Three integrase strand-transfer inhibitors (INSTIs) – raltegravir (RAL), elvitegravir (EVG), and dolutegravir (DTG) – are currently used for the treatment of HIV-positive individuals. The latter drug has demonstrated superiority in achieving virological suppression to levels inferior to 50 RNA copies/ml when compared to efavirenz  or darunavir  in treatment-naïve individuals and to RAL in treatment-experienced, INSTI-naive individuals . In addition, DTG has been shown to possess a higher genetic barrier to resistance than any other antiretroviral drug tested so far, and no resistance substitution has been detected to date in treatment-naive individuals undergoing first-line DTG therapy . However, DTG is not immune to resistance in treatment-experienced individuals who have previously experienced treatment failure with RAL or EVG [3,5,6]. Indeed, treatment failure with either RAL or EVG can lead to the emergence of Q148 substitutions, which, when associated with two or more secondary changes, are associated with a decrease in DTG clinical responsiveness . Other resistance pathways, such as the one involving the N155H substitution, can also decrease HIV susceptibility to DTG in tissue culture [7,8] and in INSTI-experienced individuals .
In the absence of pre-existing integrase substitutions, R263K seems to be a signature resistance substitution against DTG in both tissue culture and individuals [3,10]. In tissue culture selection experiments, this substitution emerged from 6 of 7 (86%) subtype B and CRF02_A/G viruses that were grown under DTG pressure, and this confers low levels of resistance against DTG [10,11]. The R263K substitution also decreased integrase enzymatic activity, viral integration, and replicative capacity [10,12], and no substitution that emerged secondary to R263K, that is, M50I, H51Y, and E138K, has been able to compensate for the enzymatic and replicative defects caused by R263K [11–13]. The presence of R263K also hinders the emergence of resistance substitutions against reverse transcriptase inhibitors in tissue culture .
The clinical relevance of the R263K substitution was confirmed by results from the Study of GSK1349572 versus Raltegravir (RAL) with Investigator Selected Background Regimen in Antiretroviral-Experienced, Integrase Inhibitor-Naive Adults (SAILING) trial in which four treatment-experienced participants experienced treatment failure with DTG-based therapy and possessed emergent HIV mutations in integrase. In two such cases, the R263K substitution was identified . These individuals received DTG for an additional 24 weeks following the detection of R263K without the appearance of additional resistance substitutions or worsening of their physical health . Given that one of these individuals possessed a subtype C virus , we characterized the R263K substitution in HIV-1 subtype C in regard to INSTI susceptibility, integrase enzymatic activity in cell-free strand-transfer assays, and viral replicative capacity in tissue culture in comparison with subtype B. Our results show that R263K in subtype C confers cross-resistance against DTG and EVG, but not against RAL. In addition, the decrease in integrase enzymatic activity, viral infectivity, and replicative capacity imparted by R263K in subtype C is more severe than that observed for subtype B.
Cells and reagents
TZM-bl, 293T, and PM1 cells were passaged as described previously . Merck & Co., Inc. (Kenilworth, New Jersey, USA), Gilead Sciences, Inc. (Foster City, California, USA) and ViiV Healthcare Ltd (Research Triangle Park, North Carolina, USA) kindly provided RAL, EVG, and DTG, respectively.
Integrase strand-transfer activity assay
We used site-directed mutagenesis to introduce the R263K substitution into the pET15b-integrase C plasmid for recombinant protein expression by using sense (5′-GGTAGTACCACGGAAGAAAGCAAAAATCATTAAGG-3′) and antisense (5′-CCTTAATGATTTTTGCTTTCTTCCGTGGTACTACC-3′) oligonucleotides . Wild-type and R263K pET15b-integrase B plasmids have been described previously . Recombinant integrase proteins were expressed in and purified from BL21 (DE3) bacterial cells and then used in strand-transfer assays using preprocessed long terminal repeat (LTR) DNA as previously described [10,12]. Following LTR-DNA coating, DNA Bind 96-well plates (Corning) were blocked with 5% BSA and washed in assay buffer, as previously described . Purified recombinant proteins that were either wild-type or carrying the R263K substitution were added and incubated at room temperature for 30 min. Biotinylated target DNA was then added and strand-transfer reactions were allowed to occur for 1 h at 37°C. Integrated biotinylated target DNA was detected after washes through the use of europium-labeled streptavidin molecules.
