Recombinant infectious molecular clones: associated clinical data, phenotypic resistance, and amino acid substitutions.
shows the PI resistance amino acid substitution patterns, phenotypic resistance test results, and replication capacities (RCs) of 14 multi-PI-resistant recombinant infectious molecular clones and the PI treatment history of the patients from whom the cloned samples were obtained. Among the 12 patients for whom the complete PI treatment history was available, the median number of PIs received was 4, and the median duration of treatment was 8 years.
Panel of prototypical multiple-PI-resistant recombinant infectious molecular clonesa
Based on their in vitro susceptibility, the clones were categorized into four groups: group 1, with resistance to the earliest-approved PIs (FPV, IDV, NFV, and SQV) and ATV; group 2, with resistance to the earliest-approved PIs, ATV, and LPV; group 3, with resistance to the earliest-approved PIs, ATV, LPV, and TPV; and group 4, with resistance to all PIs. Groups 1, 2, 3, and 4 had medians of 11.5, 15, 15.5, and 24 substitutions and medians of 4.5, 5, 5, and 10 study-defined PI resistance amino acid substitutions, respectively.
Twenty-nine of the 41 study-defined PI resistance amino acid substitutions were present in one or more of the recombinant infectious molecular clones. Substitutions V32I, L33F, M46I, I54V, V82A, I84V, and L90M were found in 5 to 10 of the 14 clones. Substitutions L10F, V11I, L24I, K43T, M46L, I47V, G48V, I54M, Q58E, G73S, and I89V were found in two to four of the clones. Substitutions D30N, I47A, I50V, F53L, I54S, G73T, T74P, V82T, V82L, and N88D were each found in one clone. The accessory PI resistance amino acid substitutions L10IV, K20RIMVT, M36IL, L63P, and A71VTI each occurred in eight or more clones. Among the 14 virus stocks for which RC results were available, the median RC was 14% (range, 3% to 94%).
Each of the 14 clones had one or more previously described compensatory NC/P1 gag
cleavage site substitutions (13
): (i) A431V was present in all clones except clones 634, 1391, and 6585; (ii) K436R was present in clone 1319; and (iii) I437N was present in clones 634, 3972, 6585, and 38129. Eight of the 14 clones had one or more previously described P1/P6 cleavage site substitutions (13
): (i) L449VF in clones 634, 3972, and 14311; (ii) R532S in clone 794; and (iii) P453LF in clones 1556, 4307, 18369, and 38129. P6 insertions containing a PTAP motif were present in clones 634 and 6585.
Correlation network analysis of PI resistance amino acid substitution patterns in the HIVDB.
Of 61,989 group M HIV-1 protease-containing viruses from 59,455 individuals, 11,351 viruses from 10,050 individuals had one or more PI resistance amino acid substitutions. Of these 11,351 variants, 9.5% were from PI-naive individuals, 20.2% were from individuals who had received one PI, 10.0% were from individuals who had received two PIs, 7.3% were from individuals who had received three PIs, 8.9% were from individuals who had received four or more PIs, and 44.1% were from PI-treated individuals for whom the exact number of PIs received was not known. A total of 1.9% of isolates were obtained prior to 1996, 31.3% were obtained between 1996 and 2000, 58.8% were obtained between 2001 and 2005, and 9.4% were obtained between 2006 and 2012. Nearly 14% of the isolates belonged to a non-B subtype, including 3.5% of isolates in subtype F, 2.8% in subtype C, 1.7% in subtype G, 1.3% in CRF01_AE, 1.2% in subtype A, 1.1% in CRF02_AG, 1.1% in subtype D, and 1.1% belonging to miscellaneous other subtypes and circulating recombinant forms (CRFs).
