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Background:  HIV-1 protease mutations rendering resistance to protease inhibitors (PIs) are phenotypically characterized mainly for B subtype viruses. The concern whether these genotypic profiles of PI resistance are suitable for other non-B clade viruses still needs to be addressed. In this work, we describe the effect of HIV-1 protease L89M substitution (recognized as a molecular signature of many non-B HIV-1 subtypes) over the phenotypic EC50  values for each of the 6 FDA-approved PI. This Leu to Met polymorphism is the same of its neighbor amino acid 90, conferring resistance to Saquinavir (SQV) and Nelfinavir (NFV).

Methods:  Site-directed mutageneses were performed over plasmid-cloned drug susceptible protease genes of subtype B and F (Bwt and Fwt), to respectively create and reverse the M polymorphism of amino acids. 89; as well for the creation of L90M mutation. The same panel of 89 or 90 mutants was created using cloned protease genes of the 2 subtypes carrying complex resistance mutation profiles for Ritonavir and Indinavir (named Bmdr and Fmdr). All the constructs were phenotyped in quadruplicates for PI susceptibility by MT-4 cell -MTT based cell viability assay. We have established the significant values of resistance by using a non-parametric Mann-Whitney U test, for comparison of the quadruplicates of EC50 for each mutagenized virus with the quadruplicates of EC50 for the Bwt L89 L90 drug-susceptible clone. Their replicative capacity were also accessed by competitive fitness assay.

Results:  The L89M mutation conferred 4.5-, 2-, 6-, 4.7-, 4.5-, and 3.4-fold increases in EC₅₀ for IDV, SQV, NFV, RTV, APV, and LPV, respectively, on Fwt 89M90L virus; comparable to the fold resistance values obtained with Bwt 89L90M virus. Surprisingly, Bwt 89M90L clone behaved phenotypically similar to the susceptible Bwt 89L90L virus for the latter 4 PI. The Fwt 89M90L also showed a replicative fitness comparable to the PI-susceptible Bwt 89L90L and were more fit than its counterpart Bwt 89M90L. Computational molecular dynamics analysis of F and B subtype proteases have pointed significant differences, mainly the hydrogen bond between the catalytic residue Asp25 and the hydroxyl group of Nelfinavir.

Conclusions:   The L89M mutation impacts phenotypic resistance to SQV, NFV, RTV, APV, and LPV differently for F and B clade viruses.

Keywords: drug resitance; hiv protease; subtype F