Scott J. Goebel

VACCINIA VIRUS HOST RANGE GENES

A gene encoding an 18-kDa polypeptide (ORF C7L) located in the vaccinia virus HindIII C fragment was shown to be functionally equivalent to previously described host range gene (ORF K1L) spanning the HindIII K/M fragment junction. Either C7L or K1L host range gene is necessary and sufficient by itself to allow replication of vaccinia virus on human cells. Deletion of both C7L and K1L genes from the wild-type vaccinia genome is required to derive a virus deficient for replication on human cells. Further, an ORF encoding a 77-kDa polypeptide derived from cowpox (CP77kDa) and previously shown to allow vaccinia to overcome the restriction for replication on Chinese hamster ovary cells could substitute for the vaccinia host range genes C7L and K1L in permitting replication of the virus on human cells. Additionally, the three unique host range genes C7L, K1L, and CP77kDa were functionally equivalent for vaccinia replication on pig kidney cells, but not on rabbit kidney cells.

Deletion of 55 open reading frames from the termini of vaccinia virus

Each copy of the inverted terminal repeat of vaccinia virus consists of 8 kb of DNA containing 9 ORFS flanked near the terminus of the genome by 4 kb of repetitive DNA which in turn contains blocks of tandem repeats. Using plasmids containing repetitive DNA as the external arm, we have generated deletions at both the left and the right termini of the vaccinia genome. We report here the engineered deletion within a single vaccinia virus of 32.7 kb of DNA (including 38 ORFS) from the left terminus and 14.9 kb of DNA (including 17 ORFS) from the right terminus.

A DNA ligase gene in the copenhagen strain of vaccinia virus is nonessential for viral replication and recombination

Biochemical and genetic analyses have been conducted to determine whether a vaccinia virus open reading frame (orf) with extensive homology to the Saccharomyces cerevisiae DNA ligase gene encodes a functional ligase activity. This orf in HindIII A, designated A50R, is capable of encoding a 552-amino-acid, 63.4-kDa polypeptide. Full-length A50R mRNA produced in vitro directed the synthesis of a polypeptide with an apparent molecular weight of 57 kDa. Significantly, translation reactions programmed with A50R mRNA were capable of ligating a 3-kb Notl restriction fragment into multimers. DNA ligase activity was not detectable when either truncated sense or full-length antisense mRNA was translated in vitro. In extracts prepared from cells infected with wt vaccinia virus, DNA ligase activity was detected as assayed by the formation of a 57 kDa ligase-AMP adduct which was expressed early in the viral replication cycle. In cells infected with a DNA ligase deletion mutant no equivalent AMP-labeled adduct was detected. Relative to wt virus, the DNA ligase deletion mutant exhibited no significant differences in homologous recombination. These results indicate that the vaccinia orf A50R encodes a functional DNA ligase expressed early in infection, but this DNA ligase is nonessential for either recombination or viral replication.

Vaccinia virus encodes a protein with similarity to glutaredoxins.

Recently, we have reported the complete nucleotide sequence of vaccinia virus (Goebel, S. J., Johnson, G. P., Perkus, M. E., Davis, S. W., Winslow, J. P., and Paoletti, E. 1990, Virology 179, 247-266). Approximately 2.2 kbp leftward of the large subunit of ribonucleotide reductase resides a 108-amino acid open reading frame, O2L (nt 62,851-62,528) with significant similarity to known glutaredoxins. The deduced amino acid sequence of open reading frame O2L is 28.7% identical to the yeast and Escherichia coli proteins and greater than 40% identical to various mammalian glutaredoxins. Similar patterns of hydrophobicity as well as alpha-helix and beta-sheet potentials suggest that O2L and the glutaredoxins share a similar secondary structure. Furthermore, a common function is inferred by the presence of a highly conserved redox-active site.

Nucleotide sequence analysis of a 10.5 kbp HindIII fragment of fowlpox virus: relatedness to the central portion of the vaccinia virus HindIII D region

The nucleotide sequence of a 10465 bp HindIII genomic fragment from fowlpox virus (FPV) is presented. Analysis of the nucleotide sequence revealed 10 potential major open reading frames (ORFs). Five of these ORFs are predicted to encode polypeptides with significant homology to hypothetical polypeptides derived from nucleotide sequence analysis of the vaccinia virus (VV) HindIII D region. Interestingly, these homologous ORFs do not occur in the same tandem arrangement in the FPV genome as they do in the VV genome. These results are discussed.