Critical Role of Viral Protein Hexon in Hypervirulent Fowl Adenovirus Serotype-4-Induced Autophagy by Interaction with BAG3 and Promotion of Viral Replication in LMH Cells.

Hepatitis-pericardial syndrome (HHS) is an acute highly infectious avian disease caused by fowl adenovirus serotype 4 (FAdV-4), characterized by fulminant hepatitis and hydropericardium in broilers. Since 2015, a widespread epidemic has occurred in China due to the emergence of hypervirulent FAdV-4 (HPFAdV-4), causing huge losses to the stakeholders. However, the pathogenesis of HPFAdV-4 and the host responses to its infection remain elusive. Here, we show that infection of leghorn male hepatocellular (LMH) cells by HPFAdV-4 induced complete autophagy in cells and that the autophagy induced by recombinant HPFAdV-4-ON1 (rHPFAdV-4-ON1), a viral strain generated by replacing the hexon gene of wild-type HPFAdV-4 (HPFAdV-4-WT) with the one of nonpathogenic strain FAdV-4-ON1, was remarkably mitigated compared to that of the rHPFAdV-4-WT control, suggesting that HPFAdV-4 hexon is responsible for virus-induced autophagy. Importantly, we found that hexon interacted with a cellular protein, BAG3, a host protein that initiates autophagy, and that BAG3 expression increased in cells infected with HPFAdV-4. Furthermore, knockdown of BAG3 by RNA interference (RNAi) significantly inhibited HPFAdV-4- or hexon-induced autophagy and suppressed viral replication. On the contrary, expression of hexon markedly upregulated the expression of BAG3 via activating the P38 signaling pathway, triggering autophagy. Thus, these findings reveal that HPFAdV-4 hexon interacts with the host protein BAG3 and promotes BAG3 expression by activating P38 signaling pathway, thereby inducing autophagy and enhancing viral proliferation, which immensely furthers our understanding of the pathogenesis of HPFAdV-4 infection. IMPORTANCE HHS, mainly caused by HPFAdV-4, has caused large economic losses to the stakeholders in recent years. Infection of leghorn male hepatocellular (LMH) cells by HPFAdV-4 induced complete autophagy that is essential for HPFAdV-4 replication. By a screening strategy, the viral protein hexon was found responsible for virus-induced autophagy in cells. Importantly, hexon was identified as a factor promoting viral replication by interaction with BAG3, an initiator of host cell autophagy. These findings will help us to better understand the host response to HPFAdV-4 infection, providing a novel insight into the pathogenesis of HPFAdV-4 infection.

Gga-miR-30c-5p Suppresses Avian Reovirus (ARV) Replication by Inhibition of ARV-Induced Autophagy via Targeting ATG5.

Avian reovirus (ARV) causes viral arthritis, chronic respiratory diseases, retarded growth, and malabsorption syndrome. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression posttranscriptionally by silencing or degrading their targets, thus playing important roles in the host response to pathogenic infection. However, the role of miRNAs in host response to ARV infection is still not clear. In this study, we show that ARV infection markedly increased gga-miR-30c-5p expression in DF-1 cells and that transfection of cells with gga-miR-30c-5p inhibited ARV replication while knockdown of endogenous gga-miR-30c-5p enhanced viral growth in cells. Importantly, we identified the autophagy related 5 (ATG5), an important proautophagic protein, as a bona fide target of gga-miR-30c-5p. Transfection of DF-1 cells with gga-miR-30c-5p markedly reduced ATG5 expression accompanied with reduced conversion of ARV-induced-microtubule-associated protein 1 light chain 3 II (LC3-II) from LC3-I, an indicator of autophagy in host cell, while knockdown of endogenous gga-miR-30c-5p enhanced ATG5 expression as well as ARV-induced conversion of LC3-II, facilitating viral growth in cells. Furthermore, knockdown of ATG5 by RNA interference (RNAi) or treatment of cells with autophagy inhibitors (3-MA and wortmannin) markedly reduced ARV-induced LC3-II and syncytium formation, suppressing viral growth in cells, while overexpression of ATG5 increased ARV-induced LC3-II and syncytium formation, promoting viral growth in cells. Thus, gga-miR-30c-5p suppressed viral replication by inhibition of ARV-induced autophagy via targeting ATG5. These findings unraveled the mechanism of how host cells combat against ARV infection by self-encoded small RNA and furthered our understanding of the role of microRNAs in host response to pathogenic infection. IMPORTANCE Avian reovirus (ARV) is an important poultry pathogen causing viral arthritis, chronic respiratory diseases, and retarded growth, leading to considerable economic losses to the poultry industry across the globe. Elucidation of the pathogenesis of ARV infection is crucial to guiding the development of novel vaccines or drugs for the effective control of these diseases. Here, we investigated the role of miRNAs in host response to ARV infection. We found that infection of host cells by ARV remarkably upregulated gga-miR-30c-5p expression. Importantly, gga-miR-30c-5p suppressed ARV replication by inhibition of ARV-induced autophagy via targeting autophagy related 5 (ATG5) accompanied by suppression of virus-induced syncytium formation, thus serving as an important antivirus factor in host response against ARV infection. These findings will further our understanding of how host cells combat against ARV infection by self-encoded small RNAs and may be used as a potential target for intervening ARV infection.

Gga-miR-30c-5p Enhances Apoptosis in Fowl Adenovirus Serotype 4-Infected Leghorn Male Hepatocellular Cells and Facilitates Viral Replication through Myeloid Cell Leukemia-1.

