The Molecular Mechanisms Underlying The Regulation Of Cellular Host Antiviral Immune Responses Through PI3K/Akt Signaling In Mammalian Reovirus Infecion

Mammalian reovirus(MRV) is a ubiquitous pathogen agent in nature. It is an important infectious zoonosis in human and nearly all mammals. MRV rarely causes severe illness in human, though several studies have shown its association with human diseases, including meningitis, neonatal hepatitis and biliary atresia. Recently, many MRV strains isolated from animals, especially in wildlife, might be generated by the reassortment of other MRV strains reported before. Considering that many wildlife species have been reported as reservoirs of pathogens, including Nipah virus, Hendra virus, Ebola virus, Zika Virus, Severe acute respiratory syndrome coronavirus(SARS) and Middle east respiratory syndrome coronavirus(MERS), which caused severe public health problem in human and animals. MRV was attracted more and more attention in research due to its potent threaten for the health of human and domestic animals.During virus infection, many key cellular processes have been modulated to cause a favorable cellular environment for virus successful propagation, such as PI3K/Akt signaling pathway. On the other hand, cell host have also developed many strategies to keep the cellular homeostasis and control viral replication, especially the antiviral innate immune system. Microbial infection activated host immune response are initiated by various germline-encoded pattern recognition receptors recognized pathogen-associated molecular patterns specific to microorganisms, and then convergent signaling pathways to raise the cellular host antiviral innate immune responses through adaptor protein MAVS and STING, followed by resisting microbes infection. Indeed, the cellular host antiviral status were activated through different routes during viruses infections, and the interferon-stimulated genes were upregulated. ISGs, as the main effector for restricting viral infection, play vital role in cellular host antiviral responses. ISGs take on a various of roles in antiviral responses. Some ISGs could inhibit virus infection directly, and several ISGs could also enhance innate pathogen sensing capabilities. Although the dsRNA structural of MRV genome should activate the cellular host type I IFN pathway, the underlying mechanism of MRV regulated cellular host antiviral responses were remains unclear.To investigate the molecular mechanism underlying the regulation of the cellular host antiviral responses during MRV infection in detail, we first analysis the influence of MRV infection on the expression of ISGs in A549 cells through double luciferase assays, real time RT-PCR and RNA interference et al. In this study. two different serotype of MRV strains, serotype 3 MRV MPC/04 and serotype 1 MRV B/03 which were isolated from wild animals, were used. Both MPC/04 and B/03 activated the ISRE promoter, and enhanced the expressions of ISGs, including IFITM1, Viperin and ISG15, respectively. Overexpression of both Viperin and ISG15 inhibited MPC/04 and B/03 replication, whereas overexpression of IFITM1 did not affect MPC/04 and B/03 replication in A549 cells. To further investigated the function role of type I IFN pathway in MRV induce ISGs expression, we cloned feline MAVS and STING and analysis its role in the regulation of type I IFN pathway. we found that the biology function of MAVS and STING in innate immune system were conserved between mammals, especially in human and feline. Both MAVS and STING induced the type I IFN activation via NF-κB and IRF3 pathways, and then induced the ISRE promoter activation and enhanced Viperin and ISG15 expression through JAK/STAT1 pathway. Overexpression of both human MAVS and STING inhibited MPC/04 and B/03 replication, respectively. However, knockdown of MAVS or STING did not reduced the activation of ISRE promoter induced by MPC/04 and B/03, respectively. Moreover, both virus activated ISRE in Vero cells, and triggered the ISRE promoter in A549 cells treated with anti-type I and II IFN receptors antibodies. All these findings indicated MRV induce the activation of ISGs did not depend on classical IFN/JAK/STAT1 pathway.To demonstrated the underlying mechanism of the activation of ISGs expressions during MRV infection, we further examined the function role of PI3K/Akt signaling through the methods of western blot assays and RNA interference et al. Both MPC/04 and B/03 induced the phosphorylation of Akt in the early stage of virus infection in A549, HEK293 T and Vero cells, and UV-inactivated viruses also induced the activation of Akt. The activation of Akt induced by MRV infection could be blocked by PI3 K inhibitors, LY294002 and Wortmannin, or siRNA targeting the PI3 K p85α subunit. Chlorpromazine, an inhibitor of clathrin-dependent endocytosis, blocked the activation of PI3K/Akt pathway induce by MPC/04 and B/03, respectively. Whereas Genistein, an inhibitor of caveolin-dependent endocytosis, did not changed the activation of PI3K/Akt pathway. Knockdown of clathrin also reduced the phosphorylation of Akt in the early stage of virus infection. Moreover, MPC/04 and B/03 also induced the phosphorylation of FAK, but not Gab1, in the early stage of virus infection. PP2, an selective inhibitor of Src-kinase, blocked the phosphorylation of FAK and Akt. Both knockdown of FAK and overexpression of dominant negative FAK impaired the activation of Akt. Our data suggested that MRV induced PI3K/Akt pathway via clathrin-dependent endocytosis and the phosphorylation of FAK. On the other hand, both MPC/04 and B/03 induced the phosphorylation of EMSY in a Akt-dependent manner at the early stage of virus infection, Overpression of EMSY, treated with LY294002, and knockdown of Akt expression inhibited the activation of ISRE promoter induced by viruses infection, respectively. Taken in all, MRV triggered the ISRE relied on the activation of PI3K/Akt/EMSY pathway.Taken together, our study demonstrated that MRV induced ISGs activation relied on PI3K/Akt/EMSY pathway, instead of classical IFN/JAK/STAT1 pathway. PI3K/Akt signaling initiated by clathrin-dependent endocytosis, and the activation of FAK. The activation of PI3K/Akt signaling resulted in the upregulation of ISGs, and resisted MRV replication in turn. This study provides us new insight into the molecular mechanism underlying the regulation of ISGs expression during virus infection, and also help us better understand the pathogenesis mechanism of MRV and development of MRV anti-tumor agent.