Direct Association and Nuclear Import of the Hepatitis B Virus X Protein with the NF-κB Inhibitor IκBα

Abstract

The X protein of hepatitis B virus (HBV) is a transcriptional activator which is required for infection and may play an important role in HBV-associated hepatocarcinogenesis. It has been suggested that X acts as a nuclear coactivator or stimulates several signal transduction pathways by acting in the cytoplasm. One of these pathways leads to the nuclear translocation of NF-κB. A recent report indicates that X activates NF-κB by acting on two cytoplasmic inhibitors of this family of transcription factors: IκBα and the precursor/inhibitor p105. We demonstrate here that X directly interacts with IκBα, which is able to transport it to the nucleus by a piggyback mechanism. This transport requires a region of IκBα (the second ankyrin repeat) which has been demonstrated to be involved in its nuclear import following NF-κB activation. Using deletion mutants, we showed that amino acids 249 to 253 of IκBα (located in the C-terminal part of the sixth ankyrin repeat) play a critical role in the interaction with X. This small region overlaps one of the domains of IκBα mediating the interaction with the p50 and p65 subunits of NF-κB and is also close to the nuclear export sequence of IκBα, therefore providing a potential explanation for the nuclear accumulation of IκBα with X. This association can also be observed upon the induction of endogenous IκBα by tumor necrosis factor alpha (TNF-α) treatment of Chang cells expressing X. In accordance with this observation, band shift analysis indicates that X induces a sustained NF-κB activation following TNF-α treatment, probably by preventing the reassociation of newly synthesized nuclear IκBα with DNA-bound NF-κB complexes.

ACKNOWLEDGMENTS

R.W. and H.S. contributed equally to this work.

We thank S. Whiteside for the gift of the C-terminal-deletion mutants of IκBα. We are grateful to R. Hellio for technical assistance with confocal microscopy. We thank S. Urban for the gift of anti-X antibody. We thank M. Hannink for the gift of the IκBα-110A3 mutant.

This research was sponsored in part by grants from ARC, ANRS, and the Ligue Nationale contre le Cancer and by Biomed contract 97-2567 and TMR contract 960026 to A.I.; grants from INSERM, EU, ARC, the Ligue Nationale contre le Cancer, and CNAM to C.B.; and grants from ARC and the Ligue Nationale contre le Cancer to C.D. R.W. is a recipient of a long-term fellowship from SIDACTION. H.S. is a recipient of a fellowship from DFG. C.G. is a recipient of a fellowship from FRM.