A genetic selection method for the transfer of HSV-1 glycoprotein B mutations from plasmid to the viral genome: preliminary characterization of transdominance and entry kinetics of mutant viruses.
A genetic selection procedure has been devised to transfer mutations from a plasmid to the viral genome. Reagents were constructed so that the recombination events that occur during cotransfection rescue a mutation in a recipient viral genome and, simultaneously, transfer a mutation in an adjacent (target) gene to the viral genome. In the example presented here the two adjacent genes are essential for viral replication, so that a transformed cell line that expresses both genes and another cell line that expresses only the target gene are required. Ideally the recipient viral genome should be deleted for the entire target gene, and the deletion should extend a short distance into the adjacent gene. In the present study UL27 (glycoprotein gB) of herpes simplex virus type 1 (KOS) is the target gene for mutation transfer and the upstream gene UL28, which specifies the ICP18.5 polypeptide, is the marker-rescue gene. A recipient virus that was deleted for DNA sequences encoding the C-terminal 74 residues of ICP18.5 and the N-terminal 711 residues of gB was constructed. All of the gB mutant plasmids used overlap the ICP18.5 deletion, and therefore, recombination events that rescue the ICP18.5 deletion must transfer a gB mutation present in codons 1-711 of the rescuing plasmid. Recombinant viruses that contain the gB mutation, unlike the parental gB-/18.5- virus, will grow on cells that express only gB (D6). This procedure has been used to transfer insertion, deletion, and chain termination mutations into the gB gene of the KOS genome. Southern blot analysis confirmed the transfer of the mutations to the viral genome. Analysis of radioactively labeled, infected cell lysates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), before and after immune precipitation with gB-specific antibodies, confirmed the presence of the mutant polypeptides in the lysates. The incorporation of the mutant polypeptides into the envelopes of purified released viruses was measured. The yield of plaque forming units per cell was determined from single step growth curves after infection of permissive D6 cells. Virus yield, a measure of transdominance, varied for the different mutants. The maximum reduction in virus yield observed was sixfold relative to the wild-type virus. Transdominance was a result of both the lack of glycoprotein processing and the presence of gB-oligomerization sites. A reduced yield was also observed for a previously isolated gB-null mutant virus, K082. The amount of gB produced in D6 cells, infected with K082, was less than that produced in KOS infected cells, which may account for the reduced yield.(ABSTRACT TRUNCATED AT 400 WORDS)
Desai
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Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pennsylvania 15261.
Homa
F L
FL
Person
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Glorioso
J C
JC
D002460:Cell Line; D004279:DNA, Viral / Q000032:analysis; D015870:Gene Expression; D005799:Genes, Dominant; D005814:Genes, Viral / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D005819:Genetic Markers; D016679:Genome, Viral; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000254:growth & development; D007700:Kinetics; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D014759:Viral Envelope Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics; D015678:Viral Structural Proteins / Q000235:genetics; D014771:Virion / Q000737:chemistry
D004279:DNA, Viral; D005819:Genetic Markers; D014759:Viral Envelope Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C040495:glycoprotein B, Simplexvirus