ABSTRACT: Hepatitis B virus (HBV) infection remains a major health issue worldwide and the leading cause of cirrhosis and hepatocellular carcinoma (HCC). It has been reported previously that HBV invasion can extensively alter transcriptome, the proteome of exosomes and host cell lipid rafts. The impact of HBV on host proteins through regulating their global post-translational modifications (PTMs), however, is not well studied. Viruses have been reported to exploit cellular processes by enhancing or inhibiting the ubiquitination of specific substrates. Nevertheless, host cell physiology in terms of global proteome and ubiquitylome has not been addressed yet. Here by using HBV-integrated HepG2.2.15 model cell line we first report that HBV significantly modify the host global ubiquitylome. As currently the most widely used HBV cell culture model, HepG2.2.15 can be cultivated for multiple generations for protein labeling, and can replicate HBV, express HBV proteins and secrete complete HBV Dane particles, which makes it a suitable cell line for ubiquitylome analysis to study HBV replication, hepatocyte immune response and HBV-related HCC progression. Our previous experimental results showed that the total ubiquitination level of HepG2.2.15 cell line was significantly higher than that of the corresponding parental HepG2 cell line. By performing a Ubiscan quantification analysis based on stable isotope labeling of amino acids in cell culture (SILAC) of HepG2.2.15 and HepG2 cell lines, we identified a total of 7188 proteins and the protein levels of nearly 19% of them were changed over 2-folds. We further identified 3798 ubiquitinated Lys sites in 1476 host proteins with altered ubiquitination in response to HBV. Our results also showed that the global proteome and ubiquitylome were negatively correlated, indicating that ubiquitination might be involved in the degradation of host proteins upon HBV integration. We first demonstrated the ubiquitination change of VAMP3, VAMP8, DNAJB6, RAB8A, LYN, VDAC2, OTULIN, SLC1A4, SLC1A5, HGS and TOLLIP. In addition, we described 5 novel host factors SLC1A4, SLC1A5, EIF4A1, TOLLIP and BRCC36 that efficiently reduced the amounts of secreted HBsAg and HBeAg. Overall, the HBV-mediated host proteome and ubiquitylome change we reported will provide a valuable resource for further investigation of HBV pathogenesis and host-virus interaction networks.
