18th International HIV Drug Resistance Workshop Basic Principles & Clinical Implications June 9–13 2009, Fort Myers, Florida, USA

RECOMBINANT VIRUSES EXPRESSING SUBTYPE B OR SUBTYPE C REVERSE TRANSCRIPTASE REVEAL NO DIFFERENCE IN THE RATE OF K65R RESISTANCE TO TENOFOVIR IN CELL CULTURE

Antivir Ther 2009; 14 Suppl 1:A23 (abstract no. 21)

C Dobard, J Lipscomb, JA Johnson, JG Garcia-Lerma and W Heneine
Centers for Disease Control and Prevention, Atlanta, GA, USA

BACKGROUND: Recent studies have shown that HIV subtype C isolates select K65R with tenofovir (TFV) in vitro more rapidly than subtype B isolates. The rapid selection of K65R was attributed to natural polymorphisms in subtype C reverse transcriptase (RT) at codons 64 and 65 that are absent in subtype B. To better understand the contribution of subtype C RT to the selection of TFV resistance in vitro, we compared the kinetics of emergence of K65R using recombinant viruses expressing the RT from subtype B or subtype C in a subtype B (HXB2) virus backbone.

METHODS: Full-length wild-type RT sequences were amplified from six HIV type-1 subtype B isolates and three subtype C isolates by RT-PCR and were used to generate recombinant viruses from a RT-deleted HXB2 backbone. In vitro selection of TFV resistance was carried out in MT4 cells under the same conditions using increasing concentrations of TFV (from 4 to 24 µM). Emergence of drug resistance was monitored in culture supernatants using conventional sequencing and a sensitive real-time PCR assay specific for the K65R mutation (detection limit = 0.4%)

RESULTS: Sequence analysis revealed all recombinant subtype C viruses contained the signature polymorphisms (bold) at RT codons 64 (AAG→AAA) and 65 (AAA→AAG). K65R was selected in all 6 recombinant subtype B viruses after a median of 45 days (range 39–51) or during culture with 24 µM of TFV. Similarly, all 3 recombinant subtype C viruses selected K65R during the same period of time (median 40 days and range 39–48 days) and TFV concentration (24 µM). The analysis of K65R by sensitive real-time PCR showed no significant difference in the rate of emergence of K65R over time.

CONCLUSIONS: In our in vitro system, the kinetics of K65R selection was similar for recombinant viruses expressing either subtype B or subtype C RT. These findings do not exclude the possibility of different selection conditions or viral regions other than the RT influencing the selection of K65R in subtype C isolates.

2009-06-09
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