Hepatitis B Virus X Protein Is a Transcriptional Modulator That Communicates with Transcription Factor IIB and the RNA Polymerase II Subunit 5

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-transferaseresin 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.

Hepatitis B virus replication in human HepG2 cells mediated by hepatitis B virus recombinant baculovirus

“…49 At the level of promoter function, transient expression of HBV genetic information in HepG2 cells has been used to evaluate the ability of various cytokines alone and in combination to alter the function of the HBV core/pregenome promoter, 47 to study the functional interaction of specific transactivation factors and cellular binding proteins with elements within the X promoter, 42 and to demonstrate that HBV X protein may facilitate transcriptional initiation of cellular and viral genes by stabilizing the association between RNA polymerase and TFIIB. 43 The HBV baculovirus infected HepG2 cell system described in the current study represents a new method for using HepG2 cells to study HBV. Indeed, the HBV baculovirus system, like 2.2.15 cells, can be used to evaluate the effect of nucleosides, cytokines, and/or novel drugs on HBV replication.…”

“…14,[40][41][42][43][44][45][46][47][48][49] In this study, we report the development of a novel transient mechanism for studying HBV in the well differentiated human hepatoblastoma cell line HepG2. We conclude the following from our studies:…”

“…Some studies involve using greater than genome length HBV DNA sequences so that all HBV gene products can be produced 14,40,45,46,48,49 whereas others have concentrated on using HBV DNA sequences which encode restricted portions of the coding regions of the genome or enhancer or promoter sequences. 14,[40][41][42][43][44][45][46][47][48][49] Recently, transient transfection of HepG2 cells has been used to understand the function of the precore gene and HBeAg. Specifically, studies have been carried out to determine the following: 1) whether preC and pregenomic RNAs are regulated by one promoter or by two separate promoters 44 ; 2) to study the effect of mutation in the precore region on production of HBeAg and on core protein 41,45,46 ; and 3) to determine the role played by the precore gene in HBV replication.…”

The hepatitis B virus X protein up-regulates tumor necrosis factor α gene expression in hepatocytes

“…37,45 HBx has been shown to possess a dual function, acting at both the nuclear and the cytoplasmic compartments. 46 The nuclear function of HBx appears to be mediated by the physical association of HBx with proteins of the transcriptional machinery 47,48 and with certain transcription factors such as CREB/ATF. 6 The cytoplasmic function of HBx involves the activation of the Ras/Raf/ ERK 49,50 and the MEKK-1/JNK 50 signalling cascades, which in turn are implicated in the activation of certain transcription factors such as AP-1 and NF-B.…”

Cloning, soluble expression, and purification of the RNA polymerase II subunit RPB5 fromSaccharomyces cerevisiae

“…7 Few studies also showed that transcriptional transactivator protein X encoded by the human hepatitis virus may specifically interact with RPB5 to stimulate transcription in virally infected cells. 5 with these specific partners do not account for the fact that RPB5 is shared by all 3 yeast RNAPs. 2,10 Earlier studies also proved the close relation of human RPB5 to the archaeal subunit H. 11 and to viral RPB5-like subunits.…”