Critical Role for Voltage-Dependent Anion Channel 2 in Infectious Bursal Disease Virus-Induced Apoptosis in Host Cells via Interaction with VP5

ABSTRACT MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression posttranscriptionally by silencing or degrading their targets and play important roles in the host response to pathogenic infection. Although infectious bursal disease virus (IBDV)-induced apoptosis in host cells has been established, the underlying molecular mechanism is not completely unraveled. Here, we show that infection of DF-1 cells by IBDV induced gga-miR-16-5p (chicken miR-16-5p) expression via demethylation of the pre-miR-16-2 (gga-miR-16-5p precursor) promoter. We found that ectopic expression of gga-miR-16-5p in DF-1 cells enhanced IBDV-induced apoptosis by directly targeting the cellular antiapoptotic protein B-cell lymphoma 2 (Bcl-2), facilitating IBDV replication in DF-1 cells. In contrast, inhibition of endogenous miR-16-5p markedly suppressed apoptosis associated with enhanced Bcl-2 expression, arresting viral replication in DF-1 cells. Furthermore, infection of DF-1 cells with IBDV reduced Bcl-2 expression, and this reduction could be abolished by inhibition of gga-miR-16-5p expression. Moreover, transfection of DF-1 cells with gga-miR-16-5p mimics enhanced IBDV-induced apoptosis associated with increased cytochrome c release and caspase-9 and -3 activation, and inhibition of caspase-3 decreased IBDV growth in DF-1 cells. Thus, epigenetic upregulation of gga-miR-16-5p expression by IBDV infection enhances IBDV-induced apoptosis by targeting the cellular antiapoptotic protein Bcl-2, facilitating IBDV replication in host cells. IMPORTANCE Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive disease in young chickens, causing severe economic losses to stakeholders across the globe. Although IBD virus (IBDV)-induced apoptosis in the host has been established, the underlying mechanism is not very clear. Here, we show that infection of DF-1 cells by IBDV upregulated gga-miR-16-5p expression via demethylation of the pre-miR-16-2 promoter. Overexpression of gga-miR-16-5p enhanced IBDV-induced apoptosis associated with increased cytochrome c release and caspase-9 and -3 activation. Importantly, we found that IBDV infection induced expression of gga-miR-16-5p that triggered apoptosis by targeting Bcl-2, favoring IBDV replication, while inhibition of gga-miR-16-5p in IBDV-infected cells restored Bcl-2 expression, slowing down viral growth, indicating that IBDV induces apoptosis by epigenetic upregulation of gga-miR-16-5p expression. These findings uncover a novel mechanism employed by IBDV for its own benefit, which may be used as a potential target for intervening IBDV infection.

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Critical role for G3BP1 in infectious bursal disease virus (IBDV)-induced stress granule formation and viral replication

Veterinary Microbiology ◽

10.1016/j.vetmic.2020.108806 ◽

2020 ◽

Vol 248 ◽

pp. 108806

Author(s):

Dianzheng Zhao ◽

Jiaxin Li ◽

Yongqiang Wang ◽

Xiaoqi Li ◽

Li Gao ◽

...

Keyword(s):

Viral Replication ◽

Disease Virus ◽

Infectious Bursal Disease Virus ◽

Critical Role ◽

Stress Granule ◽

Infectious Bursal Disease ◽

Granule Formation ◽

Induced Stress ◽

Bursal Disease

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Nonstructural Protein of Infectious Bursal Disease Virus Inhibits Apoptosis at the Early Stage of Virus Infection

Journal of Virology ◽

10.1128/jvi.80.7.3369-3377.2006 ◽

2006 ◽

Vol 80 (7) ◽

pp. 3369-3377 ◽

Cited By ~ 50

Author(s):

Meihong Liu ◽

Vikram N. Vakharia

Keyword(s):

