Genetic analysis of the herpes simplex virus type 1 UL9 gene: isolation of a LacZ insertion mutant and expression in eukaryotic cells.
HSV-1 host range mutants in complementation group 1-36 (hr27 and hr156) whose mutations map in the UL9 gene, encoding the origin binding protein, are unable to form plaques or synthesize viral DNA or late viral proteins when grown in nonpermissive Vero cells (Carmichael, E. P., Kosovsky, M. J., Weller, S. K., 1988, J. Virol. 62, 91-99). These defects are complemented efficiently by growth in the permissive cell line, S22, which contains the wild type version of several HSV genes including UL9. In this report the precise nature and location of the lesions in host range mutants hr27 and hr156 were determined by DNA sequencing; both mutants were found to contain identical single-base-pair substitutions at codons 309 and 311 in the UL9 open reading frame. This region lies within the putative helicase domain of the UL9 protein. The UL9 gene was disrupted by the insertion of an insertional mutagen ICP6::lacZ in which the Escherichia coli lacZ gene is expressed under control of the viral ICP6 promoter. Hr94, a viral mutant containing this insertion, does not form plaques or synthesize viral DNA when grown in Vero cells, although both defects are complemented efficiently on permissive cell lines. These results confirm that the UL9 gene product is essential for viral growth and DNA replication. Furthermore, since no detectable UL9 protein is synthesized in hr94-infected cells, this virus provides a useful genetic background for further structure-function analysis since no potentially interfering nonfunctional UL9 protein will be expressed. We have expressed the UL9 open reading frame under the control of the strong and inducible HSV-1 ICP6 promoter and have derived Vero cell lines containing variable copy numbers of the ICP6::UL9 construct. Cells whose copy number of this construct exceeded approximately 120 are unable to support efficient plaque formation by wild-type virus. Cell lines with low copy numbers of this construct are able to complement hr27, hr156, and hr94.
Malik
A K
AK
Department of Microbiology, University of Connecticut Health Center, Farmington 06030.
Martinez
R
R
Muncy
L
L
Carmichael
E P
EP
Weller
S K
SK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D007763:Lac Operon; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D018139:Simplexvirus / Q000235:genetics*; D014170:Transformation, Genetic / Q000235:genetics; D014709:Vero Cells; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C064565:UL9 protein, Human herpesvirus 1; D001616:beta-Galactosidase