Hepatitis B Virus pX Targets TFIIB in Transcription Coactivation

ABSTRACT

pX, the hepatitis B virus (HBV)-encoded regulator, coactivates transcription through an unknown mechanism. pX interacts with several components of the transcription machinery, including certain activators, TFIIB, TFIIH, and the RNA polymerase II (POLII) enzyme. We show that pX localizes in the nucleus and coimmunoprecipitates with TFIIB from nuclear extracts. We used TFIIB mutants inactive in binding either POLII or TATA binding protein to study the role of TFIIB-pX interaction in transcription coactivation. pX was able to bind the former type of TFIIB mutant and not the latter. Neither of these sets of TFIIB mutants supports transcription. Remarkably, the latter TFIIB mutants fully block pX activity, suggesting the role of TFIIB in pX-mediated coactivation. By contrast, in the presence of pX, TFIIB mutants with disrupted POLII binding acquire the wild-type phenotype, both in vivo and in vitro. These results suggest that pX may establish the otherwise inefficient TFIIB mutant-POLII interaction, by acting as a molecular bridge. Collectively, our results demonstrate that TFIIB is the in vivo target of pX.

ACKNOWLEDGMENTS

We thank D. Reinberg for the TFIIB mutant expression vectors, L. Runkel and H. Schaller for the X two-codon insertion mutants, and E. Schejter and D. Vaizel-Ohayon for help and advice in indirect immunofluorescent staining. We thank S. Budilovski for technical assistance.

This work was supported by the Leo and Julia Forcheimer Center for Molecular Genetics.