Virus Infection ◽

Dna Fragmentation ◽

Disease Virus ◽

Infectious Bursal Disease Virus ◽

Early Stage ◽

Nonstructural Protein ◽

Infectious Bursal Disease ◽

Knockout Mutant ◽

Bursal Disease ◽

Induced Apoptosis

ABSTRACT Infectious bursal disease virus (IBDV), the causative agent of a highly contagious disease in chickens, carries a small nonstructural protein (NS). This protein has been implicated to play a role in the induction of apoptosis. In this study, we investigate the kinetics of viral replication during a single round of viral replication and examine the mechanism of IBDV-induced apoptosis. Our results show that it is caspase dependent and activates caspases 3 and 9. Nuclear factor kappa B (NF-κB) is also activated and is required for IBDV-induced apoptosis. The NF-κB inhibitor MG132 completely inhibited IBDV-induced DNA fragmentation, caspase 3 activation, and NF-κB activation. To study the function of the NS protein in this context, we generated the recombinant rGLS virus and an NS knockout mutant, rGLSNSΔ virus, using reverse genetics. Comparisons of the replication kinetics and markers for virally induced apoptosis indicated that the NS knockout mutant virus induces earlier and increased DNA fragmentation, caspase activity, and NF-κB activation. These results suggest that the NS protein has an antiapoptotic function at the early stage of virus infection.

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VP1 and VP3 Are Required and Sufficient for Translation Initiation of Uncapped Infectious Bursal Disease Virus Genomic Double-Stranded RNA

Journal of Virology ◽

10.1128/jvi.01345-17 ◽

2017 ◽

Vol 92 (2) ◽

Cited By ~ 8

Author(s):

Chengjin Ye ◽

Yu Wang ◽

Enli Zhang ◽

Xinpeng Han ◽

Zhaoli Yu ◽

...

Keyword(s):

Disease Virus ◽

Translation Initiation ◽

Infectious Bursal Disease Virus ◽

Binding Protein ◽

Viral Proteins ◽

Host Cells ◽

Infectious Bursal Disease ◽

Genomic Rna ◽

Sense Strand ◽

Bursal Disease

ABSTRACTInfectious bursal disease virus (IBDV) is a bisegmented double-strand RNA (dsRNA) virus of theBirnaviridaefamily. While IBDV genomic dsRNA lacks a 5′ cap, the means by which the uncapped IBDV genomic RNA is translated effectively is unknown. In this study, we describe a cap-independent pathway of translation initiation of IBDV uncapped RNA that relies on VP1 and VP3. We show that neither purified IBDV genomic dsRNA nor the uncapped viral plus-sense RNA transcripts were directly translated and rescued into infectious viruses in host cells. This defect in translation of the uncapped IBDV genomic dsRNA was rescued bytrans-supplementation of the viral proteins VP1 and VP3 which was dependent on both the intact polymerase activity of VP1 and the dsRNA binding activity of VP3. Deletion analysis showed that both 5′ and 3′ untranslated regions (UTRs) of IBDV dsRNA were essential for VP1/VP3-dependent translation initiation. Significantly, VP1 and VP3 could also mediate the recovery of infectious IBDV from the authentic minus-sense strand of IBDV dsRNA. Moreover, downregulation or inhibition of the cap-binding protein eIF4E did not decrease but, rather, enhanced the VP1/VP3-mediated translation of the uncapped IBDV RNA. Collectively, our findings for the first time reveal that VP1 and VP3 compensate for the deficiency of the 5′ cap and replace eIF4E to confer upon the uncapped IBDV RNA the ability to be translated and rescued into infectious viruses.IMPORTANCEA key point of control for virus replication is viral translation initiation. The current study shows that the uncapped IBDV RNA cannot be translated into viral proteins directly by host translation machinery and is thus noninfectious. Our results constitute the first direct experimental evidence that VP1 and VP3 are required and sufficient to initiate translation of uncapped IBDV genomic RNA by acting as a substitute for cap and replacing the cap-binding protein eIF4E. Significantly, VP1/VP3 mediate the recovery of infectious IBDV not only from the plus-sense strand but also from the minus-sense strand of the IBDV dsRNA. These findings provide not only new insights into the molecular mechanisms of the life cycle of IBDV but also a new tool for an alternative strategy for the recovery of IBDV from both the plus- and the minus-sense strands of the viral genomic dsRNA.