Generation of replication-competent genetically homogenous HIV-1 variants
The pNL4.3-wild-type and -R263K plasmids have been described previously . The pMJ4IN(R263K) plasmid was generated through site-directed mutagenesis using the primers described above. The recombinant pNL4.3INC and pNL4.3INC(R263K) plasmids were produced by replacing subtype B integrase sequences in pNL4.3 with the integrase coding sequences from either pMJ4 or pMJ4IN(R263K) plasmid, respectively. All plasmids were verified by sequencing. Lipofectamine 2000 (Life Technologies, Burlington, Ontario, Canada) was used to transfect 293T cells with the above mentioned plasmids to produce genetically homogenous viral stocks. After 48 h, cell culture fluids were harvested, treated with Benzonase (Sigma–Aldrich, Oakville, Ontario, Canada) and filtered at 0.45 μm. Viral stocks were aliquoted and stored at −80°C. Viral stocks were quantified by measuring both p24 content and cell-free reverse transcriptase activity.
Susceptibility to antiretroviral compounds
HIV susceptibilities to DTG, RAL, and EVG were measured in TZM-bl cells using serial dilutions of drugs, as previously described . Cells were lyzed 48 h after infection with NL4.3-wild-type, NL4.3-R263K, MJ4-wild-type, or MJ4-R263K viruses, and luciferase production was measured using the Luciferase Assay System (Promega, Madison, Wisconsin, USA). Fifty percent effective inhibitory concentrations (EC50) and 95% confidence intervals (CIs) were calculated using GraphPad Prism 4.0 software (La Jolla, California, USA).
HIV-1 infectivity was measured through titration of the NL4.3-wild-type, NL4.3-R263K, MJ4-wild-type, and MJ4-R263K viruses in TZM-bl cells. Levels of infection were measured using the luciferase assay system described above. Fifty percentage effective concentrations (EC50) of viruses were calculated using the GraphPad Prism 4.0 Software.
Homology models of HIV-1 integrase proteins were generated using the ProtMod server (http://ffas.burnham.org) based on lead HIV-1 subtype C templates, which were initially generated using the I-TASSER protein structure prediction server [17–19]. Previously published structures were obtained through the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB) (http://www.rcsb.org/pdb/). Molecular modeling was performed as previously described , using as a template the prototype foamy virus (PFV) integrase structure in either the target capture complex (PDB ID: 4E7K)  or in complex with EVG (PDB ID: 3L2U) . The molecular visualization program PyMOL (DeLano WL, The PyMOL, Molecular Graphics System, Version 1.3 Schrödinger, LLC; DeLano Scientific, San Carlos, California, USA; http://http://www.pymol.org) was used for image processing and structural visualization. Model quality was assessed based on the root mean square deviation (RMSD) of the global homology model from the PFV lead structure. Rotamer orientations for mutated residues and key residues in the active site were carefully examined and the best backbone-dependent rotamers were selected [23,24]. All structures were verified by Ramachandran plot analysis to have greater than 89% of residues in allowed and favorable orientation [25,26].
The R263K substitution confers resistance against dolutegravir and elvitegravir
The SAILING trial reported that the presence of the R263K substitution conferred low levels of resistance against DTG or RAL (fold change < 2) on the basis of the PhenoSense Integrase phenotypic assay . However, the effect of this substitution on the susceptibility of HIV-1 subtype C viruses to EVG was not reported. We have now characterized this substitution in subtype C in regard to susceptibility to DTG, RAL, and EVG using TZM-bl reporter cells (Table 1). Our results show that the R263K substitution decreased HIV-1 subtype C susceptibility to DTG and EVG by 3.3 and 50-fold, respectively. In subtype B, R263K conferred a somewhat lesser degree of drug resistance, that is, 2.1 and 25-fold decreases in susceptibility to DTG and EVG, respectively. R263K did not confer resistance against RAL in HIV-1 subtype C in accordance with results for subtype B.
The R263K substitution decreases HIV-1 subtype C integrase strand-transfer activity
Previous results showed that R263K decreases HIV-1 subtype B integrase cell-free strand-transfer activity . We thus investigated whether the R263K substitution would have similar effects on subtype C integrase. Cell-free strand-transfer assays were performed in the presence of varying target DNA concentrations (Fig. 1). The DNA concentration that was sufficient to reach half of maximal integrase strand-transfer activity was calculated for each of the wild-type and R263K enzymes. The presence of the R263K substitution in subtype C resulted in an increase in integrase half-maximal DNA concentration from 3 to 79 nmol/l (26-fold increase). This was accompanied by a significant decrease in maximal strand-transfer activity for the R263K integrase protein (Fig. 1a) and is a far more significant effect than was observed in subtype B (Fig. 1b).
