Early transcription factor subunits are encoded by vaccinia virus late genes.

Abstract

The vaccinia virus early transcription factor (VETF) was shown to be a virus-encoded heterodimer. The gene for the 82-kDa subunit was identified as open reading frame (ORF) A8L, based on the N-terminal sequence of factor purified by using DNA-affinity magnetic beads. The 70-kDa subunit of VETF was refractory to N-terminal analysis, and so N-terminal sequences were obtained for three internal tryptic peptides. All three peptides matched sequences within ORF D6R. ORFs A8L and D6R are located within the central region of the vaccinia virus genome and are separated by about 13,600 base pairs. Proteins corresponding to the 3' ends of ORFs A8L and D6R were overexpressed in Escherichia coli and used to prepare antisera that bound to the larger and smaller subunits, respectively, of affinity-purified VETF. Immunoblot analysis of proteins from infected cells indicated that both subunits are expressed exclusively in the late phase of infection, just prior to their packaging in virus particles. The two subunits of VETF have no significant local or overall amino acid sequence homology to one another, to other entries in biological sequence data bases including bacterial a factors, or to recently determined sequences of some eukaryotic transcription factors. The 70-kDa subunit, however, has motifs in common with a superfamily of established and putative DNA and RNA helicases.Vaccinia virus, a member of the poxvirus family of large cytoplasmic DNA viruses, contains =200 genes that can be divided into at least three regulatory classes-early, intermediate, and late (for a review, see ref. 1). All of the enzymes and protein factors required for early transcription are present within the virus particle and are brought into the cytoplasm of the cell at the time of infection. Extracts of infectious virus particles are able to correctly initiate and terminate transcription of an appropriate template in vitro and also to cap and polyadenylate newly synthesized or added mRNA (2). Many of the components of the early transcriptional apparatus have been isolated and purified. These include a multisubunit DNA-dependent RNA polymerase and a multifunctional heterodimeric capping enzyme that modifies the 5' end of the nascent RNA (3, 4) and also is required for transcriptional termination (5, 6). Two additional enzymes, a poly(A) polymerase that selectively adds adenylate residues to the free 3' ends of mRNAs without apparent primer specificity (7,8) and an RNA (nucleoside-2')methyltransferase that modifies the penultimate nucleotide of the cap (9, 10), have been isolated and characterized.

Kinetic analysis of a complete poxvirus transcriptome reveals an immediate-early class of genes

“…Proteins encoded by late genes are generally required for virion morphogenesis and structure or early gene transcription factors packaged in newly made viral cores (15)(16)(17).…”

The DEK Oncoprotein Is a Critical Component of the EKLF/KLF1 Enhancer in Erythroid Cells

“…In our approach, double-stranded oligonucleotide 2 (or oligonucleotide 1 as a negative control) is 5= biotinylated at one end and attached to a streptavidin/magnetic bead column, which provides the solid-state matrix to which the protein(s) is to be bound (60,61). This column was sequentially washed in 0.2 M NaCl (which removes most of the nonspecific binding proteins) and 0.5 M NaCl before elution in 1 M NaCl, which contains almost all of the specific DNA binding responsible for the oligonucleotide 2 probe shift in the crude extract.…”

Three African swine fever virus genes encoding proteins with homology to putative helicases of vaccinia virus

“…For jl0L the closest matches were with ORF 4 of the Kluyveromyces lactis killer plasmid pGK12 (pGK12 ORF 4) (Tommasino et al, 1988) and ASFV gl0L (described here). Weaker, but significant, similarity was found with the NPH I proteins ofvaccinia virus (Rodriguez et al, 1986;Broyles & Moss, 1987a) and the entomopoxvirus of Choristoneura biennis (CbEPV) (Yuen et al, 1991) and the 70K early transcription factor subunit from vaccinia virus (Broyles & Fesler, 1990;Gershon & Moss, 1990) and fowlpox virus (Binns et al, 1990;Tartaglia et al, 1990) (Table 1). ORF gl0L also had weak similarity with vaccinia NPH I (optimized score of 121, with 18 % identity over 278 amino acids).…”

“…NPH I hydrolyses only ATP and dATP, NPH II hydrolyses all ribo-and deoxyribonucleoside triphosphates (Paoletti et al, 1974a, b;Paoletti & Moss, 1974) and the third NPH is a DNA-dependent ATPase and is the small subunit of the early transcription factor (VETF) Broyles & Fesler, 1990;Gershon & Moss, 1990).…”

“…Gene D6R encodes the small subunit of the heterodimeric VETF complex which contains DNA-dependent ATPase activity and is required for VETF binding to early promoters and for transcription (Yuen et al, 1987;Broyles & Fesler, 1990;Gershon & Moss, 1990). Gene DllL encodes NPH I, a late protein with DNA-dependent ATPase activity which is associated with the RNA polymerase complex (Rodriguez et al, 1986;Broyles & Moss, 1987b).…”