Abstract
Transcription of the DNA genome of vaccinia virus occurs in the cytoplasm and is temporally programmed by early, intermediate, and late stage-specific transcription factors in conjunction with a viral multisubunit RNA polymerase. The RNA polymerase, capping enzyme, and three factors (VITF-1, VITF-2, and VITF-3) are sufficient for in vitro transcription of a DNA template containing an intermediate stage promoter. Vaccinia virus intermediate transcription factor (VITF)-1 and -3 are virus-encoded, whereas VITF-2 was partially purified from extracts of uninfected HeLa cells. Using purified and recombinant viral proteins, we showed that the HeLa cell factor was required for transcription of linear or nicked circular templates but not of super coiled DNA. HeLa cell polypeptides of approximately 110 and 66 kDa copurified with VITF-2 activity through multiple chromatographic steps. The polypeptides were separated by SDS-polyacrylamide gel electrophoresis and identified by mass spectrometry as Ras-GTPase-activating protein SH3 domain-binding protein (G3BP) and p137, recently named cytoplasmic activation/proliferation-associated protein-1. The co-purification of the two polypeptides with transcription-complementing activity was confirmed with specific antibodies, and their association with each other was demonstrated by affinity chromatography of tagged recombinant forms. Furthermore, recombinant G3BP and p137 expressed individually or together in mammalian or bacterial cells complemented the activity of the viral RNA polymerase and transcription factors. The involvement of cellular proteins in transcription of intermediate stage genes may regulate the transition between early and late phases of vaccinia virus replication.
Highlights
The objectives of the present study were to confirm the requirement for a cellular intermediate stage transcription factor, purify the protein, and identify the gene encoding it
We discovered that virus intermediate transcription factor (VITF)-2 bound to Ni-NTAagarose and was eluted with a low concentration of imidazole
Transcription was dependent on the cellular factor, which had been partially purified on nickel-nitrilotriacetic acid (Ni-NTA)-agarose, as well as the RPOcapping enzyme complex and VITF-3
Summary
The objectives of the present study were to confirm the requirement for a cellular intermediate stage transcription factor, purify the protein, and identify the gene encoding it. We enhanced the specificity of the in vitro transcription reaction, making it entirely dependent on a factor from extracts of uninfected HeLa cells. This assay was used to monitor the presence of the transcription factor in column chromatography fractions. Polypeptides of 110- and 66-kDa co-purified as a heterodimer with VITF-2 activity and were identified as Ras-GTPase-activating protein SH3 domain-binding protein (G3BP) [21] and p137 [22] recently named cytoplasmic activation/proliferation-associated protein-1 [23]. Recombinant forms of the two polypeptides were expressed in a heteroloopen reading frame; HA, hemagglutinin; mAb, monoclonal antibody; HPLC, high pressure liquid chromatography
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