Background: The emergence of resistance mutations to protease inhibitors (PI) of the HIV-1 not only reduces the binding of the PIs to the protease but also decreases the viral replication capacity (VRC). Nevertheless, it remains to be elucidated the long-term effect of HIV-1 protease mutations on virus VRC. We observed a positive correlation between a protease sequence conservation, related to the ancestral subtype B sequence, and its in vitro viral fitness (r2 = 0.1115, p = 0.0005). Here, we aim to explore the robustness of the HIV-1 Protease.
Methods: We have studied 3 groups of viral protease sequences, two of them obtained from naive-infected patients in two different periods and a third group from infected patients treated with one or more PIs. Firstly, sequence conservation of 139 naive-infected patients from our clinical unit, 89 obtained in 1993-1994 and 50 obtained in 2006-2007 was analyzed. Secondly, VRC of 33 recombinant viruses encoding plasma-derived proteases from 11 patients from each group was measured. We calculated VRC as the slope of the natural log of viral antigen p24 production in the cell culture supernatants between days 0 and 7 post-transfection. We also measured the VRC of these 3 groups of proteases after being hypermutated in vitro using an error-prone PCR (1.56 x 10-2 nucleotide + 4.88 x 10-3). The VRC from the hypermutated recombinant viruses was related to their wt recombinant virus. (Student t-test).
Results: The average conservation of the protease sequences from the naive-infected patients dropped significantly between the sequences isolated in 1993-1994 (98%, nt; 96.5%, aa) and the ones isolated in 2006-2007 (96%, nt; 95%, aa) (p< 0.0001, nt; p< 0.0001, aa). Recombinant viruses from 1993-1994 naive-infected patients had better VRC than the 2006-2007 ones. Similarly, both groups displayed a better viral fitness than the recombinant viruses carrying proteases with resistant substitutions to PIs. Nevertheless, these differences were not significantly different. For the in vitro hypermutated recombinant viruses, the VRC significantly diminished in all of the three groups. The hypermutated recombinant viruses from the PIs resistant patients suffered the highest VRC drop.
Conclusions: Viruses under selective pressure of PIs appear to be more vulnerable to the emergence of new mutations, whereas viruses from naive-infected patients seem to be more robust independently of their conservation to ancestral sequences.