Generation of glucocorticoid-responsive Moloney murine leukemia virus by insertion of regulatory sequences from murine mammary tumor virus into the long terminal repeat.
The glucocorticoid-regulatory sequences from the murine mammary tumor virus long terminal repeat (MMTV LTR) were introduced into the LTR of Moloney murine leukemia virus (M-MuLV) by recombinant DNA techniques. The site of insertion was in the M-MuLV LTR U3 region at -150 base pairs with respect to the RNA cap site. Infectious M-MuLVs carrying the altered LTRs (Mo + MMTV M-MuLVs) were recovered by transfection of proviral clones into NIH-3T3 cells. The Mo + MMTV M-MuLVs were hormonally responsive in that infection was 3 logs more efficient when performed in the presence of dexamethasone, irrespective of the orientation of the inserted MMTV sequences. However, even in the presence of hormone, the Mo + MMTV M-MuLVs were less infectious than wild-type M-MuLV. In contrast to the large effect on infectivity, dexamethasone induced virus-specific RNA levels in chronically Mo + MMTV M-MuLV-infected cells only two- to fourfold. Fusion plasmids between the altered LTRs and the bacterial chloramphenicol acetyltransferase gene allowed the investigation of LTR promoter strength by the transient chloramphenicol acetyltransferase expression assay. The chloramphenicol acetyltransferase assays indicated that the insertion of MMTV sequences into the M-MuLV LTR reduced promoter activity in the absence of glucocorticoids but that promoter activity could be induced two- to fivefold by dexamethasone. The Mo + MMTV M-MuLVs were also tested for the possibility that viral DNA synthesis or integration during initial infection was enhanced by dexamethasone. However, no significant difference was detected between cultures infected in the presence or absence of hormone. The insertion of MMTV sequences into an M-MuLV LTR deleted of its enhancer sequences did not yield infectious virus or active promoters, even in the presence of dexamethasone.
Overhauser
J
J
Fan
H
H
D004274:DNA, Recombinant; D004279:DNA, Viral / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D005814:Genes, Viral; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics*; D008979:Moloney murine leukemia virus / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D014162:Transfection; D014779:Virus Replication
D004274:DNA, Recombinant; D004279:DNA, Viral; D003907:Dexamethasone