An ICP6::lacZ insertional mutagen is used to demonstrate that the UL52 gene of herpes simplex virus type 1 is required for virus growth and DNA synthesis.
An insertional mutagen was developed which consists of the lacZ gene of Escherichia coli under the control of the regulatory elements of the large subunit of ribonucleotide reductase (ICP6). This ICP6::lacZ cassette was used to create a mutation in a gene designated UL52 (D. J. McGeoch, M. A. Dalrymple, A. Dolan, D. McNab, L. J. Perry, P. Taylor, and M. D. Challberg, J. Virol. 62:444-453, 1988), which is predicted to encode a 114,000-molecular-weight protein. To isolate and propagate this mutant, we generated a cell line, BL-1, by cotransfection of Vero cells with pSV2neo and a plasmid containing the herpes simplex virus type 1 KOS strain BamHI L fragment (coordinates 0.708 to 0.745). An ICP6::lacZ insertion mutant, hr114, was capable of growing in BL-1 cells but not in normal Vero cells. In addition, hr114 was defective in the synthesis of viral DNA and late proteins; however, this mutant appeared to exhibit normal early gene expression. Thus, the results presented in this report show that the UL52 gene product is required for viral DNA synthesis. Furthermore, our studies indicate that the ICP6::lacZ cassette will provide a useful tool for obtaining mutants of other herpes simplex virus genes.
Goldstein
D J
DJ
Department of Microbiology, University of Connecticut Health Center, Farmington 06032-9984.
Weller
S K
SK
D000818:Animals; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D004261:DNA Replication; D004274:DNA, Recombinant; D004279:DNA, Viral / Q000096:biosynthesis*; D005814:Genes, Viral; D005816:Genetic Complementation Test; D007763:Lac Operon; D012264:Ribonucleotide Reductases / Q000235:genetics; D018139:Simplexvirus / Q000235:genetics* / Q000254:growth & development; D014764:Viral Proteins / Q000096:biosynthesis; D014779:Virus Replication
D004274:DNA, Recombinant; D004279:DNA, Viral; D014764:Viral Proteins; D012264:Ribonucleotide Reductases