Differential activation of hybrid genes containing herpes simplex virus immediate-early or delayed-early promoters after superinfection of stable DNA-transfected cell lines.
We compared the levels of gene expression obtained after herpes simplex virus (HSV) superinfection of cell lines containing integrated human beta-interferon (IFN) or chloramphenicol acetyltransferase (CAT) genes under the control of HSV immediate-early (IE) or delayed-early class promoters. DNA-transfected mouse Ltk+ cell lines harboring coselected IE175-IFN or thymidine kinase (TK)-IFN hybrid genes gave only low basal expression of human IFN. However, infection of both cell types with HSV type 1 or HSV type 2 produced abundant synthesis of IFN-specific RNA and biologically active IFN protein product. The IE175-IFN cell lines consistently gave 20- to 150-fold increases in IFN titers, and several TK-IFN cell lines yielded 100- to 500-fold induction. In the IE175-IFN cells, expression of IFN RNA also increased up to 200-fold and was detectable within 30 to 60 min after virus infection. Qualitatively similar results were obtained with hybrid G418-resistant Ltk- or Vero cell lines containing coselected IE175-CAT and TK-CAT constructs, except that there was relatively high basal expression of IE175-CAT. All three sets of IE cell lines (but not the delayed-early cell lines) responded to virus infection both in the presence of cycloheximide and with mutants defective in IE gene expression, demonstrating specific trans-activation by the pre-IE virion factor. In contrast, activation in the TK hybrid cell types required viral gene expression and the presence of a functional IE175 gene product. Up to 30-fold amplification in the copy number of the resident IFN or CAT DNA sequences also occurred within 20 h after HSV infection in IE175 hybrid cells but not in TK hybrid cells. Amplification was abolished either by treatment with phosphonacetate or by superinfection with a ts mutant unable to synthesize viral DNA, demonstrating specific HSV activation of the viral DNA replication origin (oriS) present in the IE hybrid constructs.
J D
JD
Mosca
G R
GR
Reyes
P M
PM
Pitha
G S
GS
Hayward
D000123:Acetyltransferases / Q000235:genetics; D000818:Animals; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase; D004274:DNA, Recombinant; D004279:DNA, Viral / Q000235:genetics; D005784:Gene Amplification / Q000187:drug effects; D005786:Gene Expression Regulation; D005814:Genes, Viral; D006801:Humans; D007372:Interferons / Q000235:genetics; D009154:Mutation; D010746:Phosphonoacetic Acid / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D018139:Simplexvirus / Q000235:genetics*; D013696:Temperature; D014158:Transcription, Genetic; D014162:Transfection
D004274:DNA, Recombinant; D004279:DNA, Viral; D012333:RNA, Messenger; D012367:RNA, Viral; D007372:Interferons; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase; D010746:Phosphonoacetic Acid