Multiomics Analysis Of Endocytosis Upon Hbv Infection And Identification Of SCAMP1 As A Novel Host Restriction Factor Against HBV Replication

Background: Globally,liver diseases such as severe hepatitis,cirrhosis,liver fibrosis,and hepatocellular carcinoma(HCC)are important public health problems that have a long-term impact on the life of 257 million people worldwide.In Asia,HBV is a major cause of liver diseases.As a DNA virus,HBV interacts with the NTCP receptor,endocytoses into cells with the assistance of other known or unknown host factors,affects the biological activities of infected cells,and even leads to hepatocellular carcinoma(HCC).Although many studies have proved that HBV infection changes host gene transcription or post-transcriptional modifications,the pathogenesis of HBV infection and host-virus interaction has not been fully elucidated yet.Therefore,a comprehensive exploration of HBV infectioninduced changes in host cell transcriptome,proteome,and ubiquitylome by high throughput technologies in combination with bioinformatics analysis will be crucial to clarify and understanding HBV’s replication and pathogenesis.Multiomics analysis aids in screening specific changing genes,studying their functions,and providing a theoretical basis for the follow-up research and development of drugs to treat HBV.Methods: The HBV-integrated Hep G2.2.15 and its parent Hep G2 cell lines were used as the research sample population.Transcriptome analysis,protein profiling,and ubiscan for ubiquitination profiling were carried out by RNA sequencing and stable isotope labeling by amino acids in cell culture(SILAC)in conjunction with mass spectrometry.By using bioinformatics analysis,we thoroughly explored the DEGs(differentially expressed genes),DEPs(differentially expressed proteins),and Kubsites(lysine ubiquitination sites)in the endocytosis pathway upon HBV infection.Protein-protein interaction networks were visualized with Cytoscape.The ubiquitination modifications and interactions of SCAMP1 with HBV proteins were confirmed by immunoprecipitation.ELISA,q PCR,and Northern blot analyzed the inhibitory effects of SCAMP1 on HBV replication.We performed si RNA knockdown analysis of SCAMP1 to determine the effect of its knockdown on HBV transcription and replication.Results: In this study,we comprehensively explored endocytosis-associated transcriptome,proteome,and ubiquitylome through the multi-omics analysis of Hep G2 and Hep G2.2.15 cells.We quantified 273 genes in the transcriptome;36 genes were changed over two folds which were 13.1% of quantified genes.The most significant one among the two-fold up-regulated genes was RAB31,with a fold change of ≈15.66.Similarly,across the 2-fold down-regulated genes,the most considerable one was LRP2,with the lowest fold change of ≈0.01.In proteome 190,endocytosis-associated proteins were quantified.Across these proteins,34 were changed over two folds.According to subcellular localization analysis,most of the significantly changed endocytic proteins were located in the cytoplasm.Through PPI analysis,we screened 10 hub genes in the endocytosis-associated proteome.In ubiquitylome,we quantified 221 lysine ubiquitinated sites in 77 endocytosis-associated host proteins with modified ubiquitination in response to HBV infection.We confirmed HBV-mediated ubiquitination changes in RAB8 A,DNAJB6,VAMP3,and VAMP8 proteins.Further,we correlated and compared the impact of HBV on 33 common DEGs,DEPs,and Kub-sites.We observed weak negative Pearson correlation coefficients in transcriptome-proteome,ubiquitylome-proteome,and transcriptome-ubiquitylome.Similar results were observed with the Spearman correlation coefficients.We explored the changes in SCAMPs family members upon HBV infection across the transcriptome,proteome,and ubiquitylome data sets.Our quantitative data showed that SCAMP1 was highly ubiquitinated over two-folds at lysine position K65.Immunoprecipitation confirmed the ubiquitination upregulation of endocytosis-associated secretory carrier membrane protein 1(SCAMP1).We also validated HBV-induced SCAMP1 m RNA and protein expression levels in Huh7,Hep G2,and Hep G2.2.15 cells.As SCAMP1 was found to be differentially expressed across all three omics data sets,and HBV infection has changed SCAMP1 expression at the RNA,protein,and ubiquitination levels,we choose SCAMP1 as a gene of interest to evaluate its role against HBV replication.Notably,we revealed SCAMP1 novel antiviral role against HBV transcription and replication for the first time.Overexpression of SCAMP1 efficiently inhibited HBV RNAs/pg RNA and secreted viral proteins,whereas knockdown of SCAMP1 significantly increased viral production.Mechanistically,the Enh I/XP,SP1,and SP2 promoters were inhibited by SCAMP1,which accounts for HBV X and S m RNA inhibition.At the same time,we demonstrated that SCAMP1 physically interacted with HBV L and X proteins to suppress viral replication.Conclusions: Overall,we have presented a multi-omics landscape of the HBV-host interaction through RNA-sequencing,SILAC,and Ubiscan analysis.We explored and validated HBV-induced changes in the endocytosis-associated transcriptome,proteome,and ubiquitylome.We confirmed the ubiquitination change of RAB8 A,DNAJB6,VAMP3,VAMP8,and SCAMP1.Remarkably,SCAMP’s family members,particularly SCAMP1,were differentially expressed in all three omics datasets.Our results demonstrated a novel anti-viral function of endocytosis-associated protein SCAMP1 against HBV replication and gene expression.These findings have opened new opportunities to identify potential drug targets or antiviral agents,as showcased with SCAMP1.More omics work should be performed to understand better host-virus interaction to develop novel therapeutics and potential diagnostics in combating HBV infection.