[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to investigate the development and progression of phenotypic resistance to the HIV-1-reverse transcriptase (RT) inhibitor lamivudine, and genotypic variations of HIV-1-RT occurring under lamivudine treatment in HIV-1-infected human primary monocytes-macrophages (M/M).
Cellular passages in the presence of lamivudine were performed every 2 weeks by transferring supernatants of infected M/M to fresh M/M. A fitness assay using wild-type virus and a lamivudine-resistant HIV-1 virus (harbouring the M184V RT mutation) was performed in peripheral blood mononuclear cells. Culture supernatants were tested for p24 antigen production and RT activity. The M184V RT mutant virus was obtained by site-directed mutagenesis on a CCR5-using HIV-1 backbone.
The mutagenized M184V RT virus showed full resistance to lamivudine in M/M. However, no detectable phenotypic and genotypic resistance (neither virus breakthrough, nor RT resistance-related mutations) developed in M/M infected by HIV-1 and cultured for up to seven passages in vitro (i.e. 105 days). This inefficiency of M/M to develop M184V RT mutated virus is tightly related to the low 2'-deoxynucleotide (dNTP) pool in such cells, which in turn decreases the kinetics of HIV-1-RT. Despite this, the M184V RT mutant virus replicates in M/M, although with a 30% decreased efficiency compared with the wild-type.
Our results show that the chances of development of resistance are far lower in M/M than in lymphocytes. This underlines the importance and the peculiar role of M/M as reservoirs of either wild-type or resistant strains in human organs.
[Show abstract][Hide abstract] ABSTRACT: To define the extent of amino acid protease (PR) conservation in vivo in the absence and presence of pharmacological pressure in a large patient cohort.
Plasma-derived complete protein PR sequences from a well-defined cohort of 1096 HIV-1 infected individuals (457 drug-naive and 639 under antiretroviral therapy including PR-inhibitors) were obtained and analysed, and are discussed in a structural context.
In naive patients, the PR sequence showed conservation (< 1% variability) in 68 out of 99 (69%) residues. Five large conserved regions were observed, one (P1-P9) at the N-terminal site, another (E21-V32) comprised the catalytic active-site, a third (P44-V56) contained the flap, a fourth contained the region G78-N88, and another (G94-F99) contained the C-terminal site. In PR-inhibitor treated patients, the appearance of mutations primarily associated with drug resistance determined a decrease of amino acid invariance to 45 out of 99 residues (45% conservation). The overall degree of enzyme conservation, when compared to the PR sequences in drug-naive patients, was preserved at the N- and C-terminal regions, whereas the other large conserved areas decreased to smaller domains containing, respectively, the active-site residues D25-D29, the tip of the flap G49-G52, and the G78-P81 and G86-R87 turns.
Amino acid conservation in HIV PR can be minimally present in 45 residues out of 99. Identification of these invariable residues, with crucial roles in dimer stability, protein flexibility and catalytic activity, and their mapping on the three-dimensional structure of the enzyme will help guide the design of novel resistance-evading drugs.
[Show abstract][Hide abstract] ABSTRACT: The ability of human immunodeficiency virus (HIV) strains to replicate in human target cells represents a major driving force of the progression of the disease. Despite antiretroviral treatment, HIV overcomes drug pressure by adding new (compensatory) mutations, appearing in a specific and sequential order, that modulate its replication capacity and favour viral escape. In the case of M184V (a mutation involving the catalytic site of HIV reverse transcriptase), no pathways of viral escape have been defined so far; it is thus conceivable that the mutated virus maintains a relatively low replicative capacity. At the time of interruption of specific viral pressure (lamivudine in the case of M184V), wild-type virus easily overgrows mutated strains. A deep molecular analysis (90 clones) conducted on proviral DNA of lymphocytes demonstrates that M184V strains are no longer detected in plasma and proviral DNA shortly after interruption of therapeutic regimens including lamivudine (even if a new therapeutic regimen has been started). This supports the concept that the low fitness of M184V strains is not easily compensated by additional mutations. Taken together, the results suggest that the assessment of viral fitness, either direct (through biological methods) or indirect (through the identification of specific mutations that affect the replicative capacity), may provide substantial advancements in the definition of the long-term efficacy of antiretroviral therapy.
No preview · Article · Jan 2004 · Scandinavian journal of infectious diseases. Supplementum