HBxAP/Rsf-1-mediated HBxhBubR1 interactions regulate the mitotic spindle checkpoint and chromosome instability

Hepatitis B virus (HBV) X protein (HBx), encoded by the HBV genome, is involved in the development of HBV-mediated liver cancer, whose frequency is highly correlated with chromosomal instability (CIN). We reported previously that HBx induces mitotic checkpoint dysfunction by targeting the human serine/threonine kinase BubR1 (hBubR1). However, the underlying mechanism remained unresolved. Here, we show that HBx protein-associated protein (HBxAP)/Rsf-1 associates with hBubR1 and HBx in the chromatin fraction during mitosis. Depletion of HBxAP/Rsf-1 abolished the interaction between HBx and hBubR1, indicating that HBxAP/Rsf-1 mediates these interactions. Knockdown of HBxAP/Rsf-1 with small interfering RNA did not affect the recruitment of hBubR1 to kinetochores; however, it did significantly impair HBx targeting to kinetochores. A deletion mutant analysis revealed that two Kunitz domains of HBx, the Cdc20-binding domain of hBubR1 and full-length of HBxAP/Rsf-1 were essential for these interactions. Thus, binding of HBx to hBubR1, stabilized by HBxAP/Rsf-1, significantly attenuated hBubR1 binding to Cdc20 and consequently increased the rate of mitotic aberrations. Collectively, our data show that the HBx impairs hBubR1 function and induces CIN through HBxAP/Rsf-1, providing a novel mechanism for induction of genomic instability by a viral pathogen in hepatocarcinogenesis.