Hepatitis B virus X protein induces RNA polymerase III-dependent gene transcription and increases cellular TATA-binding protein by activating the Ras signaling pathway.

Our previous studies have shown that the hepatitis B virus protein, X, activates all three classes of RNA polymerase III (pol III)-dependent promoters by increasing the cellular level of TATA-binding protein (TBP) (H.-D. Wang et al., Mol. Cell. Biol. 15:6720-6728, 1995), a limiting transcription component (A. Trivedi et al., Mol. Cell. Biol. 16:6909-6916, 1996). We have investigated whether these X-mediated events are dependent on the activation of the Ras/Raf-1 signaling pathway. Transient expression of a dominant-negative mutant Ras gene (Ras-ala15) in a Drosophila S-2 stable cell line expressing X (X-S2), or incubation of the cells with a Ras farnesylation inhibitor, specifically blocked both the X-dependent activation of a cotransfected tRNA gene and the increase in cellular TBP levels. Transient expression of a constitutively activated form of Ras (Ras-val12) in control S2 cells produced both an increase in tRNA gene transcription and an increase in cellular TBP levels. These events are not cell type specific since X-mediated gene induction was also shown to be dependent on Ras activation in a stable rat 1A cell line expressing X. Furthermore, increases in RNA pol III-dependent gene activity and TBP levels could be restored in X-S2 cells expressing Ras-ala15 by coexpressing a constitutively activated form of Raf-1. These events are serum dependent, and when the cells are serum deprived, the X-mediated effects are augmented. Together, these results demonstrate that the X-mediated induction of RNA pol III-dependent genes and increase in TBP are both dependent on the activation of the Ras/Raf-1 signaling cascade. In addition, these studies define two new and important consequences mediated by the activation of the Ras signal transduction pathway: an increase in the central transcription factor, TBP, and the induction of RNA pol III-dependent gene activity.

Download Full-text

The hepatitis B virus X protein increases the cellular level of TATA-binding protein, which mediates transactivation of RNA polymerase III genes.

Molecular and Cellular Biology ◽

10.1128/mcb.15.12.6720 ◽

1995 ◽

Vol 15 (12) ◽

pp. 6720-6728 ◽

Cited By ~ 43

Author(s):

H D Wang ◽

C H Yuh ◽

C V Dang ◽

D L Johnson

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Rna Polymerase ◽

Transient Expression ◽

Binding Protein ◽

Tata Binding Protein ◽

Transformed Cells ◽

B Virus ◽

X Gene ◽

Pol Iii

The hepatitis B virus X gene product transactivates a variety of cellular and viral genes. The mechanism for X induction of RNA polymerase (pol) III genes was investigated. By using Drosophila S-2 cells stably transformed with the X gene, the transient expression of a tRNA gene is enhanced. Comparing the transcriptional activities of extracts derived from these cells, all three types of RNA pol III promoters are stimulated by X. Interestingly, both S-2 and rat 1A cells stably transformed with the X gene produce increased cellular levels of the TATA-binding protein (TBP). By using various kinase inhibitors, it was found that the X-mediated increases in both transcription and TBP are dependent upon protein kinase C activation. Since TBP is a subunit of TFIIIB, the activity of this component fractionated from extracts derived from control and X-transformed cells was analyzed. These studies reveal that TFIIIB activity is substantially more limiting in control cells and that TFIIIB isolated from X-transformed cells has increased activity in reconstitution assays compared with TFIIIB isolated from control cells. Conversely, comparison of TFIIIC from control and X-transformed cell extracts revealed that there is relatively little change in its ability either to reconstitute transcription or to bind to DNA and that there is no change in the catalytic activity of RNA pol III. Studies were performed to determine whether directly increasing cellular TBP alone could enhance RNA pol III gene transcription. Transient expression of a TBP cDNA in rat 1A cells was capable of stimulating transcription activity from the resultant extracts in vitro. Together, these results demonstrate that one mechanism by which X mediates transactivation of RNA poll III genes is by increasing limiting TBP via the activation of cellular signaling pathways. The discovery that X increases cellular TBP, the universal transcription factor, provides a novel mechanism for the function of a viral transactivator protein and may explain the ability of X to produce such large and diverse effects on cellular gene expression.

Download Full-text

Hepatitis B virus encodes an RNA polymerase III transcript

Molecular and Cellular Biology ◽

10.1128/mcb.3.10.1774-1782.1983 ◽

1983 ◽

Vol 3 (10) ◽

pp. 1774-1782

Author(s):

D N Standring ◽

L B Rall ◽

O Laub ◽

W J Rutter

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Rna Polymerase ◽

Rna Polymerase Iii ◽

Core Antigen ◽

Transcription System ◽

Polymerase Iii ◽

B Virus ◽

Sequence Elements

We demonstrated that cloned hepatitis B virus (HBV) DNA directs the synthesis of a 700-base RNA (HBV 700) by RNA polymerase III in a cell-free transcription system. HBV 700 is the only transcript known to originate from the viral short strand and has been mapped to the region between roughly 1,635 and 954 base pairs on the viral map, between the surface and core antigen coding sequences but overlapping and opposing the putative DNA polymerase and B protein genes. The in vitro initiation sites for the HBV 700 and core antigen RNAs are only 50 bases apart, suggesting that these two genes may be coordinately regulated. Moreover, both of these initiation sites appear to lie within the approximately 300-base double-stranded region (the nick region) between the 5' end of the HBV short strand and the nick in the viral long strand. We found two unusual sequence elements in the nick region that are conserved between the human and woodchuck viruses.

