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5th International Workshop on HIV Drug Resistance & Treatment Strategies4-8 June 2001, Scottsdale, Arizona |
BACKGROUND: T-20 inhibits HIV-1 fusion to target cells by blocking the interaction of two heptad repeats (HR) in the extracellular portion of gp41. Mutations in HR-1, selected by in vitro passage of HIV-1 in the presence of T-20, confer resistance to this drug. We tested the relative fitness of T-20-resistant variants in growth competition experiments using a recombinant marker virus assay (RMVA).
METHODS: An env-deleted proviral clone with a unique BstEII site at the deletion junctions was constructed from pNL4-3. Marker virus vectors pNL4-3&DeltaenvBstEIIhisD and pNL4-3&DeltaenvBstEIIPLAP were constructed by inserting salmonella histidinol dehydrogenase (hisD) or human placental heat-stable alkaline phosphatase (PLAP) sequences in nef. Infectious recombinant viruses carrying the hisD or PLAP marker were generated by co-transfecting the appropriate marker vector together with the env gene of interest into MT-2 cells. Resulting virus stocks were tested in growth competition assays. The proportion of each species in the viral population over time was estimated by quantifying hisD and PLAP sequences using Taqman PCR. To control for possible effects on fitness of the markers’ sequences, reciprocal experiments were performed in which the mutant env gene was linked to hisD or to PLAP.
RESULTS: The I37T mutation conferred a relative loss of fitness compared to wild-type (WT) of 8.6% when linked to hisD and an 8.9% fitness loss when linked to PLAP. Likewise, the V38M mutation conferred a relative fitness loss of 8.9% when linked to hisD and 11.3% when linked to PLAP. A double mutant carrying the D36S/V38M mutations had a relative fitness of 9.45% less fit than WT when linked to hisD, and 8.3% when linked to PLAP.
CONCLUSIONS: This study demonstrated a fitness loss of approximately 10% for NL4-3 carrying T-20 resistance mutations I37T, V38M or D36S/V38M in the HR-1 domain of gp41, which is comparable to the effect on fitness of the M184V mutation for lamivudine resistance. Reduced fitness could contribute to persistent antiviral activity of T-20 in the absence of complete viral suppression. Comparison of baseline and follow-up samples from patients receiving T-20 in ongoing clinical trials may provide confirmation of these in vitro results.
PRESENTING AUTHOR: J Lu
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