Objectives: To determine the underlying biochemical mechanisms responsible for the diminished viral replicative capacity associated with K65R/M184V-containing viruses.
Methods: We studied the efficiency of (-)ssDNA synthesis by recombinant wild-type and mutated HIV-1 reverse transcriptases in cell-free assays. In addition, we determined susceptibility levels to nucleoside analog reverse transcriptase inhibitors (NRTIs) both in cell-free and cell culture assays.
Results: We observed that the K65R/M184V mutations in reverse transcriptase caused reductions in the efficiency of initiation of (-)ssDNA synthesis by increasing pausing at positions +3 and +5 as well as diminished RNA usage. These findings were confirmed in cell culture data using MT-4 cells and cord blood mononuclear cells.
Conclusions: The simultaneous presence of K65R and M184V in reverse transcriptase has a negative impact with regard to the efficiency of initiation of (-)ssDNA synthesis and RNA usage, that exceeds the effect of either mutation on its own. These mechanisms, among others, are responsible for the diminished viral replicative capacity observed in tissue culture when K65R/M184V-containing viruses are studied.
From the aMcGill AIDS Centre, Lady Davis Institute-Jewish General Hospital, Montreal, Canada
bDepartments of Experimental Medicine, Canada
cMicrobiology and Immunology, McGill University, Montreal, Quebec, Canada.
Received 24 July, 2006
Revised 17 October, 2006
Accepted 28 November, 2006
Correspondence to Dr Mark A. Wainberg, McGill AIDS Centre, Lady Davis Institute-Jewish General Hospital, 3755 Côte-Ste-Catherine Road, Montreal, Quebec, H3T 1E2, Canada. E-mail: firstname.lastname@example.org
* This paper is dedicated to Nancy and Jack Cooperberg.