Hepatitis B Virus Regulates GLUT1 Ubiquitination And Protein Level Through CSN5

Hepatitis B virus(HBV)infection is a global health problem.Although the hepatitis B vaccine can protect most people from hepatitis B virus,and a variety of antiviral drugs can improve the health of people with chronic HBV infection,HBV still cannot be completely eliminated.Patients with chronic HBV infection have chance to develop into cirrhosis and liver cancer that cannot be cured.Because the molecular mechanism of HBV infection is still unclear,it is very important to explore the interaction between HBV and host hepatocytes and to elucidate the pathogenesis of HBV.It has been reported that HBV infection causes a variety of post-translational modifications of host proteins,including protein ubiquitination that participates in regulating various physiological functions after HBV infection.However,there is no systemic study about the ubiquitination change of host proteome caused by HBV infection.We used stable-isotope labelling by amino acids in cell culture(SILAC)technology to quantify the ubiquitination of the proteome of the HepG2 cell line with or without HBV infection.Multiple host proteins with vaired ubiquitination and the related modification sites has been identified.From the results,we found that the expression of Glucose transporter isoform 1(GLUT1)was significantly up-regulated and the ubiquitination of GLUT1 was significantly down-regulated after HBV infection.Previous studies showed that tumor cells undergo metabolic reprogramming and uptake more glucose as materials for glycolysis.GLUT1 acts as a glucose transporter and is also the rate-limiting step in glycolysis.Based on these,we hypothesized that HBV may modulate one or several deubiquitinating enzymes todown-regulate the ubiquitination of GLUT1 and then up-regulate the level of GLUT1 protein,and finally increase the glucose uptake.To prove this,we first transfected GLUT1 and pHBV1.3 plasmids in HepG2 cells as well as Huh7 cells,GLUT1 ubiquitination was detected by immunoprecipitation,and GLUT1 ubiquitination was shown to be down-regulated.And we found that HBV down-regulated GLUT1-specific K48 ubiquitination.we transfected pUC18 and pHBV1.3 plasmids in HepG2 cells as well as Huh7 cells,GLUT1 was detected by western blotting,and the level of GLUT1 was shown to be up-regulated.At the same time,real-time quantitative PCR was used to detect the GLUT1 mRNA which remained unaffected.Furthermore,we found that 26 S proteasome inhibitor MG132 treatment can up-regulate the ubiquitination of GLUT1,suggesting that the ubiquitination of GLUT1 is associated with proteasomal degradation.To identify the key deubiquitination enzyme that down-regulates the ubiquitination of GLUT1,GLUT1-FLAG was overexpressed in HepG2.2.15 cell and the proteins that interact with GLUT1 were precipitated by coimmunoprecipitation,and then analyzed by mass spectrometry.COP9 signalosome subunit 5(CSN5)was found to interact with GLUT1,that is confirmed by coimmunoprecipitation.Further experiments showed that overexpression of CSN5 in HepG2.2.15 cells up-regulated the GLUT1 expression,knockdown of CSN5 down-regulated the GLUT1 expression,while exogenous transfection of CSN5 restored the expression of GLUT1.In addition,HBs has been shown to interact with GLUT1,and we found that HBs also interacts with CSN5.Co-immunoprecipitation experiments confirmed the interaction of HBs,CSN5 and GLUT1.Subsequent knockdown of HBs in HepG2.2.15 cells revealed a significant down-regulation of GLUT1 protein levels.Our results indicate that HBV might regulate the ubiquitnation and protein level of the host GLUT1 by interacting of HBs with CSN5 and GLUT1.