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1st International Workshop on HIV Drug Resistance & Treatment Strategies25-28 June 1997, St. Petersburg, Florida, USA |
NOVEL CLONAL ANALYSES OF RESISTANCE TO HIV-1 RT AND PROTEASE INHIBITORS
Antiviral Therapy 1997;2 (Suppl 5):30 (abstract no. 45)
J Martinez-Picado1,2, L Sutton2, A Hoes Helfant2, A Savara2, J Kaplan2 and RT D'Aquila2
1Institut de Recerca de la SIDA Caixa, Barcelona, Spain; and 2Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA
OBJECTIVES: (1) To determine if sequencing of multiple clones of pol PCR products improves detection of minority resistant HIV-1 variants; (2) to speed cloning of pol PCR products into new recombinant virus vectors; (3) to simplify analysis of pol genotype at multiple positions; and (4) to evaluate fidelity of proofreading PCR polymerases.
METHODS: Cycle sequencing with an automated sequencer was used for bulk and cloned amplicon templates. Two series of pol-deleted 5´ half HIV-1 genome plasmids and PCR primers were constructed. The first series is a hybrid HxB2/NL4-3 genome with deletions in pro (from HxB2 nucleotide 2259 to 2611) or pro and RT (from nucleotide 2259 to 4147); native sequence is reconstructed by cloning amplicons without ligase using uracil deglycosylase (UDG). The second NL4-3-based series allows either: (1) standard cloning of a restriction fragment which spans gag p7 through all of pro and a portion of RT, or (2) UDG cloning of a fragment which encompasses p7 and all of pro and RT (except for the 3´ 62 bp of pol). Site specific sequencing (SSS) uses T7 polymerase (Sequenase v2) to extend multiple primers of different lengths after simultaneous hybridization to a plasmid template. Each primer is extended by a single base with one of four fluorescein-labelled ddNTPs; extensions are sized on an automated sequencer.
RESULTS: In four patients failing zidovudine/lamivudine/indinavir, bulk sequences were wild-type for one to three of the agents. In one of three of these patients, a clonal analysis identified minority multiply resistant variants: 6/18 clones had permutations of six indinavir- selected pro substitutions; 1/11 clones had four zidovudine-selected RT substitutions, RT 69D and the lamivudine-selected RT 184V. Therefore, we developed clonal analyses to improve detection of minority variants. HIV sequence-specific UDG cloning is quicker and simpler than ligase-mediated cloning, and has at least a similar efficiency. Twenty SSS primers for 18 codons (pro 30, 46, 48, 50, 63, 82, 84, 90 and RT 41, 70, 74, 103, 106, 151, 181, 184, 190, 215) speed electrophoresis and data analysis. SSS results agree with standard cycle sequencing (57 templates). Misincorporation in cloned amplicon sequences occurred using XL polymerase (Perkin Elmer) in late cycles (of 40 cycle PCRs) at approximately 1.5/1000 bases.
CONCLUSIONS: Multiply-resistant mutants were not commonly found in bulk sequences from patients failing lamivudine/zidovudine/indinavir, but were found in 1/3 patients in clonal analyses. The novel deletion vectors facilitate clonal resistance analyses. Data analysis is faster with SSS than cycle sequencing and SSS offers advantages over hybridization-based genotyping. The potential for PCR misincorporation can be limited and monitored, but must be factored into interpretations of clonal analyses.
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1997-06-25
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