Elvitegravir (EVG) is a new ARV with demonstrated clinical efficacy in both treatment-naive and -experienced HIV-1-infected patients (6
). However, as shown in vitro
and in some patients failing EVG-containing regimens, primary IN mutations can develop, leading to reduced EVG susceptibility. HIV-1 IN mutations T66I, E92Q, S147G, and Q148R/K were first identified as primary EVG RAMs in cell-based resistance selection experiments (10
). In patients experiencing virologic failure on EVG-containing regimes, additional RAMs, including T66A/K, E92G, T97A, Q148H, and N155H, have been observed (7
). Thus, overall, primary EVG RAMs may develop at six amino acid positions in HIV-1 IN (). Recombinant forms of HIV-1 and IN were used to determine the effect of each primary EVG RAM on phenotypic susceptibilities to INSTIs. Fold change values relative to the WT were comparable in both types of HIV-1 drug susceptibility assays used (single cycle and multiple round) and were further confirmed in enzymatic strand transfer IN activity assays. These results demonstrated a ranked order among primary EVG RAMs whereby the most common clinically observed primary IN mutations (E92Q, Q148R, and N155H) exhibited the greatest reduction in EVG susceptibility, consistent with previous reports (10
). The finding that T97A showed only very-low-level reduced EVG susceptibility in the multicycle assay is similar to its effect on RAL susceptibility in patients experiencing virologic failure on RAL-containing regimens (32
). This special case of a primary EVG RAM suggests that additional amino acid substitutions may serve an accessory role for T97A to further reduce phenotypic susceptibility.
Structural analyses have shown that both EVG and RAL share similar scaffolds and binding modes within the IN active site leading to inhibition by disengaging the DDE catalytic triad from the viral DNA (29
). Thus, it is not surprising that both EVG and RAL exhibit largely overlapping primary INSTI resistance profiles in HIV-infected patients experiencing virologic failure on either INSTI. For RAL, three independent common primary RAMs, Y143R/H/C, Q148R/H/K, and N155H, have been identified (14
). Both N155H and Q148R/H/K are associated with the development of EVG cross-resistance, whereas Y143R/H/C mutations do not lead to decreased EVG susceptibility, likely due to the absence of a RAL-specific oxadiazole group to interact with (17
). Importantly, E92Q and T97A have also been observed in HIV-1-infected patients experiencing virologic failure on RAL, either independently or in association with N155H and Y143R/H/C, respectively (18
). While E92Q is often referred to as a secondary RAL RAM (16
), our data indicate a significant impact on EVG and RAL resistance, independent of other mutations. Small geometric differences between the interactions of EVG and those of RAL within IN may account for the observed variability in cross-resistance (T66K, E92Q, and Q148R/H/K) and why certain primary EVG RAMs (T66I/A, E92G, and S147G) retain susceptibility to RAL while certain primary RAL RAMs (Y143R/H/C) retain susceptibility to EVG.
Structural analyses have suggested that DTG shares a similar interfacial mechanism of inhibition with EVG and RAL but is able to make more intimate contacts with the viral DNA. In addition, DTG may effectively be able to readjust its position and conformation to structural changes in the active sites of EVG- or RAL-resistant IN enzymes and avoid some cross-resistance due to slower dissociation (15
). This may explain why primary high-level resistance to DTG does not readily occur in vitro
and appears difficult to establish de novo
clinically. However, recent findings indicated that the Q148R/H/K resistance pathway observed for EVG and RAL can impact cross-resistance to DTG as the number of secondary mutations increases (i.e., E138K, G140S, and N155H) (15
). The data presented in this study extend these observations by demonstrating that DTG retains activity against all individual primary EVG and RAL RAMs. Taken together, these results support clinical evidence that subjects who experience virologic failure on EVG-containing regimens respond poorly to treatment with RAL (45
) and that DTG may have utility as a second-line regimen in cases of virologic failure with EVG or RAL, although multiple mutations that include Q148R/H/K may reduce responses (46
The selective advantage of primary EVG RAMs in patients with virologic failure while on EVG-containing regimens drives both the initial appearance of these mutations and a pathway toward higher levels of resistance. While the development and prevalence of primary IN mutations may be ascribed to their effect on EVG susceptibility, their effect on viral replication fitness is equally relevant. HIV-1 with reduced EVG susceptibility from patients experiencing virologic failure on EVG typically exhibits a reduced replication capacity (36
). The results of the PhenoSense IN HIV assay showed that most primary EVG RAMs are associated with a reduction in replication capacity. Previous studies of primary RAL RAMs in recombinant forms of HIV-1 have reported similar findings (10
). Growth competition analyses further demonstrated reduced viral fitness of mutants expressing EVG RAMs and suggested a correlation between increased EVG resistance and decreased viral fitness. These results suggest that certain viral variants with primary EVG RAMs may emerge first because they are more fit and not necessarily more resistant. Indeed, previous studies of primary RAL RAMs suggested that an N155H mutant is likely to emerge before Q148R/H/K mutants because it has a substantial fitness advantage (50
). The results reported here extend this interpretation to include other common primary IN mutations (T66I, E92Q, and S147G). Moreover, of the most commonly observed viral mutants in patients experiencing virologic failure on EVG-containing regimens (6
), both the E92Q and N155H mutations appear to exhibit a selective advantage, with reasonable viral fitness and significantly reduced susceptibility to EVG.
