Resistance to HIV-1 Reverse Transcriptase Inhibitors
Session Day and Time: Tuesday 1:30 - 3:30 pm
Room: Hall A
Background: A set or subset of 5 mutations (A62V, V75I, F77L, F116Y, and Q151M) in the viral reverse transcriptase (RT) confers multi-dideoxynucleoside resistance (MDR) on HIV-1. Among the 5 mutations conferring MDR, an HIV-1 variant carrying the Q151M mutation often emerges first in HIV-1 isolated from patients (pts) receiving long-term combination chemotherapy using multiple nucleoside RT inhibitors (NRTIs).
Methods: To investigate the mechanism by which the Q151M mutation in RT requiring a two-base change (CAG→ATG) develops and to understand the reason for the relatively lengthy period of time required for its emergence under therapy with NRTIs, we compared the fitness of a series of infectious molecular clones including 2 putative intermediates (HIV-1Q151K(AAG) and HIV-1Q151L(CTG)) for HIV-1Q151M(ATG), in relation to their sensitivity to drugs. Propagation assays and competitive HIV-1 replication assays (CHRA) were employed for the evaluation of viral fitness of various clones. The data were analyzed in relation to drug sensitivity of such clones to zidovudine and didanosine. Steady-state kinetic constants of recombinant RTs were also determined.
Results: HIV-1Q151L replicated relatively poorly while HIV-1Q151K failed to replicate. When the former was propagated further, HIV-1 took 3 pathways in continuing to replicate: 1) HIV-1Q151L changed to HIV-1Q151M in 8 of 16 experiments; 2) HIV-1Q151L reverted to wild-type HIV-1 (HIV-1WT) in 4 of 16 experiments; and 3) HIV-1Q151L acquired an additional mutation M230I in 4 of 16 experiments which improved HIV-1 fitness. The relative order of replicative fitness in the absence of drugs was: HIV-1Q151M > HIV-1WT > HIV-1Q151L/M230I > HIV-1M230I >>HIV-1Q151L >>> HIV-1Q151K, HIV-1Q151K/M230I. HIV-1Q151M was less susceptible to zidovudine and didanosine, while HIV-1Q151L/M230I was as sensitive as HIV-1WT. Steady-state enzymatic assays determining kcat/Km and Ki values of various recombinant RT preparations strongly suggested that HIV-1Q151L is more replication-competent than HIV-1Q151K in the presence of zidovudine or didanosine.
Conclusions: While HIV-1Q151M most likely develops through a poorly replicating intermediate, HIV-1Q151L, it is also possible that it occurs through 2 concurrent base changes. The present data should explain the mechanism by which HIV-1Q151M emerges after long-term chemotherapy with NRTIs.