Fowl adenovirus serotype 4 (FAdV-4) is the primary causative agent responsible for the hepatitis-hydropericardium syndrome (HHS) in chickens, leading to considerable economic losses to stakeholders. Although the pathogenesis of FAdV-4 infection has gained attention, the underlying molecular mechanism is still unknown. Here, we showed that the ectopic expression of gga-miR-30c-5p in leghorn male hepatocellular (LMH) cells enhanced apoptosis in FAdV-4-infected LMH cells by directly targeting the myeloid cell leukemia-1 (Mcl-1), facilitating viral replication. On the contrary, the inhibition of endogenous gga-miR-30c-5p markedly suppressed apoptosis and viral replication in LMH cells. Importantly, the overexpression of Mcl-1 inhibited gga-miR-30c-5p or FAdV-4-induced apoptosis in LMH cells, reducing FAdV-4 replication, while the knockdown of Mcl-1 by RNAi enhanced apoptosis in LMH cells. Furthermore, transfection of LMH cells with gga-miR-30c-5p mimics enhanced FAdV-4-induced apoptosis associated with increased cytochrome c release and caspase-3 activation. Thus, gga-miR-30c-5p enhances FAdV-4-induced apoptosis by directly targeting Mcl-1, a cellular anti-apoptotic protein, facilitating FAdV-4 replication in host cells. These findings could help to unravel the mechanism of how a host responds against FAdV-4 infection at an RNA level.

GATA3 Inhibits Viral Infection by Promoting MicroRNA-155 Expression.

Recognition of viral RNAs by melanoma differentiation associated gene-5 (MDA5) initiates chicken antiviral response by producing type I interferons. Our previous studies showed that chicken microRNA-155-5p (gga-miR-155-5p) enhanced IFN-ß expression and suppressed the replication of infectious burse disease virus (IBDV), a double-stranded RNA (dsRNA) virus causing infectious burse disease in chickens. However, the mechanism underlying IBDV-induced gga-miR-155-5p expression in host cells remains elusive. Here, we show that IBDV infection or poly(I:C) treatment of DF-1 cells markedly increased the expression of GATA-binding protein 3 (GATA3), a master regulator for TH2 cell differentiation, and that GATA3 promoted gga-miR-155-5p expression in IBDV-infected or poly(I:C)-treated cells by directly binding to its promoter. Surprisingly, ectopic expression of GATA3 significantly reduced IBDV replication in DF-1 cells, and this reduction could be completely abolished by treatment with gga-miR-155-5p inhibitors, whereas knockdown of GATA3 by RNA interference enhanced IBDV growth, and this enhancement could be blocked with gga-miR-155-5p mimics, indicating that GATA3 suppressed IBDV replication by gga-miR-155-5p. Furthermore, our data show that MDA5 is required for GATA3 expression in host cells with poly(I:C) treatment, so are the adaptor protein TBK1 and transcription factor IRF7, suggesting that induction of GATA3 expression in IBDV-infected cells relies on MDA5-TBK1-IRF7 signaling pathway. These results uncover a novel role for GATA3 as an antivirus transcription factor in innate immune response by promoting miR-155 expression, further our understandings of host response against pathogenic infection, and provide valuable clues to the development of antiviral reagents for public health. IMPORTANCE Gga-miR-155-5p acts as an important antivirus factor against IBDV infection, which causes a severe immunosuppressive disease in chicken. Elucidation of the mechanism regulating gga-miR-155-5p expression in IBDV-infected cells is essential to our understandings of the host response against pathogenic infection. This study shows that transcription factor GATA3 initiated gga-miR-155-5p expression in IBDV-infected cells by directly binding to its promoter, suppressing viral replication. Furthermore, induction of GATA3 expression was attributable to the recognition of dsRNA by MDA5, which initiates signal transduction via TBK1 and IRF7. Thus, it is clear that IBDV induces GATA3 expression via MDA5-TBK1-IRF7 signaling pathway, thereby suppressing IBDV replication by GATA3-mediated gga-miR-155-5p expression. This information remarkably expands our knowledge of the roles for GATA3 as an antivirus transcription factor in host innate immune response particularly at an RNA level and may prove valuable in the development of antiviral drugs for public health.

Gga-miR-29a-3p suppresses avian reovirus-induced apoptosis and viral replication via targeting Caspase-3.

Avian reovirus (ARV) is an important pathogen causing multiple types of clinical diseases in chickens, including viral arthritis, chronic respiratory diseases, retarded growth, and malabsorption syndrome, leading to considerable economic losses to the poultry industry across the globe. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression post transcriptionally by silencing or degrading their targets, thus playing important roles in the host response to pathogenic infection. However, the role of miRNAs in host response to ARV infection is still not clear. Here, we show that infection of DF-1 cells (a chicken fibroblast cell line) with ARV markedly altered the expressions of 583 chicken miRNAs(gga-miR), and that transfection of DF-1 cells with gga-miR-29a-3p, an upregulated miRNA in ARV-infected cells, significantly suppressed ARV-induced apoptosis via directly targeting Caspase-3, retarding ARV growth in cells. In contrast, knockdown of endogenous gga-miR-29a-3p in DF-1 cells by specific miRNA inhibitor enhanced ARV-induced apoptosis and increased the content and activity of caspase-3, facilitating viral growth in cells. Consistently, inhibition of Caspase-3 activity by inhibitors decreased viral titers in cell cultures. Thus, gga-miR-29a-3p plays an important antiviral role in host response to ARV infection by suppression of apoptosis via targeting Caspase-3. This information will further our understandings of how host cells combat against ARV infection by self-encoded small RNA and increase our knowledge of the role of microRNAs in host response to pathogenic infection.