Identification and characterization of vaccinia virus late transcription factors

Min Zhu, Purdue University

Abstract

Vaccinia virus late transcription can be reconstituted in vitro with extracts from virus infected HeLa cells. These extracts have been fractionated by chromatography on phosphocellulose into three fractions, all of which were necessary for maximal transcription activity. Both biochemical and genetic approaches have begun to elucidate which proteins in these crude fractions are essential to activate late transcription. In addition to the virus encoded RNA polymerase, three late transcription factors have been identified as the 17-, 26-, and 30-kDa protein which are products of the A1L, A2L, and G8R intermediate genes, respectively. One additional factor, which has been demonstrated to activate basal level of late transcription in vitro , has been identified as the 36-kDa protein encoded by the vaccinia virus H5R gene. However, no late transcription-specific biochemical function has been ascribed to any of them. Recently, two more factors have been found in uninfected HeLa cells. One of them, named late promoter binding protein (LPBP), had late promoter-specific DNA-binding activity. It interacted with late promoter around transcription start site, and could activate transcription in vitro, suggesting that it may be the factor which specifically recognizes vaccinia virus late promoter. LPBP was purified 294-fold from cytoplasmic extract of HeLa cells. It is composed of two polypeptides of 60- and 100-kDa according to the SDS-PAGE analysis. Amino acid sequence analysis showed that the 60-kDa protein is p54 nrb/NonO, and the 100-kDa protein is PSF. These two proteins are localized in cell nuclei, and after virus infection, some of them migrate into cytoplasm. They were demonstrated to be involved in virus replication. The two proteins, expressed in bacteria, could form a hetero-complex in vitro, but the protein-DNA complex migrated different from LPBP-DNA in mobility shift assays. The bacterially produced A1, A2, G8, and H5 proteins were tested for late promoter binding activities. The A2, G8 and H5 proteins were shown to interact with 11 kDa late promoter DNA, but the interactions were not sequence specific. Homo- and hetero-complexes formed between some of them. These protein complex formation changed their affinity to late promoter, but no sequence specific protein-DNA interaction was found either.

Degree

Ph.D.

Advisors

Broyles, Purdue University.

Subject Area

Biochemistry|Molecular biology|Genetics|Microbiology

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