# HBV p22-interacting protein C1QBP inhibits viral replication through impeding nucleocapsid formation and nuclear import

## TL;DR

A host protein called C1QBP inhibits hepatitis B virus replication by targeting viral proteins for degradation and blocking DNA import into the nucleus.

## Contribution

C1QBP is identified as a novel inhibitor of HBV replication through dual mechanisms involving autolysosomal degradation and nuclear import blockade.

## Key findings

- C1QBP interacts with p22 and promotes autolysosomal degradation of the HBV core protein.
- C1QBP blocks DP-rcDNA nuclear import by binding to the capsid, reducing cccDNA formation.
- A mitochondrial-targeting-signal-lacking C1QBP mutant shows enhanced antiviral activity.

## Abstract

The circulating hepatitis B virus (HBV) e antigen (HBeAg) is known to subvert the host immune system to benefit chronic HBV infection. However, the biological function of a major intracellular form of HBeAg, specifically the precore protein intermediate of 22 kDa (p22) lacking the N-terminal signal peptide, remains largely unclear. Through pull-down and mass spectrometry analysis, we re-identified the complement C1q binding protein (C1QBP) as a p22-binding protein. Immunofluorescence results demonstrated that C1QBP was predominantly localized in the mitochondrial matrix and the leaked C1QBP interacted with p22 in the cytosol. Using co-immunoprecipitation assay, we mapped the arginine-rich, highly positively charged C-terminal domain (CTD) of p22 and the internal domain aa 74–160 of C1QBP as binding domains for p22-C1QBP interaction. By studying the impact of C1QBP on HBV replication, we found that C1QBP overexpression led to the autolysosomal degradation of HBV core protein (HBc) and significantly reduced viral nucleocapsid formation in a p22-dependent manner. Additionally, a C1QBP mutant without the mitochondrial targeting signal (MTS) exhibited a greater inhibitory effect on HBV replication compared to the wild type (wt). Although HBc and p22 share the same CTD sequence, C1QBP does not bind to wt HBV capsid. However, disrupting capsid assembly by HBc-Y132A mutant or CAM-A (class A capsid assembly modulator) treatment enables HBc-C1QBP interaction. Moreover, C1QBP binds to the CTD of HBc on the cytoplasmic deproteinated relaxed circular DNA (DP-rcDNA)-containing capsid that is partially disassembled, hindering DP-rcDNA nuclear import and subsequent covalently closed circular DNA (cccDNA) formation. Collectively, our study suggests that C1QBP inhibits HBV replication through dual mechanisms, proposing a novel therapeutic approach for managing chronic HBV infection.

Hepatitis B virus (HBV) remains a major global public health burden, causing chronic liver infections that can lead to severe diseases such as cirrhosis and liver cancer. HBV produces only seven proteins, among which the biological function of a major intracellular precursor of the circulating viral e antigen (HBeAg), namely p22, remains poorly understood. In our study, we re-identified a host protein, the complement C1q binding protein (C1QBP) leaked from mitochondria, as an intracellular binding partner of p22. Further investigation revealed two mechanisms by which C1QBP regulates HBV replication: first, C1QBP promotes the autolysosomal degradation of the viral core protein in a p22-dependent manner; second, C1QBP blocks the nuclear import of nucleocapsid by competitively binding to the nuclear localization signal of viral capsid protein exposed on the nucleocapsid containing deproteinated mature viral DNA genome, subsequently reducing the production of viral cccDNA in the nucleus. Our findings not only shed light on the virus-host interaction between p22 and C1QBP but also indicate the potential of C1QBP as an antiviral strategy for treating hepatitis B.