Mutated K65R recombinant reverse transcriptase of human immunodeficiency virus type 1 shows diminished chain termination in the presence of 2',3'-dideoxycytidine 5'-triphosphate and other drugs.

Abstract

A lysine-to-arginine substitution at amino acid 65 (K65R) in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is associated with resistance to 2',3'-dideoxycytidine (ddC), 2',3'-dideoxyinosine (ddI), and the (-) enantiomer of 2',3'-dideoxy-3'-thiacytidine (3TC). To further characterize the molecular basis of such resistance, we expressed the p66/pSi heterodimer of wildtype RT, K65R mutated RT, and a doubly mutated (K65R/M184V) RT in Escherichia coli and assessed the characteristics of nucleotide incorporation and chain termination in cell-free reverse transcription reactions in the presence and absence of various nucleoside triphosphate analogs. These reactions employed a HIV RNA template (HIV-PBS) that contained the primer binding sequence (PBS) and the U5 and R regions of HIV-1 genomic RNA and an oligodeoxynucleotide (dPR) complementary to the HIV-1 PBS as primer. The K65R and K65R/M184V RTs showed significantly decreased chain-termination effects during polymerization with the 5'-triphosphates of ddC, 3TC, 2',3'-dideoxyadenosine, and AZT (3'-azido-3'-deoxythymidine) in comparison with wild-type RT. Detailed analysis with ddCTP and wild-type RT revealed that chain termination occurred at all guanines in the RNA template. However, the frequency of dideoxynucleoside triphosphate (ddNTP)-induced chain termination was decreased at certain guanines but not others in reactions catalyzed by K65R RT. Both the K65R mutant RT and wild-type RT had similar processive activity. These results indicate that decreased chain termination of K65R RT in the presence of ddNTPs is consistent with data obtained in viral replication assays.

Role of Murine Leukemia Virus Reverse Transcriptase Deoxyribonucleoside Triphosphate-Binding Site in Retroviral Replication and In Vivo Fidelity

“…This alteration in turn can lead to changes in fidelity. This hypothesis is supported by evidence from mutations introduced at positions K65 and Q151 of recombinant HIV-1 RT, which exhibited decreased mispair extension in vitro as well as resistance to a number of dideoxynucleoside analogs such as 2Ј,3Ј-dideoxy-3Ј-thiacytidine, 3Ј-azido-3Ј-deoxythymidine, 2Ј,3Ј-dideoxycytidsine, and 2Ј,3Ј-dideoxyinosine (14,18,47). Finally, a number of mutations introduced at residue Y115 of HIV-1 RT were shown to affect the frequency of misinsertions and mispair extensions (35,36).…”

Drug Resistance and Drug Combination Features of the Human Immunodeficiency Virus Inhibitor, BCH-10652 [(±)-2′-Deoxy-3′-Oxa-4′-Thiocytidine, dOTC]

“…This K65R mutation has been observed previously in vitro and confers resistance to 3TC, ddC (Gu et al, 1994), PMEA (Gu et al, 1995a;Taylor et al, 1996) and abacavir (Tisdale et al, 1997). The 65R/184V double mutant has also been previously observed in a patient on ddC therapy (Gu et al, 1995b). This virus with dual codon changes at positions 65 and 184 showed only low-level resistance to dOTC (<5-fold).…”

Selection of Resistance-Conferring Mutations in HIV-1 by the Nucleoside Reverse Transcriptase Inhibitors (+/-)dOTC and (+/-)dOTFC

“…Crystallographic and modelling studies showed that the side chain of lysine at position 65 contacts the γ-phosphate of the incoming nucleotide and stacks over arginine at position 72 (Huang et al, 1999). In vitro, RT containing the K65R mutation displayed decreased affinity for the triphosphates of 3TC and ddC, resulting in diminished incorporation and chain termination (Arion et al, 1996;Gu et al, 1995).…”