Characterization Of The Mechanisms Of MARCH8 Restriction Effects On IAV Proliferation And Of The Viral Antagonism

Membrane-associated RING-CH(MARCH)family is a class of membrane E3 ubiquitin ligases.The family members contain a C4HC3 RING-finger domain in the N-terminal cytoplasmic tail that potentially interacts with an E2 enzyme which modifies substrate molecules with ubiquitin links.MARCH8 is one of 11 members of the recently discovered cellular MARCH family.MARCH8 downregulates several host transmembrane proteins,including major histocompatibility complex(MHC)-II,CD86,interlukin(IL)-1 receptor accessory protein,and plays important roles in immune regulation.In recent years,MARCH8 has been indicated as an anti-viral protein.MARCH8 is originally characterized through an RNAi genentic screening and shown to be a host restriction factor against HIV.In the following research,it is reported that MARCH8 impedes replications of both VSV and HIV.MARCH8 blocks the incorporation of envelope glycoprotein into virus particles by downregulating it from the cell surface,resulting in a substantial reduction in the efficiency of viral entry,suggesting a broad-spectrum inhibition effects of enveloped viruses by MARCH8.The restriction effects of MARCH8 on other enveloped viruses and the molecular mechanisms are unclear.IAV is a common respiratory envelope virus.In addition to the annual seasonal popularity,IAV has also caused several crazes,causing serious casualties and public panic.Because of the potential outburst risk,IAV has always been a research hotspots and highlights in virology,immunology and epidemiology.In this study,IAV is chosen as the research object to explore the influence of MARCH8 on virus replication.It is found that MARCH8 can inhibit the replication of H1N1 A/PR8/34 virus.By detecting the expression of IAV VLPs(viral like particles),we found that MARCH8 does not affect the cellular expression of two major viral envelope glycoprotein HA and NA,as well as the incorporation in VLPs,but specifically downregulated of the cellular expression of envelope protein M2.MARCH8 overexpression and siRNA knockdown tests showed that MARCH8 downregulates the expression of M2 protein and its distribution on plasma membrane during IAV infection.Further study of the mechanism revealed that MARCH8 downregulated M2 expression depending on its E3 activity.MARCH8 mediates ubiquitination of M2,connects K63-polyubiquitin chain to it and degrades M2 through lysosomes.Immunofluorescence assay showed that MARCH8 and M2 were located in the early endosomes and late endosomes.We found that the lysine at 78(K78)is essential for MARCH8 mediated M2 degradation through mutations of multiple lysine sites in the M2 cytoplasmic tail(CT)region.Using reverse genetics method,the A/PR8/34 virus carrying M2 K78R mutation can escape the inhibitory effect of MARCH8 on M2.We further studied the inhibitory effect of MARCH8 on different IAV strains.It was found that the inhibition effect of MARCH8 on H1N1 A/WSN/33 virus replication was modest.Further detection revealed that M2 expression was not downregulated by MARCH8 in A/WSN/33 infected cells,while MARCH8 expression lessened.By overexpressing different viral protein of A/WSN/33 virus,it is found that the NS 1 protein of A/WSN/33 virus could down regulate the expression of MARCH8 and antagonize the effect of MARCH8 on the downregulation of M2,and the NS 1 protein of A/PR8/34 virus did not have this function.By comparing the amino acid sequences,7 amino acid divergence were found between the NS 1 proteins from the two H1N1 strains,and the capacity of binding CPSF30 was found to be a necessary condition for the downregulation of MARCH8 expression by A/WSN/33 NS1 through amino acid replacement.In summary,this study found that MARCH8 could inhibit A/PR8/34 virus replication firstly.And demonstrated that MARCH8 targeted the envelope protein M2,which mediates M2 degradation through E3 ubiquitin ligase activity.The K78 site in the CT region of M2 is a necessary condition for MARCH8 to mediate its degradation.Meanwhile,it is found that A/WSN/33 virus has evolved a function of antagonizing MARCH8,which uses NS1 to antagonize the role of MARCH8 in regulating M2.This study not only enriches the understanding of the physiological function of MARCH8,but also suggests that it may be an important host anti IAV strategy,which provides a new target for further study of the pathogenesis of IAV.