Construction and characterization of herpes simplex type 1 viruses without introns in immediate early gene 1.
Herpes simplex virus type 1 (HSV-1) encodes at least 70 distinct genes in a DNA genome sequence of about 150 kb. In contrast to most cellular genes and those of several other DNA viruses, the overwhelming majority of HSV-1 transcripts are not spliced. One exception is immediate early (IE) gene 1, which contains two introns in the Vmw110 coding region. This study investigated the possibility that IE-1 intron sequences have a role during HSV-1 infection. IE-1 genes lacking the first, second or both introns were constructed by site-directed deletion mutagenesis and recombined into the viral genome. Viruses lacking the IE-1 introns were essentially indistinguishable from the parent virus in terms of growth, particle to p.f.u. ratio or viral polypeptide expression in a variety of cell types. The lack of introns did not affect the time-course or efficiency of expression of Vmw110 either during normal infection or in cycloheximide reversal experiments. In contrast, in transfection assays, the loss of both intron sequences resulted in the elimination of the ability of a plasmid-encoded IE-1 to activate gene expression. These results imply that in certain situations the introns in IE-gene 1 may contribute to the efficient expression of Vmw110 but such an effect is not readily apparent using a recombinant virus in the tissue culture systems tested. In the course of this work a mutant of Vmw110 was fortuitously isolated which had lost the majority of an extremely acidic section of the polypeptide; this mutation appeared to have little effect on Vmw110 function.
Everett
R D
RD
MRC Virology Unit, Glasgow, U.K.
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids; D015183:Restriction Mapping; D018139:Simplexvirus / Q000235:genetics* / Q000254:growth & development / Q000502:physiology; D014162:Transfection; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics
D004279:DNA, Viral; D009841:Oligonucleotides; D014764:Viral Proteins; D003513:Cycloheximide