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Vaccinia Virus Intermediate Stage Transcription Is Complemented by Ras-GTPase-activating Protein SH3 Domain-binding Protein (G3BP) and Cytoplasmic Activation/Proliferation-associated Protein (p137) Individually or as a Heterodimer
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,...
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Published in: | The Journal of biological chemistry 2004-12, Vol.279 (50), p.52210-52217 |
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Main Authors: | , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | 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 â¼110 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. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M411033200 |