Abstract
Hepatitis B virus infection is associated with hepatocellular carcinoma, claiming 1 million lives annually worldwide. To understand the carcinogenic mechanism of hepatitis B virus-encoded oncoprotein HBx, we explored the function of HBx interaction with its cellular target HBXIP. Previously, we demonstrated that viral HBx and cellular HBXIP control mitotic spindle formation, regulating centrosome splitting. By using various fragments of HBx, we determined that residues (137)CRHK(140) within HBx are necessary for binding HBXIP. Mutation of the (137)CRHK(140) motif in HBx abolished its ability to bind HBXIP and to dysregulate centrosome dynamics in HeLa and immortal diploid RPE-1 cells. Unlike wild-type HBx, which targets to centrosomes as determined by subcellular fractionation and immunofluorescence microscopy, HBx mutants failed to localize to centrosomes. Overexpression of viral HBx wild-type protein and knockdown of endogenous HBXIP altered centrosome assembly and induced modifications of pericentrin and centrin-2, two essential proteins required for centrosome formation and function, whereas HBXIP nonbinding mutants of HBx did not. Overexpression of HBXIP or fragments of HBXIP that bind HBx neutralized the effects of viral HBx on centrosome dynamics and spindle formation. These results suggest that HBXIP is a critical target of viral HBx for promoting genetic instability through formation of defective spindles and subsequent aberrant chromosome segregation.
Highlights
Hepatocellular carcinoma [1,2,3]
HBXIP has been identified as an adaptor for Survivin, a BIR family chromosomal passenger protein involved in controlling apoptosis and cell division [7], with HBXIP collaborating with cytosolic Survivin to suppress apoptosis [8]
HBx Dysregulates HBXIP Control of Mitotic Spindle Formation interaction with HBXIP performing a mutagenesis analysis to identify the domains within cellular HBXIP and viral HBx viral proteins required for their interactions
Summary
Hepatocellular carcinoma [1,2,3]. HBV-associated hepatocellular carcinoma kills over 1 million people annually, ranking it among the most lethal cancers. To test the effects of HBXIP and HBXIP-(1– 64) on spindle formation, HeLa cells were fixed at 48 h after transfection with plasmids encoding HBx WT viral protein, in combination with FLAGtagged HBXIP full-length FLAG-HBXIP-(1– 64) fragment, or pcFLAG vector and stained with anti-␣-tubulin (green) antipericentrin (red) antibodies, and with DNA-binding fluorochrome (DAPI).
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