Identification of trans-dominant HIV-1 rev protein mutants by direct transfer of bacterially produced proteins into human cells.
A synthetic rev gene containing substitutions which introduced unique restriction sites but did not alter the deduced amino acid sequence was used as a vehicle to construct mutations in rev. Insertion or substitution mutations within a domain of Rev resulted in proteins able to inhibit the function of Rev protein in trans. Rev function was monitored in a cell line, HLfB, which contained a rev- mutant provirus. HLfB cells require the presence of rev for virus production, which was conveniently monitored by immunoblot detection of p24gag. Trans-dominant mutants were identified after expression in bacteria and delivery into HLfB cells by protoplast fusion. In addition, the trans-dominant phenotype was verified by expression of the mutant proteins in HLfB cells after cotransfection. These studies define a region between amino acid residues 81 and 88 of rev, in which different mutations result in proteins capable of inhibiting Rev function.
National Cancer Institute-Frederick Cancer Research Facility, BRI-Basic Research Program, MD 21701-1013.
B
B
Mermer
B K
BK
Felber
M
M
Campbell
G N
GN
Pavlakis
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015694:Gene Products, rev / Q000096:biosynthesis / Q000235:genetics*; D005813:Genes, Synthetic; D005814:Genes, Viral; D015757:Genes, rev; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D015534:Trans-Activators / Q000235:genetics*; D014162:Transfection; D054321:rev Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D015534:Trans-Activators; D054321:rev Gene Products, Human Immunodeficiency Virus