Patients exhibiting virologic failure on EVG or RAL therapy typically harbor a complex dynamic population of primary IN mutations that are generally not found on the same HIV-1 genome. The following combinations of primary INSTI RAMs have, however, been found to colocalize in rare exceptions: T97A+Y143R/C in RAL failures, S147G+Q148R/H/K in EVG failures, and E92Q+N155H in both RAL and EVG failures (14
). To understand the underlying typical mutual exclusivity among primary EVG RAMs, dual combinations were introduced into recombinant viral vectors. As expected, the addition of a second primary IN mutation further reduced EVG and RAL susceptibility relative to that of either mutation alone. In contrast, DTG retained susceptibility against most double primary IN mutants but showed significantly reduced susceptibility in dual combinations with Q148R. While the Q148R+N155H mutant has been shown to reduce DTG susceptibility (15
), the finding that other primary EVG RAMs (T66I or E92Q) in combination with Q148R also have the same effect has not been reported. The addition of a second primary IN mutation also further reduced replication capacity and, where studied, viral fitness. The extent of this reduction for the E92Q+N155H mutant was least among all dual combinations, in agreement with previous assessments of replication capacity (16
) and the observed coexistence of these primary INSTI RAMs in resistant viral variants. While an earlier report found the E92Q+N155H mutant to be more fit than the N155H mutant in growth competition experiments (51
), the discordance in these results could be attributable to differences in the viral backbone utilized (HIV-1LAI/IIIB
, respectively). With prolonged selective pressure to EVG or RAL, Q148R/H/K with compensatory secondary mutations (E138K or G140S/A) tends to replace earlier primary INSTIs RAMs as the dominant species (19
). The results presented here suggest that viruses bearing an additional primary IN mutation fail to gain a similar selective advantage despite a further reduction in phenotypic susceptibility. Thus, genotypic switching among primary EVG RAMs over a period of virologic failure likely represents active emergence of distinct and progressively dominant viral variants.
Not surprisingly, most primary EVG RAMs surround the catalytic pocket of IN and are proximal to the catalytic DDE triad and other active-site components (29
). Whereas T66 and T97 lie slightly outside the active site, D64 and E152 surround N155, D64 and D116 surround E92, and all three acidic residues of the catalytic triad are close to S147 and Q148. Since the Q148 residue lies within the catalytic β4-α2 loop, Q148R/H/K mutations would change the flexibility of this loop, which may (i) hamper metal ion binding by D64 and E152 and chelation by EVG (54
), (ii) sterically affect binding of the viral DNA 5′ end (55
), and (iii) weaken the backbone-backbone H-bond interaction between Q148 and E152 (34
). In contrast, an S147G mutation within the same catalytic loop may have a less dramatic impact on loop flexibility, potentially affecting viral DNA binding, strand separation, and interactions of EVG with adjacent residues. Residue 155 lies within the α4 helix, and an N155H mutation here has been predicted to (i) perturb a salt bridge with the phosphate of the terminal 3′ adenosine, (ii) affect coordination of the metal ion by E152, and (iii) widen the base of the catalytic pocket (34
). Although T66 lies within the β2 sheet distal from the DDE triad, its proximity to the viral DNA 3′ end and N155 suggests that T66I/A/K mutations may (i) sterically affect viral DNA binding and/or (ii) metal ion coordination through N155. The E92 residue lies within a β3-α1 loop less than 4 Å from a metal ion and another 1 to 2 Å from the isobutyl substituent of EVG. An E92Q/G mutation, which results in the loss of a negative charge, may (i) stabilize the metal ion by making hydrogen bonding with a coordinating water molecule less favorable and (ii) remove any repulsion charge with the hydroxyl in the isobutyl substituent of EVG (10
). Finally, although T97 lies within the α1 helix farthest away from the IN active site, a T97A mutation would eliminate a hydroxyl moiety and the H-bond interaction with N120, thus unblocking the α2 helix and creating an empty space in the active site. This structural change may (i) permit small movement of the viral DNA, (ii) affect proper placement of the β4-α2 catalytic loop (32
), and/or (iii) perturb coordination of a metal ion with D116 in a manner similar to that of E92Q. Taken together, all primary EVG RAMs are hypothesized to subtly change the IN active-site environment by affecting local secondary structure and network interactions involved in coordination of the metal ions and/or the viral DNA. These interpretations are in agreement with the higher koff
rates for INSTI dissociation from IN-DNA complexes and reductions in phenotypic susceptibility to INSTIs (43
In summary, the key primary EVG RAMs observed in HIV-1-infected patients experiencing virologic failure on EVG-containing regimens (T66I/A/K, E92Q/G, T97A, S147G, Q148R/H/K, and N155H) have been defined and characterized. All primary IN mutations showed both reduced EVG susceptibility and reduced viral fitness. Moreover, these attributes correlated with both reduced activities and inhibition sensitivity of the HIV-1 IN enzyme. Multiple primary EVG RAMs detected by population sequencing commonly represent mixtures of multiple primary IN mutations that are not capable of coexisting on the same HIV-1 genome, likely due to more severely attenuated viral fitness. Patients who experience initial virologic failure on EVG-containing regimens typically transition toward EVG RAMs that confer greater phenotypic resistance and greater viral fitness with the accumulation of compensatory secondary IN mutations. Thus, as with other classes of ARVs, prolonged virologic failure under continuous EVG-selective pressure should be avoided in order to minimize resistance development within the class of INSTIs.