The R263K substitution decreases HIV-1 subtype C infectiousness
Decreases in integrase cell-free strand-transfer activity have been correlated with decreases in viral infectivity. We performed infectivity assays using MJ4 subtype C viruses into which the R263K substitution had been introduced by site-directed mutagenesis (Fig. 2a). Subtype B NL4.3 wild-type and R263K viruses were used as controls (Fig. 2b). TZM-bl reporter cells were infected with various concentrations of either wild-type or R263K-containing viruses, and the concentration of each virus that was required to attain half-maximal infectiousness was calculated. The results show that the R263K substitution decreased HIV-1 subtype C infectiousness by 70% compared with wild type.
The R263K substitution decreases HIV-1 subtype C replicative capacity
To determine if the decrease in infectiousness observed with the R263K substitution in TZM-bl reporter cells also resulted in diminished long-term replicative capacity, we measured viral replication of wild-type and R263K variants in PM1 cells over 12 days (Fig. 3). Given the relative inability of MJ4 viruses to grow in tissue culture (not shown), we first cloned HIV-1 subtype C integrase coding sequences from the MJ4 wild-type and R263K proviruses into the pNL4.3 plasmid to replace the subtype B integrase coding sequence. We then produced recombinant B/C viruses that express either wild-type (NL4.3INC(WT)) or R263K (NL4.3INC(R263K)) subtype C integrase and monitored replication in PM1 cells over time. The results show that replication of the R263K variant was delayed by more than 3 days compared to wild type. Only after 12 days was a rebound in viral growth seen and, even then, the growth of the R263K variant was only about 50% of that observed with wild-type virus at day 5. The R263K substitution was less detrimental to HIV-1 subtype B viral growth (Fig. 3b).
Impact of the R263K substitution on subtype C integrase structure by homology modeling
Homology models of the wild-type and R263K subtype C integrase proteins were generated using the I-TASSER server with the crystal structures of PFV integrase bound to DNA or INSTIs as templates [21,22,27–30]. No significant differences in overall secondary structure (Fig. 4a) or in the relative position of the D64D116D152 catalytic triad were observed between the wild-type and the R263K enzymes (Fig. 4b). On the basis of the homology model, the R263 residue in wild-type integrase is within 3 Å of LTR-DNA (Fig. 4c) – consistent with its role in DNA interaction. Three positions involved in EVG resistance, that is, P145, Q146, and S147, are within 6 Å from position 263 and may functionally link this residue to EVG binding within the catalytic site through multiple inter-residue interactions (Fig. 4d).
Previous tissue culture selection studies with DTG in subtypes B and C yielded the R263K and G118R substitutions, respectively, in integrase [10,20]. However, the R263K substitution was reported in a subtype C participant in the SAILING clinical trial . Tissue culture studies do not always parallel what is seen in clinical settings, and discrepancies may sometimes be due to polymorphisms that affect integrase strand-transfer activity and resistance against INSTIs [11,20]. For example, both subtype C isolates that were used in our selections with DTG contained an isoleucine (I) at position 50 instead of the methionine (M) that is commonly found in subtype B [10,11], and M50I in subtype B can contribute to DTG resistance in association with R263K . Unfortunately, the contribution of subtype C-specific polymorphisms in the emergence of R263K under DTG pressure remains unclear.
The two subtype B and C participants in the SAILING trial, whose viruses developed R263K, did not worsen physically, nor were additional resistance substitutions ever detected, even though these persons remained on DTG therapy . An explanation for this clinical result is that R263K diminishes viral integration, infectiousness, and replicative capacity [10,31], and that no compensatory mutations for R263K have ever been observed [11–13]. Furthermore, R263K delays the emergence of resistance substitution against reverse transcriptase inhibitors , and exposure to DTG seems to restrict HIV-1 genetic evolution to a greater extent than does RAL, even if the R263K substitution is present . Given that decreases in integration, infectiousness, and replicative capacity likely contribute to the inability of HIV-1 to become highly resistant to DTG in treatment-naive individuals , we have now studied the effects of R263K in subtype C.
Increases in half-maximal target DNA concentrations are indicative of diminished ability of integrase to interact with target DNA . Our results show that R263K was even more detrimental to HIV-1 subtype C than subtype B integrase strand-transfer activity, with a 26-fold increase in the target DNA concentration to achieve half-maximal strand-transfer activity and a 40% decrease in relative maximal activity (Fig. 1a), compared with only a 2-fold increase in half-maximal target DNA concentration and a 15% decrease in maximal strand-transfer activity for subtype B [10,12,13] (Fig. 1b, Table 2). These findings are consistent with reports of residues 262–264 and 273 being involved in DNA binding [10,34–39], as is the finding that the presence of R263K decreased HIV-1 subtype C infectiousness by 70% compared to wild type (Fig. 2), whereas R263K in subtype B only resulted in a 40% decrease [12,13]. Moreover, the decrease in infectiousness associated with R263K (Fig. 2a) translated into a decrease in HIV-1 subtype C replicative capacity (Fig. 3a). Thus, the R263K substitution appears to be more deleterious in subtype C than in subtype B.
