Identification of p90 Ribosomal S6 Kinase 2 as a Novel Host Protein in HBx Augmenting HBV Replication by iTRAQ‐Based Quantitative Comparative Proteomics

PURPOSE: The aim of this study was to screen for novel host proteins that play a role in HBx augmenting Hepatitis B virus (HBV) replication. EXPERIMENTAL DESIGN: Three HepG2 cell lines stably harboring different functional domains of HBx (HBx, HBx‐Cm6, and HBx‐Cm16) were cultured. ITRAQ technology integrated with LC‐MS/MS analysis was applied to identify the proteome differences among these three cell lines. RESULTS: In brief, a total of 70 different proteins were identified among HepG2‐HBx, HepG2‐HBx‐Cm6, and HepG2‐HBx‐Cm16 by double repetition. Several differentially expressed proteins, including p90 ribosomal S6 kinase 2 (RSK2), were further validated. RSK2 was expressed at higher levels in HepG2‐HBx and HepG2‐HBx‐Cm6 compared with HepG2‐HBx‐Cm16. Furthermore, levels of HBV replication intermediates were decreased after silencing RSK2 in HepG2.2.15. An HBx‐minus HBV mutant genome led to decreased levels of HBV replication intermediates and these decreases were restored to levels similar to wild‐type HBV by transient ectopic expression of HBx. After silencing RSK2 expression, the levels of HBV replication intermediates synthesized from the HBx‐minus HBV mutant genome were not restored to levels that were observed with wild‐type HBV by transient HBx expression. CONCLUSION AND CLINICAL RELEVANCE: Based on iTRAQ quantitative comparative proteomics, RSK2 was identified as a novel host protein that plays a role in HBx augmenting HBV replication.