Generation of a herpes simplex virus type 2 variant devoid of XbaI sites: removal of the 0.91 map coordinate site results in impaired synthesis of glycoprotein G-2.
Herpes simplex virus (HSV) type 2 (strain HG52) has four XbaI sites at map coordinates (m.c.) 0.45, 0.7, 0.91 and 0.94, i.e. two in the unique long and two in the unique short regions of the genome. Previously, we had isolated a genome containing only the 0.45 and 0.94 XbaI sites. Here we report the isolation of a mutant (JH2611) in which all four XbaI sites have been removed using an enrichment selection procedure, without any loss of viability. Removal of each site has been shown to be due to a base change or small undetectable deletion/insertion of less than 100 bp. In HSV-1, the XbaI site at 0.45 m.c. is in an open reading frame coding for a polypeptide of 14K. Both the 0.7 and 0.94 m.c. HSV-2 XbaI sites are in intergenic positions. The 0.91 m.c. XbaI site has been shown to be within the coding sequence of the glycoprotein gG-2. Synthesis of gG-2 by JH2611 and two other mutants, JH2610 (formerly HG52X163X3) and JH2609 (formerly HG52X163X21), in which the 0.91 m.c. site has been deleted was analysed by immunoprecipitation using the gG-2-specific monoclonal antibodies AP1 and LP5 and the anti-peptide serum 14713. In the mutants JH2610 and JH2611 neither gG-2 nor its precursor were detected but the monoclonal antibodies detected two polypeptides migrating above the normal positions of gG-2 and the gG-2 precursor; these were not precipitated by the anti-peptide serum. With the mutant JH2609 neither gG-2 nor its precursors could be detected by either the monoclonal antibodies or the anti-peptide serum. The results strongly suggest that gG-2 is non-essential for the growth of HSV-2 in vitro.
Harland
J
J
MRC Virology Unit, Institute of Virology, Glasgow, U.K.
Brown
S M
SM
D002872:Chromosome Deletion; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000235:genetics; D015245:Deoxyribonuclease BamHI; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific; D005814:Genes, Viral; D007118:Immunoassay; D009154:Mutation; D018139:Simplexvirus / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics*
D004279:DNA, Viral; D014759:Viral Envelope Proteins; C045464:glycoprotein G, herpes simplex virus type 2; D004262:DNA Restriction Enzymes; D015245:Deoxyribonuclease BamHI; D015246:Deoxyribonuclease EcoRI; C025580:endodeoxyribonuclease XBAI; D015252:Deoxyribonucleases, Type II Site-Specific