Use of site-directed mutagenesis to generate a herpes simplex virus type 1 strain 17+ mutant lacking seven HindIII restriction endonuclease cleavage sites.
The genome of herpes simplex virus type 1 (HSV-1) strain 17+ contains ten HindIII and four XbaI restriction endonuclease (RE) cleavage sites. We have previously reported the isolation of an HSV-1 mutant, 1702, devoid of all the four XbaI sites. Here we report the isolation of HSV-1 mutants lacking seven of the HindIII sites plus the four XbaI sites. In order to destroy the various HindIII sites, mutagenic oligonucleotides were synthesized and introduced in to the plasmids containing HSV-1 restriction endonuclease fragments spanning these HindIII sites. All the seven HindIII sites were removed by site-directed mutagenesis. Two methods of site-directed mutagenesis were used: 1) the HindIII site at 0.91 map coordinates (mc) of HSV-1 strain 17+ genome was deleted using a gapped, heteroduplex molecule of DNA, and 2) uracil-rich single-stranded DNA templates were used in in vitro mutagenesis reactions to remove the HindIII sites at 0.08, 0.1, two at 0.18, 0.26 and 0.64 mc. These HindIII site deletions were then marker transferred back in to the 1702 genome to generate virus mutants devoid of specific HindIII sites. No other deletions and/or insertions were observed within the viral genomes of mutant viruses as allowed by restriction endonuclease analysis of their 32P-labelled DNAs. All the HindIII site-deletion mutants, 1721-1733, showed comparable growth properties and polypeptide profiles to those of the parental 17+ and 1702 viruses.
Fareed
M U
MU
Institute of Virology, University of Glasgow, UK. fareed@zeus.bwh.harvard.edu
Brown
S M
SM
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D006224:Cricetinae; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D015247:Deoxyribonuclease HindIII / Q000378:metabolism*; D018259:Herpesvirus 1, Human / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017384:Sequence Deletion; D014764:Viral Proteins / Q000032:analysis
D004279:DNA, Viral; D014764:Viral Proteins; D015247:Deoxyribonuclease HindIII