Nucleic Acids Research, 1990, Vol. 18, No. 8 2037-2044
© 1990
MOLECULAR BIOLOGY |
Identification of trans-dominant HIV-1 rev protein mutants by direct transfer of bacterially produced proteins into human cells
National Cancer Institute-Frederick Cancer Research Facility, BRI-Basic Research Program Frederick, MD, 21701-1013, USA
* To whom correspondence should be addressed at National Cancer Institute, Frederick Cancer Research Facility, BRI-Basic Research Program, PO Box B/Building 539 Room 121, Frederick, MD 21701-1013, USA
Received January 3, 1990. Revised March 19, 1990. Accepted March 19, 1990.
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 Revresulted in proteins able to inhibit the function of Revprotein 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.
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