Twenty-five percent of sequences had one study-defined PI resistance amino acid substitution, 17% had two PI resistance amino acid substitutions, 16% had three PI resistance amino acid substitutions, 14% had four PI resistance amino acid substitutions, 11% had five PI resistance amino acid substitutions, and 17% had six or more PI resistance amino acid substitutions. The 11,351 isolates had 3,139 unique patterns of PI resistance amino acid substitutions. L90M was the most common PI resistance amino acid substitution, occurring in 51% of isolates with one or more PI resistance amino acid substitutions; substitutions I54V, V82A, M46I, I84V, L33F, G73S, M46L, and L10F occurred in 10% to 34% of isolates; substitutions D30N, L24I, N88D, V32I, F53L, Q58E, I47V, G48V, L89V, V82T, L76V, I54L, I54M, G73T, I50V, V82F, T74P, and N88S occurred in 2% to 9% of isolates; and substitutions G73C, I54A, V82S, I54T, I54S, V82C, I50L, G48M, G73A, I47A, and V82L occurred in <2% of isolates. Although most PI resistance amino acid substitutions increased in frequency with each calendar year, the L33F, V11I, I47V, Q58E, L10F, I84V, K43T, and I54ML substitutions demonstrated the greatest increase in prevalence per year since 2000.
shows those amino acid substitution pairs with the highest levels of pairwise correlation using the Spearman correlation coefficient (rho). The two most highly correlated substitutions, D30N and N88D, displayed no significant positive correlations with other PI resistance amino acid substitutions. In contrast, most of the other PI resistance amino acid substitutions were correlated (rho > 0.1; P < 1e−16) with two or more other PI resistance amino acid substitutions. The V32I, L33F, I47V, V82A, and I84V substitutions were significantly correlated with 10 to 11 other PI resistance amino acid substitutions; M46I, I54V, I54M, L89V, and L90M were correlated with 7 to 9 other PI resistance amino acid substitutions; and L10F, K43T, F53L, I54L, G73S, and L90M were correlated with 5 to 6 other PI resistance amino acid substitutions. The I50L, Q58E, V82FL, N83D, and N88S substitutions were not correlated with any other PI resistance amino acid substitution.
Positively correlated PI resistance amino acid substitutions ranked by Spearman's correlation coefficient
shows the protease inhibitor amino acid substitution patterns of group 1 to 4 recombinant infectious molecular clones superimposed on the correlation network created from the adjacency matrix of amino acid substitution correlations in more than 10,000 published protease sequences. As indicated in Materials and Methods, the following amino acid substitutions at the same position were represented by a single amino acid: I47VA, G48VM, I54TAS, I54ML, G73STCA, and V82ATS. The graphs do not show the PI resistance amino acid substitutions I50L, Q58E, V82FL, N83D, and N88S, which were not correlated with any other PI resistance amino acid substitutions. Substitutions D30N and N88D, which correlated only with one another, were not shown either.
Fig 1 Protease inhibitor (PI) amino acid substitution patterns in the panel of 14 multiple-PI-resistant recombinant infectious clones. (A) Group 1 clones; (B) group 2 clones; (C) group 3 clones; (D) group 4 clones. The amino acid substitutions within a clone (more ...)
Three of the four group 1 clones and the pan-PI-resistant group 4 clones had cluster indexes of between 80 and 99%, indicating that the medians of their pairwise shortest-path distances were lower than those of matched random substitution patterns (). In contrast, the group 2 and group 3 clones had clustering indexes that did not differ from matched random substitution patterns.
lists the 27 maximal cliques generated from the matrix of significantly correlated pairs of PI resistance amino acid substitutions. Two maximal cliques contained 2 amino acid substitutions, 7 contained 3 amino acid substitutions, and 18 contained 4 or 5 amino acid substitutions. Nine of the 27 substitution patterns comprising a maximal clique were found in a clinical virus sample without any other study-defined PI resistance amino acid substitution. The remaining 18 patterns occurred only in the presence of one or more additional study-defined PI resistance substitutions. The substitutions comprising all but four of the cliques were present in one or more of the recombinant infectious molecular clones.
Presence of maximal cliques of PI resistance amino acid substitutions created from strongly correlated pairs of PI resistance amino acid substitutions in the panel of recombinant infectious molecular clonesa