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, α27 and AT1b. The α27 plasmid encodes the HSV-1 immediate early gene, α27; 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, α27, contains the HSV-1 immediate early gene, α27, so the possibility that other immediate early genes could substitute for α27 was tested. Inhibition of RSV-CAT gene expression was also achieved by cotransfection of AT1a and AT1b with either an α0- or α4-containing plasmid, suggesting that the role of the α27-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 α27 that interfere with gene expression.


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