Download Full-text

Regulation of RNA Polymerase I-Dependent Promoters by the Hepatitis B Virus X Protein via Activated Ras and TATA-Binding Protein

Molecular and Cellular Biology ◽

10.1128/mcb.18.12.7086 ◽

1998 ◽

Vol 18 (12) ◽

pp. 7086-7094 ◽

Cited By ~ 62

Author(s):

Horng-Dar Wang ◽

Alpa Trivedi ◽

Deborah L. Johnson

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Promoter Activity ◽

Tata Binding Protein ◽

S2 Cells ◽

X Protein ◽

Rna Pol Ii ◽

Pol Ii ◽

Pol I ◽

B Virus

ABSTRACT The hepatitis B virus (HBV) X protein is essential for viral infectivity, and evidence indicates that it is a strong contributor to HBV-mediated oncogenesis. X has been shown to transactivate a wide variety of RNA polymerase (Pol) II-dependent, as well as RNA Pol III-dependent, promoters. In this study, we have investigated the possibility that X modulates RNA Pol I-dependent rRNA transcription. In both human hepatoma Huh7 and Drosophila Schneider S2 cell lines, X expression stimulated rRNA promoter activity. Extracts prepared from X-expressing cells stably transfected with anX gene also exhibited an increased ability to transcribe the rRNA promoter. The mechanism for X transactivation was examined by determining whether this regulatory event was dependent on Ras activation and increased TATA-binding protein (TBP) levels. Our previous studies have demonstrated that X, and the activation of Ras, produces an increase in the cellular levels of TBP (H.-D. Wang, A. Trivedi, and D. L. Johnson, Mol. Cell. Biol. 17:6838–6846, 1997). Expression of a dominant negative form of Ras blocked the X-mediated induction of the rRNA promoters, whereas expression of a constitutively activated form of Ras mimicked the enhancing effect of X on rRNA promoter activity. When TBP was overexpressed in either Huh7 or S2 cells, a dose-dependent increase in rRNA promoter activity was observed. To analyze whether the increase in TBP was modulating rRNA promoter activity indirectly, by increasing activity of RNA Pol II-dependent promoters, a Drosophila TBP cDNA was constructed with a mutation that eliminated its ability to stimulate RNA Pol II-dependent promoters. Transient expression of wild-type TBP in S2 cells increased the activities of specific RNA Pol I- and Pol II-dependent promoters. Expression of the mutant TBP protein failed to enhance the activity of the RNA Pol II-dependent promoters, yet the protein completely retained its ability to stimulate the rRNA promoter. Furthermore, the addition of recombinant TBP to S2 extracts stimulated rRNA promoter activity in vitro. Together, these results demonstrate that the HBV X protein up-regulates RNA Pol I-dependent promoters via a Ras-activated pathway in two distinct cell lines. The enhanced promoter activity can, at least in part, be attributed to the X- and Ras-mediated increase in cellular TBP, a limiting transcription component.

Download Full-text

Hepatitis B virus encodes an RNA polymerase III transcript.

Molecular and Cellular Biology ◽

10.1128/mcb.3.10.1774 ◽

1983 ◽

Vol 3 (10) ◽

pp. 1774-1782 ◽

Cited By ~ 9

Author(s):

D N Standring ◽

L B Rall ◽

O Laub ◽

W J Rutter

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Rna Polymerase ◽

Rna Polymerase Iii ◽

Core Antigen ◽

Transcription System ◽

Polymerase Iii ◽

B Virus ◽

Sequence Elements

Download Full-text

Strategies to Inhibit Hepatitis B Virus at the Transcript Level

Viruses ◽

10.3390/v13071327 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1327

Author(s):

Bingqian Qu ◽

Richard J. P. Brown

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Gene Transcription ◽

Antigen Expression ◽

Transcript Level ◽

Viral Gene ◽

Small Interfering Rnas ◽

Viral Gene Transcription ◽

Hbv Vaccination ◽

B Virus

Approximately 240 million people are chronically infected with hepatitis B virus (HBV), despite four decades of effective HBV vaccination. During chronic infection, HBV forms two distinct templates responsible for viral transcription: (1) episomal covalently closed circular (ccc)DNA and (2) host genome-integrated viral templates. Multiple ubiquitous and liver-specific transcription factors are recruited onto these templates and modulate viral gene transcription. This review details the latest developments in antivirals that inhibit HBV gene transcription or destabilize viral transcripts. Notably, nuclear receptor agonists exhibit potent inhibition of viral gene transcription from cccDNA. Small molecule inhibitors repress HBV X protein-mediated transcription from cccDNA, while small interfering RNAs and single-stranded oligonucleotides result in transcript degradation from both cccDNA and integrated templates. These antivirals mediate their effects by reducing viral transcripts abundance, some leading to a loss of surface antigen expression, and they can potentially be added to the arsenal of drugs with demonstrable anti-HBV activity. Thus, these candidates deserve special attention for future repurposing or further development as anti-HBV therapeutics.