These differences may be due, in part, to the presence of different natural polymorphisms in various subtypes. To assess this, we compared the homology structures of subtype C wild-type and R263K integrases in silico (Fig. 4). Although arginine and lysine are both positively charged, arginine is more bulky, less hydrophobic, and possesses a higher pKa than does lysine [40,41], and the positive charge in lysine is localized to a primary amine group (ε-amine) that is limited to one possible hydrogen bond (H-bond), unlike the charge dispersal in arginine over a guanidinium group [42,43]. Delocalization of the positive charge in the guanidinium group of arginine allows the formation of multiple H-bonds in three possible directions . When overlaying DNA ligands from the respective crystal structures were superimposed on the wild-type integrase model, the R263 side chain was within 3 Å of LTR-DNA and from the flexible loop of the catalytic core domain (CCD) (i.e. residues 140–150) (Fig. 4c and d). Residues P145, Q146, and S147 are close to the three acidic residues of the catalytic triad, D64D116E152, and are located in a conserved area of the enzyme . Although a potential loss in interaction with neighboring residues may not strongly impact on the flexibility of the catalytic loop, the presence of R263K might affect DNA binding, hinder metal ion binding by D64 and E152, and alter interactions of EVG-bound integrase with adjacent positions (Fig. 4d).
Residues 145, 146, and/or 147 have been involved in various levels of resistance against INSTIs. The S147G substitution has been selected in the presence of EVG both in patients and in vitro[45,46]. Although substitutions at positions P145S and Q146P have not been reported in published sequences from patients receiving RAL, EVG, or DTG, they have been observed in vitro with EVG [45,47]. HIV-1-containing S147G was eight-fold more resistant against EVG than wild type, but remained susceptible to RAL and DTG [45,48]. The P145S substitution alone conferred greater than 350-fold resistance to EVG, but not against RAL or DTG , whereas a substitution at position Q146 conferred low-level resistance to all three INSTIs. Residues P145 and Q146 are known to contact EVG through hydrophobic interactions and van der Waals forces, respectively (Fig. 4d). The R263K substitution in subtype C may either decrease EVG binding or increase the dissociation of EVG from integrase through interaction with the 145–147 residues, thus explaining the high levels of resistance against this drug that have been observed in tissue culture (Table 1).
We have also shown that R263K in HIV-1 subtype C conferred low levels of resistance against DTG (3.3-fold; Table 1) and high levels of resistance against EVG (50-fold), that is, higher levels than were reported for subtype B (2.3 and 21.8-fold for DTG and EVG, respectively ). However, R263K was innocuous to RAL in both subtypes (Table 1) – a result that may be due to the fact that RAL, EVG, and DTG do not occupy an identical physical space within the catalytic core domain of integrase . The structural changes that are associated with subtype-specific polymorphisms may be sufficient to modulate the binding of members of the INSTI family of drugs.
In conclusion, this study showed that the R263K substitution confers higher levels of resistance against DTG, but is more detrimental to HIV-1 integrase activity and viral infectiousness in subtype C than in subtype B virus (Table 2). These findings help to explain why the viruses of individuals experiencing treatment failure with DTG with the R263K substitution did not acquire additional substitutions following the detection of this substitution at week 24 in the SAILING trial. In addition, our results suggest that individuals who possess a R263K substitution in either HIV-1 subtype C or B integrase should not be treated with EVG. Furthermore, our results suggest that individuals with subtype C viruses will probably have at least as good a response to DTG as patients infected by subtype B viruses. Given the outstanding clinical trial results obtained with DTG to date, our results may justify the scaling up of DTG use in resource-limited countries in which HIV-1 subtype C is endemic.
The project was supported by the Canadian Institutes for Health Research (CIHR).
Authors’ contributions: T.M. designed and performed cell-based experiments, analyzed data, and wrote the manuscript; P.K.Q. designed and performed cell-free experiments and analyzed data; S.H. performed the molecular homology modeling; N.O., Y.H., J.L., and D.N.S. performed experiments; and M.A.W. supervised the project and revised the manuscript. All authors read and approved the final manuscript.
Conflicts of interest
P.K.Q. was the recipient of a CIHR doctoral fellowship. D.N.S. was the recipient of a CIHR doctoral scholarship. S.H. is a recipient of a doctoral studentship from the Fonds de la Recherche du Québec en Santé (FRQS).
The authors declare no competing financial interests.
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