Inhibition of transient gene expression with plasmids encoding herpes simplex virus type 1 UL55 and alpha genes.
Herpes simplex virus type 1 (HSV-1) subgenomic sequences from 0.743 to 0.782 map units have been molecularly cloned as plasmid AT1 and shown to inhibit stable DNA-mediated gene transformation of Ltk- cells with the HSV-1 thymidine kinase (tk) gene. Here it is shown that AT1 also inhibits transient gene expression. Expression from the chloramphenicol acetyltransferase (CAT) gene under the control of either the HSV-1 tk gene or the Rous sarcoma virus (RSV) promoter was inhibited when cotransfected into Ltk- and CV-1 cells with equimolar amounts of AT1. AT1 was subcloned as three overlapping plasmids called AT1a, alpha 27 and AT1b. The alpha 27 plasmid encodes the HSV-1 immediate early gene, alpha 27; AT1a possesses sequences that specify an open reading frame in HSV-1 strain KOS used in these studies, although the significance of this open reading frame is unknown; AT1b possesses the sequences for UL55 and UL56, also genes for which no function has been reported. No single subclone or pair of subclones demonstrated significant inhibition of transient gene expression. Cotransfection of all three subclones did result in inhibition of RSV-CAT gene expression, suggesting that information from each subclone is necessary. One of the three subclones, alpha 27, contains the HSV-1 immediate early gene, alpha 27, so the possibility that other immediate early genes could substitute for alpha 27 was tested. Inhibition of RSV-CAT gene expression was also achieved by cotransfection of AT1a and AT1b with either an alpha 0- or alpha 4-containing plasmid, suggesting that the role of the alpha 27-containing plasmid can be replaced by other alpha genes with trans-regulating capability. Finally, AT1a and AT1b linker insertion mutants have been constructed and used to study the role these plasmids play in mediating inhibition. These results suggest that AT1 contains HSV-1 functions in addition to that of alpha 27 that interfere with gene expression.
Block
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Department of Microbiology, Jefferson Medical College, Philadelphia, Pennsylvania 19107.
Jordan
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Farkas
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Hughes
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D000818:Animals; D002460:Cell Line; D002472:Cell Transformation, Viral; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D015870:Gene Expression; D005814:Genes, Viral; D007739:L Cells / Q000201:enzymology; D051379:Mice; D016366:Open Reading Frames; D010957:Plasmids; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase; D013937:Thymidine Kinase