Download Full-text

RMP, a Novel RNA Polymerase II Subunit 5-Interacting Protein, Counteracts Transactivation by Hepatitis B Virus X Protein

Molecular and Cellular Biology ◽

10.1128/mcb.18.12.7546 ◽

1998 ◽

Vol 18 (12) ◽

pp. 7546-7555 ◽

Cited By ~ 65

Author(s):

Dorjbal Dorjsuren ◽

Yong Lin ◽

Wenxiang Wei ◽

Tatsuya Yamashita ◽

Takahiro Nomura ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Binding Protein ◽

Host Protein ◽

Dependent Manner ◽

Binding Region ◽

X Protein ◽

B Virus

ABSTRACT To modulate transcription, regulatory factors communicate with basal transcription factors and/or RNA polymerases in a variety of ways. Previously, it has been reported that RNA polymerase II subunit 5 (RPB5) is one of the targets of hepatitis B virus X protein (HBx) and that both HBx and RPB5 specifically interact with general transcription factor IIB (TFIIB), implying that RPB5 is one of the communicating subunits of RNA polymerase II involved in transcriptional regulation. In this context, we screened for a host protein(s) that interacts with RPB5. By far-Western blot screening, we cloned a novel gene encoding a 508-amino-acid-residue RPB5-binding protein from a HepG2 cDNA library and designated it RPB5-mediating protein (RMP). Expression of RMP mRNA was detected ubiquitously in various tissues. Bacterially expressed recombinant RMP strongly bound RPB5 but neither HBx nor TATA-binding protein in vitro. Endogenous RMP was immunologically detected interacting with assembled RPB5 in RNA polymerase in mammalian cells. The central part of RMP is responsible for RPB5 binding, and the RMP-binding region covers both the TFIIB- and HBx-binding sites of RPB5. Overexpression of RMP, but not mutant RMP lacking the RPB5-binding region, inhibited HBx transactivation of reporters with different HBx-responsive cis elements in transiently transfected cells. The repression by RMP was counteracted by HBx in a dose-dependent manner. Furthermore, RMP has an inhibitory effect on transcriptional activation by VP16 in the absence of HBx. These results suggest that RMP negatively modulates RNA polymerase II function by binding to RPB5 and that HBx counteracts the negative role of RMP on transcription indirectly by interacting with RPB5.

Download Full-text

Transcription of hepatitis B virus by RNA polymerase II.

Molecular and Cellular Biology ◽

10.1128/mcb.3.10.1766 ◽

1983 ◽

Vol 3 (10) ◽

pp. 1766-1773 ◽

Cited By ~ 51

Author(s):

L B Rall ◽

D N Standring ◽

O Laub ◽

W J Rutter

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Simian Virus 40 ◽

Virus Genome ◽

Simian Virus ◽

Free System ◽

B Virus

We employed an in vitro cell-free transcription system to locate RNA polymerase II promoters on the hepatitis B virus genome. The strongest promoter precedes the surface antigen (HBsAg) gene, which is comprised of a long (500 base pairs) presurface region as well as the mature HBsAg coding sequence. The origin of this transcript was localized by using truncated templates and S1 endonuclease mapping. The activity of the promoter was confirmed in transfection experiments in which the complete HBsAg gene was introduced into monkey kidney cells via a simian virus 40 expression vector. A second RNA polymerase II promoter preceding the HBcAg gene was also active in the cell-free system. The presence of multiple promoters in the hepatitis B virus genome suggests that the relative levels of viral-specific proteins detected in liver and serum may reflect differential or regulated promoter efficiency.

Download Full-text

Relative Affinities of Nucleotide Substrates for the Yeast tRNA Gene Transcription Complex

Zeitschrift für Naturforschung C ◽

10.1515/znc-1992-3-426 ◽

1992 ◽

Vol 47 (3-4) ◽

pp. 320-322 ◽

Cited By ~ 1

Author(s):

Przemyslaw Szafranski ◽

W. Jerzy Smagowicz

Keyword(s):

Rna Polymerase ◽

Gene Transcription ◽

Trna Gene ◽

Rna Polymerase Iii ◽

Rna Polymerases ◽

Yeast Trna ◽

Polymerase Iii ◽

Michaelis Constants ◽

Auxiliary Protein

Abstract Apparent Michaelis constants for nucleotides in transcription of yeast tRN Agene by hom ologous RNA polymerase III with auxiliary protein factors, were found to be remarkably higher in initiation than in elongation of RNA chain. This supports presumptions regarding topological similarities between catalytic centers of bacterial and eukaryotic RNA polymerases.

Download Full-text