The Mechanism Research Of Membrane-associated RING-CH 8 Mediate Coronaviruses S Protein Degradation

Coronaviruses are important human respiratory pathogens that cause severe pneumonia,even death,and currently no antiviral drugs are available.Their genomes encode 16 non-structural proteins(NSPs),and NSP3 is the largest and possesses multiple functional domains,including papain-like protease(PLpro)domain.PLpro has deubiquitination and deISGylation activities.However,the exact role of its deubiquitination function during virus infection remains to be determined.Ubiquitination is an important post-translational modification and involved in many critical cellular processes.Ubiquitination is initiated by El ubiquitin-activating enzyme,followed by E2 ubiquitin-conjugating enzyme,and finalized by E3 ubiquitin-ligase enzyme.E3 determines the substrate specificity.During the virus-host co-evolution,while many viruses employ ubiquitination to facilitate their propagation,many hosts are also evolved to use ubiquitination to limit virus infection.Membrane-associated RING-CH(MARCH)protein 8(MARCH8)is a member of RING-domain protein family E3 ligases and has inhibitory effect on HIV and VSV infection.However,effect of MARCH8 on coronavirus infection is still unknown.In this study,we found that overexpression of MARCH8 led to significant reduction of expression of SARS-CoV and MERS-CoV S protein in cells,compared to controls,and overexpression of MARCH8 also resulted in markedly decrease of the levels of SARS-CoV and MERS-CoV S proteins incorporation into pseudovirons.Interestingly,overexpression of MARCH8 only had minimal effect on MHV S expression and incorporation into pseudovirions.Using mutagenesis,we further showed that enzymatic activity of MARCH8 was required for degradation of SARS-CoV and MERS-CoV S proteins,and K1349 of MERS-CoV S protein was one of ubiquitination sites targeted by MARCH8.Finally,we found that MARCH8-mediated S protein degradation of SARS-CoV and MERS-CoV relied on lysosome pathway,and multiple lysine residues in ubiquitin were involved.In conclusion,our results showed that MARCH8 limited SARS and MERS coronavirus infection by promoting coronavirus S protein degradation through ubiquitination.