Isolation of a herpes simplex virus type 1 mutant deleted for the essential UL42 gene and characterization of its null phenotype.
We isolated a cell line, designated V9, stably transformed with the herpes simplex virus type 1 (HSV-1) UL42 gene, which is one of seven genes required in trans for the replication of plasmids containing an HSV origin of replication (C. A. Wu, N. J. Nelson, D. J. McGeoch, and M. D. Challberg, J. Virol. 62:435-443, 1988). V9 cells inducibly expressed the product of the UL42 gene, the 65-kDa DNA-binding protein (65KDBP), and were used as a permissive host to construct a mutant virus deleted for this essential gene. The UL42 deletion mutant, designated Cgal delta 42, displayed a tight early phenotype in nonpermissive Vero cells producing no infectious progeny, viral DNA, or late gene products but accumulated selected immediate-early and early transcripts with kinetics similar to those of wild-type virus. Wild-type levels of viral DNA and infectious progeny were produced in permissive V9 cells, despite the fact that V9 cells infected with Cgal delta 42 accumulated less than 1% of the UL42 RNA and protein found in Cgal+ virus-infected V9 or Vero cells. These results indicate that only small quantities of the 65KDBP are required for the synthesis of HSV DNA and the production of infectious virus. Although we could find no evidence that the superinduction of the 65KDBP in V9 cells infected with Cgal+ repressed expression of HSV-1 genes as observed in cells expressing another DNA-binding protein, ICP8 (P. K. Orberg and P. A. Schaffer, J. Virol. 61:1136-1146, 1987), the induction of the 65KDBP in V9 cells correlated with an approximately 2-h-earlier shift in the expression of genes from all three kinetic classes. The availability of the UL42 mutant should facilitate the construction of more subtle UL42 mutants which will be useful in the elucidation of the interrelationship between the 65KDBP and other DNA replication proteins as well as in the characterization of additional important functional domains.
Johnson
P A
PA
Center for Molecular Genetics, University of California, San Diego, La Jolla 92093.
Best
M G
MG
Friedmann
T
T
Parris
D S
DS
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002472:Cell Transformation, Viral; D002872:Chromosome Deletion; D004247:DNA / Q000096:biosynthesis; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics; D005814:Genes, Viral; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D010641:Phenotype; D010957:Plasmids; D015183:Restriction Mapping; D018139:Simplexvirus / Q000235:genetics* / Q000254:growth & development; D014709:Vero Cells
D004279:DNA, Viral; D015345:Oligonucleotide Probes; D004247:DNA