Characterization Of The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Structural And Accessory Proteins:Focusing On IFN Antagonism And Autophagy

Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly identified human pathogen associated with severe acute respiratory disease occasionally accompanied by renal failure. As of June 6,2015, the World Health Organization has been notified of 1,190 laboratory-confirmed cases of infection with MERS-CoV, including 444 related deaths. Although there is no definite evidence that MERS-CoV can be transmitted from human-to-human, the animal surveillance, genomic analysis and epidemiology study raises concern regarding a possible outbreak similar to the outbreak caused by the severe acute respiratory syndrome-related coronavirus (SARS-CoV) in 2002-2003.IFN and IFN-induced cellular antiviral responses are the first line of defense against viral infection, and plays a vital role in the early antiviral response. Autophagy is an intrinsic lysosome-dependent degradation pathway among eucaryon, which also plays an important role against viral infection, both antiviral and proviral. During infection, virus have evolved mutiple mechanisms to combat IFN respons, induce or inhibit aotophagy to promote replication via the viral proteins. Previous studies have shown that MERS-CoV infection failed to elicit a strong type Ⅰ or Ⅲ IFN or proinflammatory innate immune responses in ex vivo respiratory tissue cultures, and its replication was susceptible to IFN alpha, indicating that MERS-CoV is able to cope with the antiviral effect of type I IFN system. The relationship between antophagy and coronavirus replication is not clear, however, coronavirus replication indeed induces autophagy and several proteins of coronavirus have been shown to induce autophagy. Identification of the connection between MERS-CoV structural and accessory proteins and innate immunity and autophagy will help us to have a better understanding of the pathogenesis and immune evasion mechanisms, provide theoretical basis and new strategies for antiviral drug development.Given this reason, this study characterizes the functions of the MERS-CoV structural and accessory proteins focusing on IFN antagonism and autophagy, the main results are as follows:1. Expression and subcellular localization of MERS-CoV structural proteins and accessory proteins ORF3, ORF4a, ORF4b, ORF5.Firstly, we synthesized the genes of the MERS-CoV structural and accessory proteins according to the published sequences in NCBI. Subsequently individual structural protein and accessory protein encoding plasmids were constructed with an HA-tag at the N-terminus of each protein, and confirmed the correct expression by WB and IFA.Furthermore, the subcellular localization was observed by Confocal assay. S was detected throughout the typical secretory pathway of mammalian cells, from the ER to the plasma membrane; E and M mainly localise to the Golgi apparatus; and N exclusively localises to the cytoplasm.ORF3 demonstrates punctate cytoplasmic distribution and co-localization with the ER and ERGIC; ORF4a is detected throughout the cytoplasm; ORF4b protein predominantly localizes to the nucleus with a small part dispersed throughout the cytoplasm; ORF5 co-localizes with the Golgi apparatus and ERGIC. These results serve as the basis of further study.2、 ORF 4a, ORF 4b, ORF 5 and M proteins antagonize the type Ⅰ interferon response.We screened the IFN antagonism of MERS-CoV structural and accessory proteins by dual reporter gene assay and semi-qPCR ampliation of IFNβ mRNA, and found that ORF 4a, ORF 4b, ORF 5 and M proteins could antagonize the type Ⅰ interferon response. All of them could inhibit the function of IRF3 and additionally NF-κB by ORF4a. Furthermore, using dual reporter gene assay and semi-qPCR ampliation of ISG54 and ISG56, ORF4a, ORF4b and M were shown to inhibit the IFN signaling, suggesting that ORF4a, ORF4b and M not only inhibited the synthesis of IFN, but also the the IFN signaling. However, ORF5 could only inhibit the synthesis of IFN.3、ORF4b inhibits the induction of interferon both in the cytoplasm and nucleus, and in the cytoplasm it fulfill the function through a direct interaction with IKKε/TBKI.By co-immunoprecipitation and confocal assay, we identified that ORF4b could could specifically bind to MDA5, IKKε and TBK in the RLR pathway. ORF4b had a stronger interaction with the amino-terminal CARD domain, but did not affect formation of the complex between MDA5 and MAVS. In addition, ORF4b specifically reduced IKKs and TBK1 mediated IRF3 phosphorylation and nuclear translocation, while ORF4b could not affect the interaction between IKKε/TBK1 and their IRF substrates but suppress the formation of the IKKs and MAVS complex. Utilizing the transducer proteins in the RLR pathway as IFN inducers, we found both ORF4b and ORF4b (A2-38) could inhibit the RIG-I, MDA5, MAVS, IKKε, TBK1 induced activation of IFN-β while ORF4b inhibited IRF3 as well, suggesting that ORF4b inhibited the type Ⅰ IFN signaling pathway both in cytoplasm and in the nucleus.4. E and M proteins can induce incomplete autophagy independent of the mTOR pathway.By fluorescence microscopy, transmission electron microscopy and western-blot, we confirmed that E and M proteins could induce autophagy. Moreever, by monitoring the p62 expression level, LC3 turnover assay, mRFP-GFP-LC3 delivery assay and colocalization of GFP-LC3 and LAMP1, we further confirmed E and M proteins induce incomplete autophagy by inhibiting the autophagosome-lysosome fusion. By analyzing the phosphorylation level of 4E-BP1 and S6K, we found E and M proteins induced autophagy was mTOR/4E-BP1and mTOR/S6K independent. Furthermore, E and M proteins were shown to activate the IRE1-XBP1 pathaway of unfolded protein response(UPR).In conclusion, this study has found that MERS-CoV accessory proteins ORF4a, ORF4b, ORF5 and structural protein M function as interferon antagonism. Among them, ORF4b directly binds to IKKε and TBK1 in the cytoplasm to inhibit the IFN regulatory factor 3 (IRF3) phosphorylation and subsequent IFN-β production; additionally it also inhibit the induction of IFN in the nucleus via some mechanism. Furthermore, MERS-CoV E and M proteins induce incomplete autophagy by inhibiting the autophagosome-lysosome fusion. These recults will help us to have a better understanding of the pathogenesis and immune evasion mechanisms, provide theoretical basis and new strategies for developing immunoprophylaxis and antiviral drugs.