Hepatitis B virus is a stealth virus that minimizes proteomic and secretomic changes in primary human hepatocytes.

Hepatitis B virus (HBV) is a hepatotropic DNA virus that infects over 250 million people worldwide and causes serious liver diseases. HBV infection can modulate host cellular processes, potentially inducing proteomic changes in hepatocytes. In this study, we investigated how acute HBV infection alters the proteome and secretome of primary human hepatocytes, a physiologically relevant in vitro model that retains essential liver-specific functions. Protein-level changes in cell lysates and culture supernatants were quantified 8 days post-infection using data-independent acquisition MS. We used HBV infection in the presence of the entry inhibitor bulevirtide as a control to separate the effects of productive infection from those caused by inoculum-associated components. Despite robust infection, active HBV replication induced only subtle changes in host protein levels. Orthogonal validation of MS-identified candidates confirmed reticulocalbin-2 as a novel host factor downregulated during productive HBV infection. The functional role of candidate proteins identified by MS was assessed in vitro by siRNA-mediated knockdown and measurement of viral replication markers. Knockdown had no impact on viral RNA or antigen levels, suggesting that the observed proteomic changes may reflect stress responses or broader modulation of the hepatic microenvironment. Our findings support the concept of HBV as a stealth virus and underscore the importance of carefully controlled experimental systems for studying host responses to infection in vitro.