The Functional Study Of OTULIN Ubiquitination After HBV Infection

Hepatitis B virus(HBV)is an incompletely closed circular double-stranded DNA virus with strong self-replicating ability,its ccc DNA generated in the host cell as the self-replicating template cannot be eliminated by existing drugs.Therefore,the treatment of patients with chronic HBV infection is still a worldwide problem.Blocking the self-replication of HBV is the key to completely cure patients with HBV infection.The relationship between ubiquitination modification and HBV replication is few reported.As one of the protein modifications,ubiquitination plays essential roles in various cellular activity which requires the participation of ATP,E1,E2,E3 and ubiquitin molecules.In addition,ubiquitin chains or monoubiquitin molecules on substrate proteins can be removed by deubiquitinating enzyme.Among ubiquitination modifications,polyubiquitination is more common than monoubiquitination.Polyubiquitination could be further divided into eight different types of ubiquitin chains linked through M1,K6,K11,K27,K29,K33,K48,or K63,respectively.In order to study the role of ubiquitination modification during HBV infection and host immune response,we previously employed SILAC quantitative proteomics technology to compare the host protein level and ubiquitination level in Hep G2.2.15 cells and Hep G2 cells.Results showed that the protein level of OTULIN in Hep G2.2.15 cells was significantly lower than in Hep G2 cells,but the ubiquitination level of OTULIN in Hep G2.2.15 cells was significantly higher than in Hep G2 cells.Furthermore,OTULIN K66 was identified as the only lysine whose ubiquitination level was changed in our proteomics analysis.OTULIN protein is known to be a deubiquitinating enzyme in 2013 which contains two considerably important sites W96 and C129.With W96 mutated to alanine,OTULIN cannot undergo ubiquitination.C129 is critical for the deubiquitinating activity of OTULIN since its mutation causes OTULIN unable to deubiquitinate M1 ubiquitin chains.So far,most of the OTULIN-related research involves its deubiquitinating activity or ubiquitination catalyzed by other E3 s,the role of OTULIN in the process of HBV infection and host immune response has not been reported yet.Based on our SILAC quantitative proteomics results,HBV infection could down-regulate the protein level of OTULIN and up-regulate the polyubiquitination level of OTULIN.Experiments were conducted in three liver cancer cell lines including Hep G2.2.15,Hep G2,and Huh7 cells to further verify this conclusion.IP experiments showed that HBV infection could up-regulate ubiquitination level of OTULIN in all three cell lines,while IP experiments in Hep G2 cells confirmed K66 as the up-regulating site of OTULIN ubiquitination upon HBV1.3 transfection.Western Blot experiments demonstrated that HBV infection down-regulated OTULIN protein level in Hep G2.2.15 cells,Hep G2 cells,and Huh7 cells.Subsequently,the results of q RT-PCR,Western Blot,and ubiquitination experiments showed that HBV infection was unable to affect OTULIN transcription but could lead to K48-linked ubiquitination of OTULIN at K66,causing its degradation by 26 S proteasome.To further study the potential role of OTULIN degradation during HBV infection,OTULIN was overexpressed,knocked down,or knocked out in HBV1.3-transfected Huh7 cells,results showed that OTULIN overexpression could promote HBV replication while OTULIN knocked-down and knocked-out could reduce HBV replication.Next,we studied the mechanism of OTULIN to regulate HBV replication.Results show that the promotion effect of OTULIN on HBV replication does not rely on its deubiquitinating activity but the ubiquitination modification at OTULIN K66 which may promote the replication of HBV by inhibiting the expression of IL-6/STAT3.Our results indicate that HBV could promote K48-linked polyubiquitination and subsequent proteasome degradation of OTULIN,resulting in reduced HBV replication.The detailed molecular mechanism needs further investigation.