Mechanism Of O-GlcNAcylation Of SAMHD1 Protein To Inhibit HBV Replication

Objective:Hepatitis B virus(Hepatitis B virus,HBV)infection is an important global health problem,about 240 million people infected with hepatitis B virus around the world^[1],which can lead to liver cirrhosis,liver failure and liver cancer^[1].The current two types of HBV infection treatment drugs(Interferon(Interferon)and nucleoside/nucleotide analogs(NAs))have very limited efficacy(1 year treatment of HBs Ag negative conversion rate is usually<5%,prolonged treatment of HBs Ag negative The conversion rate is usually<10%)^[2],coupled with the emergence of HBV resistant mutants,the therapeutic effect of nucleotide analogs is often not sustainable.Therefore,it is of great significance to find new antiviral targets.It is reported that pattern recognition molecules,several metabolic pathways and metabolites play an important role in regulating host innate immune response^[3-5].Recent studies have emphasized the role of HBP pathway in host innate immunity.The 2%-5%glucose entering the cell is converted into the final product of HBP,uridine diphosphate N-acetylglucosamine(UDP-GlcNAc)^[6],which acts as a donor of O-linked?-N-acetylglucosamine(O-GlcNAc)modification^[7].HBP plays an active role in the antiviral immunity of hosts against vesicular stomatitis virus(VSV)^[8],influenza virus^[9]and hepatitis C virus^[10].SAM domain and HD domain-containing protein 1(SAMHD1)is a restrict factor induced by interferon and plays an important role in innate immune response^[11].The host restriction factor SAMHD1 has d GTP/GTP-dependent triphosphate hydrolase activity(d NTP hydrolase activity),which limits the replication of HIV-1 by reducing the intracellular d NTP concentration^[12];at the same time,the SAMHD1 protein also has nuclease activity and can directly Degrade the viral RNA genome and inhibit the replication and transcription of the virus.Studies have found that the d NTP hydrolase activity of SAMHD1 can specifically inhibit HBV DNA synthesis and virus replication,but does not affect the transcription and gene expression of HBV ccc DNA^[13,14].The post-translational modification study of SAMHD1 protein found that SAMHD1 can undergo phosphorylation,acetylation,oxidation,and ubiquitination modifications,which may play multiple regulatory effects on its antiviral function.Our previous studies have shown that Cyclin E2-CDK2 mediates SAMHD1 phosphorylation to eliminate its inhibitory effect on HBV replication^[14].However,it is not clear whether SAMHD1 has OGT-catalyzed O-GlcNAcylation,and whether O-GlcNAcylation of SAMHD1 affects its antiviral function.This study will first detect changes in glucose metabolism,HBP pathway and O-GlcNAc modification in cell models of HBV infection and clinical specimens,observe the influence of intervention of O-GlcNAc modification on HBV replication,and explore the up-regulation of HBP pathway and O-GlcNAc after HBV infection.The molecular mechanism of GlcNAc glycosylation modification,and further clarify the mechanism of action in the antiviral natural immunity of virus infection through metabolic reprogramming to promote the host restriction factor SAMHD1 O-GlcNAc modification.Methods:1.Changes in glucose metabolism,HBP pathway and O-GlcNAc modification in HBV infected cells:The cellular lysis were collected in HepG2 cells overexpressing Ad HBV1.3,and applied to detect the levels of glucose and UDP-GlcNAc by non-target and target metabolite analysis.As UDP-GlcNAc is the only substrate for proteins O-GlcNAcylation,we focus on the effect of HBV infection on HBP pathway.The levels of protein O-GlcNAcylation after HBV infecting cells were determined by Western blot.The protein levels of GFPT1,OGT and OGA,the key enzymes of HBP,were detected by Western blot,and the expressing level of glucose transporter 1(GLUT1)was detected by q RT-PCR and immunofluorescence after HBV infection.2.The effect of intervention of O-GlcNAc modification on HBV replication:Furthermore,the O-GlcNAcylation level were determined by Western blot,and the change of HBV DNA levels was detected by q PCR or Southern blot in Hep AD38,HepG2-NTCP,HepG2 cells treated with WZB117(GLUT1 inhibitor),ST045849(GFPT1 inhibitor),and Thiamet G(OGA inhibitor)after HBV infection.The level of O-GlcNAc was detected by Western blot,and the level of HBV DNA was detected by q PCR or Southern blot in Hep AD38,HepG2-NTCP,HepG2 cells infected by HBV after knocking down GFPT1,OGT,and OGA.3.Identify the key