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. 2001 May;75(9):4453-8.
doi: 10.1128/JVI.75.9.4453-4458.2001.

Inhibition of host transcription by vesicular stomatitis virus involves a novel mechanism that is independent of phosphorylation of TATA-binding protein (TBP) or association of TBP with TBP-associated factor subunits

H Yuan  1 , S Puckett, D S Lyles
Affiliations

Inhibition of host transcription by vesicular stomatitis virus involves a novel mechanism that is independent of phosphorylation of TATA-binding protein (TBP) or association of TBP with TBP-associated factor subunits

H Yuan et al. J Virol. 2001 May.

Abstract

The matrix (M) protein of vesicular stomatitis virus (VSV) is a potent inhibitor in vivo of transcription by all three host RNA polymerases (RNAP). In the case of host RNA polymerase II (RNAPII), the inhibition is due to lack of activity of the TATA-binding protein (TBP), which is a subunit of the basal transcription factor TFIID. Despite the potency of M protein-induced inhibition in vivo, experiments presented here show that M protein cannot directly inactivate TFIID in vitro. Addition of M protein to nuclear extracts from uninfected cells did not inhibit transcription activity, indicating that the inhibition is indirect and is mediated through host factors. The host factors that are known to regulate TBP activity include phosphorylation by host kinases and association with different TBP-associated factor (TAF) subunits. However, TBP in VSV-infected cells was found to be assembled normally with its TAF subunits, as shown by ion exchange high-pressure liquid chromatography and sedimentation velocity analysis. A normal pattern of phosphorylation of TBP in VSV-infected cells was also observed by pH gradient gel electrophoresis. Collectively, these data indicate that M protein inactivates TBP activity in RNAPII-dependent transcription by a novel mechanism, since the known mechanisms for regulating TBP activity cannot account for the inhibition.

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Figures

FIG. 1
FIG. 1
Effect of M protein on transcription by nuclear extracts in vitro. (A) Nuclear extracts from uninfected or VSV-infected cells prepared at 6 h postinfection were mixed in the indicated amounts and incubated in an in vitro transcription reaction with a plasmid DNA template containing the adenovirus MLP. The transcription products were analyzed by an RNase protection assay using a radiolabeled riboprobe, an autoradiograph of which is shown, and quantitated by densitometry as indicated. The position of the MLP-initiated product is indicated. (B) Nuclear extracts were prepared from VSV-infected HeLa cells at 3, 6, and 9 h postinfection. The amount of M protein in the nuclear extracts was determined by Western blot, using the indicated amounts of purified VSV as a standard. (C) Nuclear extract from uninfected cells was mixed with M protein purified from VSV virions by ion exchange chromatography (lane 3). Nuclear extract with no additions (lane 1) or with column buffer used to elute M protein (lane 2) were used as controls. Transcription in vitro was assayed as in panel A. (D) Nuclear extract from uninfected cells was mixed with in vitro translation reactions containing M protein (lanes 2 to 4) or N protein (lanes 5 to 7) or with no additions (lane 1). In vitro transcription reactions contained 8 μl of nuclear extract and 1 μl (lanes 2 and 5), 2.5 μl (lanes 3 and 6), or 5.5 μl (lanes 4 and 7) of translation mixture in a total volume of 25 μl.
FIG. 2
FIG. 2
Ion exchange HPLC of TBP-containing transcription factors. Nuclear extracts were prepared from VSV-infected or uninfected HeLa cells at 6 h postinfection and chromatographed on an ion exchange HPLC column eluted with a NaCl concentration gradient. (A) Column fractions were assayed for TBP content by Western blot. (B) Densitometry of the Western blots in panel A was used to determine the percent of total TBP in each fraction for nuclear extracts from infected (dark bars) or uninfected cells (light bars). The concentration gradient of NaCl used to elute the column is also shown.
FIG. 3
FIG. 3
Sedimentation analysis of TBP-containing transcription factors. Nuclear extracts were prepared from VSV-infected (dark bars) or uninfected (light bars) HeLa cells at 6 h postinfection and sedimented on 10 to 30% sucrose gradients. Gradient fractions and the pelleted material (P) were assayed for TBP content by Western blots as in Fig. 2, and the percentage of total TBP in each fraction was determined by densitometry.
FIG. 4
FIG. 4
Analysis of TBP phosphorylation by pH gradient gel electrophoresis. Nuclear extracts were prepared from VSV-infected (I) or uninfected (U) HeLa cells at 6 h postinfection and chromatographed on an ion exchange HPLC column eluted with a NaCl concentration gradient as in Fig. 2. Column fractions were combined into pool 1 (fractions 17 through 19), pool 2 (fractions 21 through 23), and pool 3 (fractions 24 through 26) and electrophoresed on a pH gradient gel. TBP was detected by Western blot and migrated as three forms, with TBP1 being the fastest migrating form and TBP3 the slowest.
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References

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