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Insertions between Codons 35 and 38 of the Protease Gene in HIV-1 Strains from Patients Failing Antiretroviral Therapy M. A. WINTERS*1, E. KIM1, S. CHOU2, A. WARFORD1, R. KAGAN3, R. FENWICK3, L. KOVARI4, and T. C. MERIGAN1.
1Stanford Univ., CA; 2Univ. of Oregon, Portland; 3Quest Diagnostics, San Juan Capistrano, CA; and 4Wayne State Univ., Detroit, MI Objective: To evaluate protease gene mutations in patients failing antiretroviral therapy.
Methods: Genotyping was performed by population-based sequencing of plasma-derived HIV RNA. Following RT-PCR, amplicons were sequenced in both directions by cycle sequencing using standard dideoxyterminator methods. Structure analysis was performed using homology protein structure modeling.
Results: Eight of 8396 patients genotyped over the past 2 years were found to have HIV strains that possess an insert in the protease gene. The inserts were composed of 1, 2, or 5 amino acids that mapped between codons 35 and 38. The genotypes of the inserts were typically a duplication of neighboring sequences. Four strains had at least 2 major resistance-associated protease gene mutations (G48V, I54V, V82A, I84V, and/or L90M) with an average of 10 other amino acid changes. The 4 other strains did not possess any major protease gene mutations, but had an average of 7 other changes compared to consensus B. The inserts appeared to be selected during protease inhibitor therapy, as available pre-therapy samples did not show evidence of the insert, and 522 sequences in various databases from protease naive patients did not possess protease gene inserts. Molecular modeling of the patient protease enzymes indicates that the inserts exist in a region on the surface of the molecule that possesses structural flexibility. The presence of the insert also resulted in structure alterations at distant regions of the molecule. Experiments are in progress to assess the impact of these genotypic features and structural changes on drug susceptibility.
Conclusions: A diverse set of inserts in the protease gene of HIV-1 can develop during protease inhibitor therapy. While the drug susceptibility profile is unclear, the drug-selected origins of these complex strains suggest that the inserts are likely to be involved in changes in protease inhibitor susceptibility and/or protease enzyme kinetics.
Key Words: HIV, mutations, protease
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