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Proteomics Analysis of Host Cells Infected with Infectious Bursal Disease Virus

Molecular & Cellular Proteomics ◽

10.1074/mcp.m700396-mcp200 ◽

2007 ◽

Vol 7 (3) ◽

pp. 612-625 ◽

Cited By ~ 84

Author(s):

Xiaojuan Zheng ◽

Lianlian Hong ◽

Lixue Shi ◽

Junqing Guo ◽

Zhen Sun ◽

...

Keyword(s):

Disease Virus ◽

Infectious Bursal Disease Virus ◽

Host Cells ◽

Infectious Bursal Disease ◽

Proteomics Analysis ◽

Bursal Disease

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MicroRNA gga-miR-130b Suppresses Infectious Bursal Disease Virus Replication via Targeting of the Viral Genome and Cellular Suppressors of Cytokine Signaling 5

Journal of Virology ◽

10.1128/jvi.01646-17 ◽

2017 ◽

Vol 92 (1) ◽

Cited By ~ 19

Author(s):

Mengjiao Fu ◽

Bin Wang ◽

Xiang Chen ◽

Zhiyuan He ◽

Yongqiang Wang ◽

...

Keyword(s):

Disease Virus ◽

Viral Genome ◽

Infectious Bursal Disease Virus ◽

Host Cells ◽

Infectious Bursal Disease ◽

Cytokine Signaling ◽

Type I ◽

Bursal Disease ◽

Suppressors Of Cytokine Signaling

ABSTRACTMicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression posttranscriptionally through silencing or degrading their targets, thus playing important roles in the immune response. However, the role of miRNAs in the host response against infectious bursal disease virus (IBDV) infection is not clear. In this study, we show that the expression of a series of miRNAs was significantly altered in DF-1 cells after IBDV infection. We found that the miRNA gga-miR-130b inhibited IBDV replication via targeting the specific sequence of IBDV segment A and enhanced the expression of beta interferon (IFN-β) by targeting suppressors of cytokine signaling 5 (SOCS5) in host cells. These findings indicate that gga-miR-130b-3p plays a crucial role in host defense against IBDV infection.IMPORTANCEThis work shows that gga-miR-130b suppresses IBDV replication via directly targeting the viral genome and cellular SOCS5, the negative regulator for type I interferon expression, revealing the mechanism underlying gga-miR-130-induced inhibition of IBDV replication. This information will be helpful for the understanding of how host cells combat pathogenic infection by self-encoded small RNA and furthers our knowledge of the role of microRNAs in the cell response to viral infection.

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Identification and Molecular Characterization of the RNA Polymerase-Binding Motif of Infectious Bursal Disease Virus Inner Capsid Protein VP3

Journal of Virology ◽

10.1128/jvi.77.4.2459-2468.2003 ◽

2003 ◽

Vol 77 (4) ◽

pp. 2459-2468 ◽

Cited By ~ 33

Author(s):

Antonio Maraver ◽

Roberto Clemente ◽

Jose Francisco Rodríguez ◽

Eleuterio Lombardo

Keyword(s):

Rna Polymerase ◽

Capsid Protein ◽

Disease Virus ◽

Infectious Bursal Disease Virus ◽

Critical Role ◽

Rnase A ◽

Infectious Bursal Disease ◽

Binding Motif ◽

Bursal Disease

ABSTRACT Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is the causative agent of one of the most important infectious poultry diseases. Major aspects of the molecular biology of IBDV, such as assembly and replication, are as yet poorly understood. We have previously shown that encapsidation of the putative virus-encoded RNA-dependent RNA polymerase VP1 is mediated by its interaction with the inner capsid protein VP3. Here, we report the characterization of the VP1-VP3 interaction. RNase A treatment of VP1- and VP3-containing extracts does not affect the formation of VP1-VP3 complexes, indicating that formation of the complex requires the establishment of protein-protein interactions. The use of a set of VP3 deletion mutants allowed the mapping of the VP1 binding motif of VP3 within a highly charged 16-amino-acid stretch on the C terminus of VP3. This region of VP3 is sufficient to confer VP1 binding activity when fused to an unrelated protein. Furthermore, a peptide corresponding to the VP1 binding region of VP3 specifically inhibits the formation of VP1-VP3 complexes. The presence of Trojan peptides containing the VP1 binding motif in IBDV-infected cells specifically reduces infective virus production, thus showing that formation of VP1-VP3 complexes plays a critical role in IBDV replication.

