analisi del contributo delle poliproteine GAG e POL nello sviluppo della resistenza in pazienti HIV-1 positivi sottoposti a terapia antiretrovirale

The introduction in the mid 1990s of Antiretroviral Therapy in the cure of AIDS has dramatically decreased the morbidity and mortality rate and has significantly extended the lifespan and the quality of life of HIV-1 positive patients. Today there are more than 20 drugs licensed for clinical use, targeting different steps of viral life cycle including viral entry (coreceptor antagonists and fusion inhibitors), reverse transcription (nucleoside, NRTIs, and non-nucleoside inhibitors, NNRTIs, of the viral reverse transcriptase), integration (integrase inhibitors) and viral maturation (protease inhibitors, PIs). Current Guidelines recommend the use of combination therapies, including drugs of almost two classes, in particular one or two NRTIs and one NNRTIs or one PIs, those are considered the standard of care for the treatment of HIV infection. Despite the success of pharmacological combination strategy, the emergence of drug resistance is still a major factor contributing to the therapy failure. The mechanisms of resistance mainly involve mutations directly altering the interaction of viral enzymes and inhibitors (Menendez-Arias, 2010). Nowadays, most of these mutations, defined drug resistance mutations, are well characterized (Johnson et al., 2010) and are used in standard genotypic tests as predictive clues of treatment failure, but in some cases such limited information is not sufficient to explain the virological failure. Emerging studies reveal that, besides the ones encoding Gag and Pol polyproteins, other regions might contribute to the development of resistance. In particular, some specific cleavage sites and non-cleavage site mutations (Ho et al., 2008; Parry et al., 2009; Dam et al., 2009; Nijhuis et al., 2007), as well as frameshift-regulating site mutations in Gag (Doyon et al., 1998), increase cleavage sites accessibility and polyprotein processing, thus compensating for the catalytic loss of function of the viral Protease (PR) induced by primary resistance mutations. Moreover, some residues in the HIV-1 Reverse Transcriptase (RT) are critical for proteolytic processing of Gag-Pol precursors (Nishitsuji et al., 2011; Chiang et al., 2012). Although the augmenting consciousness of the relevance of other viral protein domains, besides PR and RT enzymes, in the prediction and acquisition of resistance to the antiviral formulations, little is known about the function of such specific mutations in the viral life cycle. Moreover, the available data are not unanimous in defining the relative importance of each analyzed region, for example within the Gag protein (Dam et al., 2009; Parry et al., 2007). In this context, we are interested in studying the functional role of HIV-1 Pr55Gag protein as natural substrates of PR and its contribution in resistance mechanisms. We optimized PCR amplification experimental settings and sequencing conditions of the gag gene from clinical isolates of different subtypes. In collaboration with Professor Parisi, University of Padua, we analyzed the gag sequence derived from clinical samples of HIV-1 infected patients failing PR Inhibitors (PIs) and RT Inhibitors (RTIs), selected among a cohort of five infectious diseases units located in Veneto in Northeastern Italy. In order to determine the contribution of the Pr55Gag protein to the resistance mechanisms and its specific function in the viral life cycle, we designed a cloning strategy that allows to analyze the differential contribution of N-terminal and/or C-terminal regions of Gag in the presence or in the absence of the mutated PR-RT. Indeed, each patient derived PCR products could be inserted in an HIV-1 proviral genome. In particular, we used a modified version of the previously described env-complementation system, in which an env-deleted provirus expresses a reporter gene under the transcriptional control of the viral LTR and it is capable of one single round of replication. The HIV-1 envelope glycoprotein is given in trans along with the Rev protein. Among all the patient samples sequenced so far, we selected one characterized by resistant mutations only in the PR coding region to analyze the effect of the mutated N-terminal region of Pr55Gag protein. Our results indicated that: (i) the patient-derived mutations in the N-terminal region of Pr55Gag enhance the RT activity and the p24 content in the supernatant of producing cells in comparison to the wild type context; (ii) the patient-derived MA/CA cleavage site amino acid sequence doesn’t affect the processing ability of the wild type PR; (iii) the infectivity of virions carrying the patient-derived N-terminal region of Pr55Gag is reduced. Our results would contribute to better characterize the role of Gag and the relations with PR and RT in resistance development, their relevance in viral replication and evolution in the presence or in the absence of drugs