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1st International Workshop on HIV Drug Resistance & Treatment Strategies25-28 June 1997, St. Petersburg, Florida, USA |
IMPACT OF THE P236L DELAVIRDINE RESISTANCE MUTATION ON HIV-1 REPLICATION AND REVERSE TRANSCRIPTASE FUNCTION
Antiviral Therapy 1997;2 (Suppl 5):18 (abstract no. 28)
LM Demeter1, P Gerondelis1,2, RH Archer1, C Palaniappan3, RC Reichman1,2 and R Bambara2,3
1Infectious Diseases Unit; 2Department of Microbiology and Immunology; and 3Department of Biochemistry and Biophysics, University of Rochester, New York, USA
OBJECTIVES: P236L confers high-level resistance to delavirdine and other bisheteroarylpiperazine non-nucleoside reverse transcriptase (RT) inhibitors, but sensitizes HIV-1 to nevirapine and pyridinones. P236L is selected for during passage in vitro of HIV-1 in the presence of delavirdine, but is only infrequently seen in clinical isolates from patients receiving delavirdine. We studied the impact of the P236L mutation on HIV-1 replication in vitro.
METHODS: Oligonucleotide site-directed mutagenesis was used to introduce P236L into the RT-coding sequence of pNL4-3. The absence of extraneous mutations was verified by sequence analysis. Virus stocks of wild-type and P236L NL4-3 were generated by transient transfection of A293 cells, followed by co-cultivation with H9 cells for 48h. Virus stocks were titrated in H9 cells. H9 cells were infected in parallel with either wild-type or P236L NL4-3 at a m.o.i. of 0.001. Serial p24 determinations were performed daily for 7 days after infection. In addition, competition experiments were performed by serially passaging a 50:50 mixture of P236L NL4-3 and wild-type NL4-3 in H9 cells. The ratio of P236L NL4-3 to wild-type NL4-3 after each passage was estimated using automated sequencing with dye-labelled primers. The p66 and p51 sequences derived from pNL4-3 were cloned into expression vectors containing amino-terminal hexahistidine extensions to facilitate purification. Using these constructs, we have expressed heterodimeric RT with and without the P236L mutation in Escherichia coli.
RESULTS AND CONCLUSIONS: We found an approximately fivefold decrease in the p24 concentration in P236L cultures compared with wild-type cultures that was initially evident at day 4 and persisted through day 7. In addition, we were unable to detect the presence of the P236L mutant after two passages of the wild-type/P236L mixture. These results suggest that the P236L mutation significantly impairs HIV-1 replication, and may offer an explanation for the infrequent occurrence of this mutation in clinical isolates during delavirdine therapy. Preliminary studies suggest that wild-type and P236L recombinant RT have similar specific activities for polymerization. Studies to determine the effect of this mutation on the ratio of RNase H/polymerase activity, RT fidelity and processivity are in progress.
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1997-06-25
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