FREQUENCY AND INFECTIOUSNESS OF HIV-1 PROTEASE INHIBITOR RESISTANCE VARIANTS.

CAROBENE M.; RUBIO A.; TURK G.; SARACCO M.; WAINBERG M.; SALOMÓN H.

Paris, Francia

Conferencia; 2nd IAS Conference On HIV Pathogenesis And Treatment; 2003

International AIDS Society

Introduction: The most common mechanism for the generation of drug resistance in HIV-1 infected patients treated with protease inhibitors (PIs) is through the emergence and accumulation of multiple amino acid substitutions within the viral protease (PR). This emergence is a stepwise process and is promoted by ongoing viral replication in the presence of subinhibitory concentrations of these specific inhibitors. The aim of the present work is to analyze the frequency of appearance of mutations in PR selected by most of the PIs currently in use (SQV, IDV, NFV, RTV, APV), in patients undergoing treatment failure, and to perform in vitro assays to evaluate viral infectivity of recombinant clones harboring some PI resistance associated mutations. Methods: Viral RNAs extracted from plasma samples of 470  Argentinean patients undergoing treatment failure were sequenced and the frequency of PI resistance-associated mutations was assessed. Recombinant clones carrying drug resistance-associated mutations at positions 84 (I84V), 90 (L90M) and 84/90 (I84V/L90M) of the PR were constructed  by cloning a fragment containing the PR gene from the sequence of BH10 reference strain and using site-directed mutagenesis procedures. Afterwards,  mutant fragments were put back into  BH10 and transfection of COS 7 cells  was performed to yield infectious particles. Every cloning and mutanegesis step was confirmed by sequencing. Infectivity of recombinant clones was evaluated by Single Cycle Infectivity assay, using GHOST/CXCR4 cells as target for infection, and flow cytometry. Results: Frequency analysis of mutations present in the sequence of the analyzed samples showed that mutations in positions 84 and 90 of the PR region, that causes and/or contributes to clinical  and phenotypic resistance to SQV, IDV, NFV, RTV and  APV, were found in only 4% of the samples (n=19). In these cases, several other mutations in PR were identified as well. The recombinant clones carrying I84V, L90M and I84V/L90M mutations were dramatically impaired i.e. 68-75% in infectiousness compared to wild-type virus (WT). Conclusions: The low frequency of appearance of mutations at positions 84 and 90  in the PR of  our samples may be due to low fitness and/or infectiousness of the variants harboring these mutations.