Abstract
Hepatitis B virus X protein (HBx) transactivates viral and cellular genes through a wide variety of cis-elements. However, the mechanism is still obscure. Our finding that HBx directly interacts with RNA polymerase II subunit 5 (RPB5), a common subunit of RNA polymerases, implies that HBx directly modulates the function of RNA polymerase (Cheong, J. H., Yi, M., Lin, Y., and Murakami, S. (1995) EMBO J. 14, 142-150). In this context, we examined the possibility that HBx and RPB5 interact with other general transcription factors. HBx and RPB5 specifically bound to transcription factor IIB (TFIIB) in vitro, both of which were detected by either far-Western blotting or the glutathione S-transferase-resin pull-down assay. Delineation of the binding regions of these three proteins revealed that HBx, RPB5, and TFIIB each has two binding regions for the other two proteins. Co-immunoprecipitation using HepG2 cell lysates that express HBx demonstrated trimeric interaction in vivo. Some HBx substitution mutants, which had severely impaired transacting activity, exhibited reduced binding affinity with either TFIIB or RPB5 in a mutually exclusive manner, suggesting that HBx transactivation requires the interactions of both RPB5 and TFIIB. These results indicated that HBx is a novel virus modulator that facilitates transcriptional initiation by stabilizing the association between RNA polymerase and TFIIB through communication with RPB5 and TFIIB.
Highlights
Human hepatitis B virus (HBV)1 is one major risk factor associated with primary hepatocellular carcinoma
Hepatitis B virus X protein (HBx), RNA polymerase II subunit 5 (RPB5), and transcription factor IIB (TFIIB) Associate with Each Other in Vitro and in Vivo—Specific HBx-RPB5 interaction is involved in HBx transactivation, suggesting that RPB5, as a communicating subunit of RNA polymerase, interacts with general transcription factors [18]
Our results imply that trimeric interaction or complex formation is required for HBx transactivation, since the HBx mutations, which reduced the binding ability either to TFIIB or RPB5, markedly impaired transactivation activity
Summary
Human hepatitis B virus (HBV)1 is one major risk factor associated with primary hepatocellular carcinoma. The bound proteins were eluted with elution buffer (10 mM Tris-HCl, pH 7.4, 2% SDS, 0.3 M NaCl, and 1 mM EDTA), separated by SDS-PAGE, transferred onto nitrocellulose membranes, and Western blotted using second antibodies to detect HBx, TFIIB, or RPB5. We investigated whether or not RPB5 interacts with other general transcription factors by means of far-Western blotting using bacterially expressed GST fusion forms of TBP, TFIIB, and CTD (Fig. 1A).
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