The Study Of Host Restriction Factor SERINC5 Inhibiting Action To Hepatitis B Virus And Related Mechanisms

Hepatitis B virus(HBV)infection is a serious public health problem and a major cause of cirrhosis and primary hepatocellular carcinoma.Worldwide there are more than 240 million people infected with hepatitis b virus,the situation in China is particularly serious,there are more than 80 million adults for HBV carriers,the control of HBV infection requirements are very urgent.It is of great clinical significance and value to further study the mechanism of interaction between HBV infection and host and to find new therapeutic targets.HBV is a hepatotropic virus and the smallest partially double-stranded DNA virus that replicates via reverse transcription,and the genome length was about 3.2 kb.Upon HBV infection,relaxed circular(rc DNA)is delivered into the nucleus,where it forms covalently closed circular DNA(ccc DNA),which serves as a template for the transcription of pre-genomic RNA(pg RNA).HBV is an enveloped virus that expresses three co-terminal proteins: large(L),middle(M),and small(S),which are important in the viral life cycle.The complex interaction between the virus and the host suggests that the hope of developing new antiviral drugs lies in the host restrictive factors.Over the past decade,host restriction factors such as APOBEC3,TRIM5?,tetherin/BST-2,SAMHD1,Mx2 and GBP5 were discovered as host cell barriers against human immunodeficiency virus(HIV)replication.These proteins which alwalys have a broad spectrum of antiviral capabilities interfere with nearly all stages of viral replication,including viral uncapsid,nucleic acid entry,genome replication,virion release,and fusion.In 2015,two elegant studies discovered that the host cell proteins serine incorporator 5(SERINC5)impair HIV-1 infectivity,and HIV-1 Nef and the glycogag protein of murine leukemia virus(MLV)antagonize their restriction by downregulating SERINC expression on the cell surface and preventing their incorporation into virions.Does SERINC5 have a similar effect on other enveloped virions? We applied SERINC5 to HBV,which also has an envelope,to explore the regulatory effect of SERINC5 on HBV replication,and obtained the following experimental results and conclusions:1.SERINC5 potently inhibits HBV virions secretion;Hep G2 cells were transfected with increased amounts of SERINC5,SERINC3 or SERINC1 plus a HBV expression vector,and increased ectopic SERINC5 inhibited the level of HBV surface antigen(HBs Ag)in the supernatant in a dose-dependent manner.In contrast,increased SERINC3 and SERINC1 did not inhibit the level of HBV surface antigen.SERINC5 also inhibited HBV virions-associated DNA in the supernatant which were concentrated by immunoprecipitation with polyclonal antiHBs antibodies conjugated to protein G.HBV DNA from intracellular core particles and the total RNA levels in cell lysates were further detected by Southern blot and Northern blot.The results showed that SERINC5 had no effect on the levels of HBV core particle-associated DNA and the total RNA.We got the same result in Hep G2.2.15 cells and Hep G2-NTCP cells.Moreover,the knockdown of SERINC5 in Hep G2 cells increased the levels of secreted HBs Ag and HBV virions-associated DNA in the supernatant compared to that in si NC cells,but not HBV core particleassociated DNA and the total RNA in the cell lysates.2.SERINC5 affects the glycosylation of LHB,MHB and SHB proteins;To explore the mechanism by which SERINC5 inhibits HBV virion secretion,we investigated the effects of SERINC5 on HBV proteins.The mobility of LHB,MHB and SHB was different due to differences in the N-linked glycosylation patterns of LHB,MHB and SHB proteins in the presence of SERINC5.Obviously,the nonglycosylation of LHB,MHB and SHB was increased in the presence of SERINC5.3.SERINC5 inhibits the secretion of HBV virus particles by affecting the glycosylation of HBV envelope proteins;We designed the glycosylation site mutants of HBV envelope proteins and found that the glycosylation changes of LHB,MHB and SHB proteins caused by SERINC5 were consistent with the expression forms of the mutants at the glycosylation sites of LHB,MHB and SHB proteins,as well as the expression forms of the wild-type LHB,MHB and SHB proteins after the addition of the glycosylation inhibitor Tunicamycin.A experiment of virus secretion was used to confirm the effection of SERINC5 on HBV secretion.So in our opinions,SERINC5 restricts HBV through affecting glycosylation of LHB,MHB and SHB proteins,thus inhibiting the secretion of complete virion particles.4.Co-localization of SERINC5 with LHB protein in the Golgi;N-linked oligosaccharides are processed within the ER,from which they are transported through the Golgi apparatus for required for post-translational modifications and the trafficking of proteins into extracellular fluid.We next tested whether SERINC5 co-localizes with LHB protein in the ER or Golgi apparatus,thereby interfering with the glycosylation of HBV envelope proteins.We observed that LHB protein alone did not localize within the ER or Golgi apparatus,while SERINC5 alone was distributed in the Golgi but not the ER.However,LHB protein were translocated into the Golgi in the presence of SERINC5 and completely colocalized with SERINC5 in the Golgi in the cytoplasm.A co-immuniprecipitation assay also showed that SERINC5 specifically interacted with LHB,MHB and SHB proteins.These data indicated that SERINC5 may affect the localization of HBV envelope proteins in cells,thereby affecting their glycosylation.5.Functional domains of SERINC5 are required for HBV restrictionSERINC5 is a transmembrane protein that contains 10 putative transmembrane helices.To identify the functional domains of SERINC5 important for HBV restriction,we constructed a series of truncated SERINC5 mutants,and found that 4th to 6th domains in SERINC5 are necessary for HBV suppression.We also examined the effect of phosphorylation or N-glycosylation of SERINC5 on its anti-HBV activity and found phosphorylation or N-glycosylation were not necessary for HBV restriction.In summary,we demonstrated that SERINC5 restricts HBV through affecting glycosylation of LHB,MHB and SHB proteins,thus inhibiting the secretion of virion particles.This may be caused by SERINC5 altering the subcellular localization of LHB protein in cells so that it co-locates with SERINC5 in the Golgi apparatus.This study demonstrated that SERINC5 is a potential anti-HBV intrinsic factor.Therefore,the stimulation or upregulation of SERINC5 may be a novel approach for the development of anti-HBV strategies.