Nucleotide sequence and transcript organization of a region of the vaccinia virus genome which encodes a constitutively expressed gene required for DNA replication

Abstract

A vaccinia virus (VV) gene required for DNA replication has been mapped to the left side of the 16-kilobase (kb) W HindIII D DNA fragment by marker rescue of a DNA-temperature-sensitive mutant, tsU7, using cloned fragments of the viral genome. The region of W DNA containing the tsl7 locus (3.6 kb) was sequenced. This nucleotide sequence contains one complete open reading frame (ORF) and two incomplete ORFs reading from left to right. Analysis of this region at early times revealed that transcription from the incomplete upstream ORF terminates coincidentally with the complete ORF encoding the ts17 gene product, which is directly downstream. The predicted proteins encoded by this region correlate well with polypeptides mapped by in vitro translation of hybrid-selected early mRNA. The nucleotide sequences of a 1.3-kb Bglll fragment derived from ts17 and from two ts17 revertants were also determined, and the nature of the tsU7 mutation was identified. S1 nuclease protection studies were carried out to determine the 5' and 3' ends of the transcripts and to examine the kinetics of expression of the ts17 gene during viral infection. The ts17 transcript is present at both early and late times postinfection, indicating that this gene is constitutively expressed. Surprisingly, the transcriptional start throughout infection occurs at the proposed late regulatory element TAA, which immediately precedes the putative initiation codon ATG. Although the biological activity of the tsl7-encoded polypeptide was not identified, it was noted that in tsl7-infected cells, expression of a nonlinked VV immediate-early gene (thymidine kinase) was deregulated at the nonpermissive temperature. This result may indicate that the ts17 gene product is functionally required at an early step of the W replicative cycle.

Vaccinia virus-encoded ribonucleotide reductase: sequence conservation of the gene for the small subunit and its amplification in hydroxyurea-resistant mutants

“…Si nuclease mapping and primer extension. S1 mapping using 5'-end-labeled DNA fragments was performed as previously described (30). For primer extension, 20 ,ug of total RNA was annealed to 3 to 5 ng of a 5'-end-labeled synthetic oligonucleotide, RD1, in a 15-Ill reaction mixture containing 10 mM Tris (pH 8.0), 0.25 M KCI, and 1 mM EDTA.…”

The Genome of Melanoplus sanguinipes Entomopoxvirus

“…Although more divergent than other ChPV D5R homologues, MSV089 contains regions of similarity throughout the protein, including the extended I (A) type of nucleoside triphosphate binding motif (55). D5R is known to be essential for viral DNA replication and is involved in homologous recombination (159).…”

Superinfection exclusion of vaccinia virus in virus-infected cell cultures

“…To test if either DNA replication or late viral gene expression was required from the primary infecting virus in order to exclude superinfection, we analyzed the ability of a DNA-ts virus to mediate exclusion at the permissive and nonpermissive temperatures. A series of cell monolayers was infected with the ts mutation C17, in gene D5R (Condit and Motyczka, 1981;Niles et a/., 1986;Seto et a/., 1987;Roseman and Hruby, 1987;Evans and Traktman, 1987), at either 3 lo or 40", and a standard superinfection assay was carried out. In Fig.…”