RESUMO
During avian influenza virus (AIV) infection, host defensive proteins promote antiviral innate immunity or antagonize viral components to limit viral replication. UFM1-specific ligase 1 (UFL1) is involved in regulating innate immunity and DNA virus replication in mammals, but the molecular mechanism by which chicken (ch)UFL1 regulates AIV replication is unclear. In this study, we first identified chUFL1 as a negative regulator of AIV replication by enhancing innate immunity and disrupting the assembly of the viral polymerase complex. Mechanistically, chUFL1 interacted with chicken stimulator of IFN genes (chSTING) and contributed to chSTING dimerization and the formation of the STING-TBK1-IRF7 complex. We further demonstrated that chUFL1 promoted K63-linked polyubiquitination of chSTING at K308 to facilitate chSTING-mediated type I IFN production independent of UFMylation. Additionally, chUFL1 expression was upregulated in response to AIV infection. Importantly, chUFL1 also interacted with the AIV PA protein to inhibit viral polymerase activity. Furthermore, chUFL1 impeded the nuclear import of the AIV PA protein and the assembly of the viral polymerase complex to suppress AIV replication. Collectively, these findings demonstrate that chUFL1 restricts AIV replication by disrupting the viral polymerase complex and facilitating type I IFN production, which provides new insights into the regulation of AIV replication in chickens.
Assuntos
Vírus da Influenza A , Influenza Aviária , Interferon Tipo I , Ubiquitina-Proteína Ligases , Replicação Viral , Animais , Galinhas/genética , Imunidade Inata , Vírus da Influenza A/metabolismo , Vírus da Influenza A/fisiologia , Influenza Aviária/metabolismo , Nucleotidiltransferases , Replicação Viral/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The RING finger (RNF) family, a group of E3 ubiquitin ligases, plays multiple essential roles in the regulation of innate immunity and resistance to viral infection in mammals. However, it is still unclear whether RNF proteins affect the production of IFN-I and the replication of avian influenza virus (AIV) in ducks. In this article, we found that duck RNF216 (duRNF216) inhibited the duRIG-I signaling pathway. Conversely, duRNF216 deficiency enhanced innate immune responses in duck embryonic fibroblasts. duRNF216 did not interacted with duRIG-I, duMDA5, duMAVS, duSTING, duTBK1, or duIRF7 in the duck RIG-I pathway. However, duRNF216 targeted duTRAF3 and inhibited duMAVS in the recruitment of duTRAF3 in a dose-dependent manner. duRNF216 catalyzed K48-linked polyubiquitination of duck TRAF3, which was degraded by the proteasome pathway. Additionally, AIV PB1 protein competed with duTRAF3 for binding to duRNF216 to reduce degradation of TRAF3 by proteasomes in the cytoplasm, thereby slightly weakening duRNF216-mediated downregulation of IFN-I. Moreover, although duRNF216 downregulated the IFN-ß expression during virus infection, the expression level of IFN-ß in AIV-infected duck embryonic fibroblasts overexpressing duRNF216 was still higher than that in uninfected cells, which would hinder the viral replication. During AIV infection, duRNF216 protein targeted the core protein PB1 of viral polymerase to hinder viral polymerase activity and viral RNA synthesis in the nucleus, ultimately strongly restricting viral replication. Thus, our study reveals a new mechanism by which duRNF216 downregulates innate immunity and inhibits AIV replication in ducks. These findings broaden our understanding of the mechanisms by which the duRNF216 protein affects AIV replication in ducks.
Assuntos
Patos , Imunidade Inata , Virus da Influenza A Subtipo H5N1 , Influenza Aviária , Transdução de Sinais , Ubiquitina-Proteína Ligases , Replicação Viral , Animais , Patos/imunologia , Patos/virologia , Replicação Viral/imunologia , Transdução de Sinais/imunologia , Influenza Aviária/imunologia , Influenza Aviária/virologia , Virus da Influenza A Subtipo H5N1/imunologia , Virus da Influenza A Subtipo H5N1/fisiologia , Imunidade Inata/imunologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologia , Fibroblastos/imunologia , Fibroblastos/virologia , Proteínas Aviárias/imunologia , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Ubiquitinação , Proteína DEAD-box 58/metabolismo , Proteína DEAD-box 58/imunologiaRESUMO
Duck Tembusu virus (DTMUV) is an emerging pathogen that poses a serious threat to the duck industry in China. Currently, polymerase chain reaction (PCR), quantitative PCR (qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) are commonly used for DTMUV detection. However, these methods require complex steps and special equipment and easily cause false-positive results. Therefore, we urgently need to establish a simple, sensitive and specific method for the clinical field detection of DTMUV. In this study, we developed an RT-LAMP-based CRISPR-Cas12a assay targeting the C gene to detect DTMUV with a limited detection of 3 copies/µL. This assay was specific for DTMUV without cross-reaction with other common avian viruses and only required some simple pieces of equipment, such as a thermostat water bath and blue/UV light transilluminator. Furthermore, this assay showed 100% positive predictive agreement (PPA) and negative predictive agreement (NPA) relative to SYBR Green qPCR for DTMUV detection in 32 cloacal swabs and 22 tissue samples, supporting its application for clinical field detection.