Homeobox protein MSX-1 restricts hepatitis B virus by promoting ubiquitin-independent proteasomal degradation of HBx protein

Hepatitis B virus (HBV) X protein (HBx) is a key factor for regulating viral transcription and replication. We recently characterized homeobox protein MSX-1 (MSX1) as a host restriction factor that inhibits HBV gene expression and genome replication by directly binding to HBV enhancer II/core promoter (EnII/Cp) and suppressing its promoter and enhancer activities. Notably, HBx expression was observed to be repressed more drastically by MSX1 compared to other viral antigens. In this work, we report that in addition to transcriptional repression, MSX1 also post-transcriptionally downregulates HBx protein stability. Mechanistically, MSX1 induces ubiquitin-independent proteasomal degradation of HBx, which is mediated through HBx C-terminal domain. Furthermore, this effect on HBx degradation correlates with MSX1-induced upregulation of DNAJA4 and CRYAB expression. Similar to MSX1, both DNAJA4 and CRYAB promote HBx degradation and repress HBV gene expression and genome replication. In chronic hepatitis B (CHB) patients, immune active phase (IA) is associated with higher intrahepatic expression of MSX1, DNAJA4 and CRYAB, and lower serum HBV markers compared to immune tolerant (IT) phase. Finally, HBV infection is significantly suppressed by MSX1 overexpression in both NTCP-overexpressing cell and humanized liver mouse models. These results demonstrate additional and novel mechanisms of MSX1-mediated repression of HBV, and establish MSX1 as a multi-functional HBV restriction factor with therapeutic potential.