por Birnaviridae/enzimologia , Infecções por Birnaviridae/metabolismo , Infecções por Birnaviridae/patologia , Morte Celular , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Doenças dos Peixes/enzimologia , Doenças dos Peixes/metabolismo , Doenças dos Peixes/virologia , Genisteína/farmacologia , Oligopeptídeos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Salmão/metabolismo , Fatores de Tempo
Acta Vet Hung ; 54(2): 281-93, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16841765


The influence of two infectious bursal disease vaccines on the activities of hepatic microsomal enzymes aniline hydroxylase, ethylmorphine N-demethylase, NADPH-cytochrome c reductase, aryl sulphotransferase and p-nitrophenol UDP-glucuronyltransferase was investigated in chickens. The vaccines contained attenuated Winterfield 2512 and VMG-91 strains, respectively. The activities of enzymes were determined on postvaccination days 0, 2, 5 and 7. At the same time, post-mitochondrial supernatant, cytosolic and microsomal pellet protein concentrations were determined. As expected, the antibody titres against infectious bursal disease virus in the serum were increased in both tested groups in relation to each administered vaccine. Using RT-PCR, the presence of the VP2 gene fragment of virus in the liver of chicken was demonstrated 4 and 6 h after vaccination. The results of this study suggest that the two commercial vaccines modulate the activities of five enzymes tested, and that the two attenuated vaccines applied triggered induction and/or inhibition of phases I and II of biotransformation enzyme activities.

Infecções por Birnaviridae/veterinária , Galinhas , Vírus da Doença Infecciosa da Bursa/imunologia , Microssomos Hepáticos/enzimologia , Doenças das Aves Domésticas/prevenção & controle , Vacinas Virais/farmacologia , Animais , Animais Recém-Nascidos , Anticorpos Antivirais/sangue , Infecções por Birnaviridae/enzimologia , Infecções por Birnaviridae/prevenção & controle , Feminino , Vírus da Doença Infecciosa da Bursa/patogenicidade , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Doenças das Aves Domésticas/enzimologia , Distribuição Aleatória , Vacinação/métodos , Vacinação/veterinária , Vacinas Atenuadas/farmacologia