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Triplet amino acids located at positions 145/146/147 of the RNA polymerase of very virulent infectious bursal disease virus contribute to viral virulence

Journal of General Virology ◽

10.1099/vir.0.060194-0 ◽

2014 ◽

Vol 95 (4) ◽

pp. 888-897 ◽

Cited By ~ 23

Author(s):

Li Gao ◽

Kai Li ◽

Xiaole Qi ◽

Honglei Gao ◽

Yulong Gao ◽

...

Keyword(s):

Amino Acids ◽

Glutamic Acid ◽

Disease Virus ◽

Infectious Bursal Disease Virus ◽

Chicken Embryo Fibroblast ◽

Critical Role ◽

Infectious Bursal Disease ◽

Effective Prevention ◽

Viral Virulence ◽

Bursal Disease

Infectious bursal disease virus (IBDV) causes an economically significant disease of chickens worldwide. The emergence of very virulent IBDV (vvIBDV) has brought more challenges for effective prevention of this disease. The molecular basis for the virulence of vvIBDV is not fully understood. In this study, 20 IBDV strains were analysed phylogenically and clustered in three branches based on their full-length B segments. The amino acid triplet located at positions 145/146/147 of VP1 was found highly conserved in branch I non-vvIBDVs as asparagine/glutamic acid/glycine (NEG), in branch II vvIBDVs as threonine/glutamic acid/glycine (TEG) and in branch III vvIBDVs as threonine/aspartic acid/asparagine (TDN). Further studies showed that the three amino acids play a critical role in the replication and pathogenicity of vvIBDV. Substitution of the TDN triplet with TEG or NEG reduced viral replication and pathogenicity of the vvIBDV HuB-1 strain in chickens. However, the replication of the attenuated IBDV Gt strain was reduced in chicken embryo fibroblast cells, whilst it was enhanced in the bursa by substituting NEG with TEG or TDN. The exchange of the three amino acids was also found to be capable of affecting the polymerase activity of VP1. The important role of segment B in the pathogenicity of IBDV was confirmed in this study. These results also provided new insights into the mechanism of the virulence of vvIBDVs and may offer new targets for their attenuation to develop potential vaccines using reverse genetics.

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Cyclophilin A Interacts with Viral VP4 and Inhibits the Replication of Infectious Bursal Disease Virus

BioMed Research International ◽

10.1155/2015/719454 ◽

2015 ◽

Vol 2015 ◽

pp. 1-10 ◽

Cited By ~ 2

Author(s):

Nian Wang ◽

Lizhou Zhang ◽

Yuming Chen ◽

Zhen Lu ◽

Li Gao ◽

...

Keyword(s):

Disease Virus ◽

Infectious Bursal Disease Virus ◽

Nonstructural Protein ◽

Chicken Embryo Fibroblast ◽

Cyclophilin A ◽

Host Cells ◽

Infectious Bursal Disease ◽

Cell Protein ◽

Small Interfering Rnas ◽

Bursal Disease

Nonstructural protein VP4, a serine protease of infectious bursal disease virus (IBDV) that catalyzes the hydrolysis of polyprotein pVP2-VP4-VP3 to form the viral proteins VP2, VP4, and VP3, is essential to the replication of IBDV. However, the interacting partners of VP4 in host cells and the effects of the interaction on the IBDV lifecycle remain incompletely elucidated. In this study, using the yeast two-hybrid system, the putative VP4-interacting partner cyclophilin A (CypA) was obtained from a chicken embryo fibroblast (CEF) expression library. CypA was further confirmed to interact with VP4 of IBDV using co-immunoprecipitation (CO-IP), GST pull-down, and confocal microscopy assays. Moreover, we found that the overexpression of CypA suppressed IBDV replication, whereas the knock-down of CypA by small interfering RNAs promoted the replication of IBDV. Taken together, our findings indicate that the host cell protein CypA interacts with viral VP4 and inhibits the replication of IBDV.

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