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. 1998 Aug;72(8):6880-3.
doi: 10.1128/JVI.72.8.6880-6883.1998.

A new vaccinia virus intermediate transcription factor

P Sanz  1 , B Moss
Affiliations

A new vaccinia virus intermediate transcription factor

P Sanz et al. J Virol. 1998 Aug.

Abstract

Transcription of the vaccinia virus genome is mediated by a virus-encoded multisubunit DNA-dependent RNA polymerase in conjunction with early-, intermediate-, and late-stage-specific factors. Previous studies indicated that two virus-encoded proteins (capping enzyme and VITF-1) and one unidentified cellular protein (VITF-2) are required for specific transcription of an intermediate promoter template in vitro. We have now extensively purified an additional virus-induced intermediate transcription factor with a native mass of approximately 100 kDa.

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Figures

FIG. 1
FIG. 1
Evidence for a new intermediate transcription factor. In vitro transcription was carried out as previously described (13) for 30 min at 37°C in a total volume of 20 μl with 0.1 μg of uncleaved plasmid (containing the G8R intermediate promoter followed by a template lacking G residues), ribonucleoside triphosphates including [α-32P]UTP, and additional protein components as indicated in the figure. The RNA was analyzed on a 4% polyacrylamide gel, which was then dried and autoradiographed. Symbols: −, no addition; +, 1× addition; ++, 10× addition. (A) Protein components of the reaction mixture were the 0.15 M NaCl fraction from the second DEAE-cellulose column (0.15 M), a total uninfected HeLa cell extract (HeLa), and the pooled 1 M NaCl fractions from the first and second DEAE-cellulose columns (VITF-X). (B) Protein components were an extract of purified vaccinia virions (VV), a total extract of uninfected HeLa cells (HeLa), and VITF-X purified by DEAE-cellulose, phosphocellulose, SP Sepharose, and single-stranded DNA agarose chromatography.
FIG. 2
FIG. 2
Elution of VITF-X from the final two chromatography columns. Samples (20 μl) from HS (A) or CM (C) column fractions were analyzed by SDS-PAGE on 10% or 4 to 20% polyacrylamide gels, respectively, and silver stained. Samples—1 μl from the HS column (B) and 5 μl from the CM column (D)—were tested for VITF-X activity as described in the legend to Fig. 1. Fraction numbers are indicated at the top of each panel, and the positions and masses (in kilodaltons) of markers are indicated on the left. In panel C, dots are placed next to the 65-, 50-, 45-, and 35-kDa bands in the most active fraction. In panel A, the dot in lane 65 is a silver staining artifact.
FIG. 3
FIG. 3
Gel filtration of VITF-X. Partially purified VITF-X was applied to a 1.6- by 60-cm S300 Sephacryl column equilibrated with 0.15 M NaCl, 40 mM Tris-HCl (pH 8.0), 5% glycerol, 5 mM imidazole, and 0.5 mM phenylmethylsulfonyl fluoride. Samples—0.1 μl of loading material (L) or 2 μl of column fractions (numbered)—were assayed for VITF-X activity as described in the legend to Fig. 1. The masses (in kilodaltons) and elution positions of standard proteins (thyroglobulin, 669 kDa; ferritin, 440 kDa; aldolase, 158 kDa; serum albumin, 67 kDa; and ovalbumin, 43 kDa) (Pharmacia) used to calibrate the column are indicated at the top.
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References

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