molecules that undergo O-GlcNAc modification after HBV infection and their role in antiviral natural immunity:Hep AD38 cells were depleted of tetracycline,lysed,and incubated with anti-O-GlcNAc antibody to enrich the O-GlcNAcylation proteins,and then identified by LC-MS/MS.The immune-related proteins were screened by GO analysis.Furthermore,Co-IP assay and immunofluorescence assay were used to detect the proteins interacting with OGT,and the main regions of combination.The combination of IP assay and LC-MS/MS anaylsis were performed to detect O-GlcNAcylation site.The modified sites were verified by IP and s WGA assay.The effect of HBV infection on SAMHD1 tetramerization was detected by oligomerization assay,and the d NTPase activity of SAMHD1 WT and S93A protein was detected by HPLC assay.SAMHD1 wild-type(WT)or S93A variant were transfected into SAMHD1-knockout Hep AD38,HepG2 and HepG2-NTCP cells.The effect of S93A mutation on HBV replication was detected by real-time quantitative PCR or Southern blot.SAMHD1-knockout THP-1 cells were transfected with SAMHD1 WT or S93A mutants variant,and treated with single-round replicated pseudoviruses containing HIV-1 with luciferase expression gene.The inhibitory effect of SAMHD1 S93A mutation on HIV-1 was detected by luciferase assay.4.To verify the O-GlcNAc modification of SAMHD1 in HBV transgenic mice and clinical specimens:The UDP-GlcNAc level of patients with chronic HBV infection was detected by LC-MS/MS.The levels of O-GlcNAcylation of HBV transgenic mice and chronic HBV patients in lever was detected by Western blot,and the O-GlcNAcylation of SAMHD1 in liver tissue of chronic HBV patients was detected by s WGA pull-down.Results:1.Non-target and target metabolism analysis found that the levels of glucose and UDP-GlcNAc were significantly increasing after HBV infection.The protein O-GlcNAcylation were significantly enhanced after HBV infection by Western blot anaylsis.The protein level of GFPT1,OGT,OGA,the key enzyme of HBP,showed no difference between control and HBV infection.The results indicated that HBV infection upregulated the expression of glucose transporter 1(GLUT1),which promoted glucose uptake,provided a substrate for HBP synthesis of UDP-GlcNAc,and led to the increase of protein O-GlcNAcylation.2.WZB117(GLUT1 inhibitor),DON(GFPT1 inhibitor),ST045849(OGT inhibitor)decreased the protein O-GlcNAcylation and upregulated the HBV replication.However,thiamet G,an inhibitor of OGA,enhanced the protein O-GlcNAcylation and significantly inhibit the replication of HBV.The levels of protein O-GlcNAcylation was decreased,and the HBV replication was promoted after shGFPT1 or shOGT treatment.However,OGA knocking down could enhance the protein O-GlcNAcylation and significantly inhibit the replication of HBV.3.LC-MS/MS had identified O-GlcNAc-modified 1034 proteins.GO analysis had screened the immune-related protein SAMHD1.Furthermore,Co-IP assay and immunofluorescence assay found that the SAMHD1 was interacted with OGT,and the region of interaction with OGT was 1-150aa.The Ser93 was found to be the O-GlcNAcylation site of SAMHD1 by the combination of IP assay and LC-MS/MS anaylsis.IP and s WGA assay showed S93A mutation decreased the levels of SAMHD1 O-GlcNAcylation,indicating that the Ser93 of SAMHD1 is the main site of O-GlcNAcylation.Oligomerization assay found enhancement of SAMHD1 tetramerization after HBV infection,and HPLC assay showed the decrease of d NTPase activity of prokaryotic expression of SAMHD1 S93A protein.The mutation of SAMHD1 S93A eliminated the effect of it on the inhibition of HBV and HIV-1 replication.4.The levels of UDP-GlcNAc were increased in plasma of patients with chronic HBV infection by LC-MS/MS analysis.Western blot found that the levels of O-GlcNAcylation were increased in the liver tissue of HBV transgenic mice and patients with chronic HBV infection.The levels of SAMHD1 O-GlcNAcylation was significantly increased in liver tissue of chronic HBV infection by s WGA detection.Conclusion:HBV infection increases the levels of UDP-GlcNAc of the HBP pathway and O-GlcNAcylation.HBP-mediated O-GlcNAcylation could inhibit HBV replication.Mass spectrometry showed that host restriction factor SAMHD1 was modified by O-GlcNAc,and Ser93 site was the key site of O-GlcNAcylation.The Ser93site of SAMHD1 O-GlcNAcylation positively regulated anti-HBV immune response in vitro and in vivo.These findings reveal the relationship between HBP,O-GlcNAcylation and innate antiviral immunity targeting SAMHD1.