Viral fitness cost prevents HIV-1 from evading dolutegravir drug pressure
© Mesplède et al; licensee BioMed Central Ltd. 2013
Received: 14 November 2012
Accepted: 20 February 2013
Published: 22 February 2013
Clinical studies have shown that integrase strand transfer inhibitors can be used to treat HIV-1 infection. Although the first-generation integrase inhibitors are susceptible to the emergence of resistance mutations that impair their efficacy in therapy, such resistance has not been identified to date in drug-naïve patients who have been treated with the second-generation inhibitor dolutegravir. During previous in vitro selection study, we identified a R263K mutation as the most common substitution to arise in the presence of dolutegravir with H51Y arising as a secondary mutation. Additional experiments reported here provide a plausible explanation for the absence of reported dolutegravir resistance among integrase inhibitor-naïve patients to date.
We now show that H51Y in combination with R263K increases resistance to dolutegravir but is accompanied by dramatic decreases in both enzymatic activity and viral replication.
Since H51Y and R263K may define a unique resistance pathway to dolutegravir, our results are consistent with the absence of resistance mutations in antiretroviral drug-naive patients treated with this drug.
Although highly active antiretroviral therapy (HAART) is lifesaving for people infected by the human immunodeficiency virus (HIV), the long-term efficacy of HAART is limited by drug resistance . This problem exists in both resource-limited  and developed countries . The addition of integrase strand transfer inhibitors (INSTIs) to the arsenal of antiretroviral drugs represents an important advance for the treatment of HIV-positive patients [4–9]. Raltegravir (RAL) and elvitegravir (EVG) are the first INSTIs approved for therapy [10, 11] while dolutegravir (DTG) is in advanced phase 3 clinical trials . Although both RAL and EVG, the first INSTIs, are susceptible to virological failure due to the emergence of resistance mutations within the integrase coding sequence [6, 13, 14], major resistance mutations have not been reported in drug-naive patients who were treated in clinical trials with the second-generation drug, DTG [6, 12, 15, 16]. However, in vitro drug selection experiments performed in our laboratory have identified a R263K mutation within the integrase coding region as a resistance mutation when subtype B viruses were cultivated in the presence of DTG and also showed that H51Y commonly emerged as a secondary mutation . Both of these mutations have also been selected in vitro with EVG and metabolites of EVG although neither is considered to be an important mutation for the latter drug [18–20]. In addition, H51Y was detected in highly treatment-experienced patients failing EVG-containing regimens .
The current work was carried out to further characterize resistance against INSTIs and especially DTG. A common pattern of resistance involving INSTIs and members of other drug classes, including some protease inhibitors (PIs) and nucleoside reverse transcriptase inhibitors (NRTIs), is that a first mutation imparts a minimal level of drug resistance that is accompanied by a loss of enzymatic activity, as well as a diminution in viral replication capacity. We show here that the H51Y mutation in combination with R263K increased resistance to DTG, over that conferred by R263K alone, and was accompanied by a dramatic decrease in integrase strand transfer enzymatic activity, viral replicative fitness, and the ability of HIV DNA to integrate into host cell genomes. In contrast, H51Y on its own did not affect any of these various activities. In view of the possibility that H51Y and R263K may define a unique resistance pathway against DTG, our results provide an explanation for the absence of drug resistance mutations in drug-naive patients who have been treated with DTG.
The addition of H51Y to R263K increases resistance against dolutegravir
Effects of the H51Y and R263K mutations on IC 50 s and 95% confidence intervals for dolutegravir (DTG), raltegravir (RAL), and efavirenz (EFV)
5.714 to 8.324
9.833 to 13.46
0.964 to 1.897
7.707 to 11.17
11.62 to 15.50
0.957 to 1.970
29.91 to 180
10.06 to 15.38
0.763 to 1.712
77.43 to 167.2
15.51 to 38.00
0.852 to 1.723
Effects of the H51Y and R263K mutations on HIV replication capacity and susceptibility to dolutegravir (DTG), raltegravir (RAL), and elvitegravir (EVG) as measured by the PhenoSense® Integrase assay (Monogram Biosciences)
The addition of H51Y to R263K decreases integrase strand-transfer activity
The addition of H51Y to R263K decreases HIV replication capacity
The addition of H51Y to R263K decreases HIV integration
In silicostudies of H51Y mutant integrase
In vitro selection experiments with both DTG and another second-generation compound termed MK-2048 have identified several mutations within the integrase coding region, including G118R, S153Y, and R263K that confer low-level resistance to these drugs [17, 24–27]. Additionally, during selection studies in primary human cord blood mononuclear cells, it appeared as though a secondary mutation H51Y was often present . Here, we have shown that the combination of mutations at positions H51Y and R263K define a unique resistance pathway for DTG. Typically, the emergence of a first resistance mutation usually results in a decrease in relevant enzymatic activity, as has been observed for multiple drugs in different drug classes, whereas secondary mutations are often compensatory and partially or totally restore both enzymatic function and viral fitness [28, 29]. In contrast, we demonstrate that the combination of R263K together with H51Y simultaneously increased levels of resistance against DTG while diminishing both viral replicative capacity and integrase strand transfer activity. Perhaps, more importantly, this combination of mutations further diminished the ability of viral DNA to integrate within the host cell genome beyond the deficit associated with the R263K mutation alone. The H51Y substitution on its own did not affect either DTG drug resistance, viral replication capacity, or integrase strand transfer enzymatic activity, as tested both biochemically and in cell-based assays. We believe that the fitness cost of the H51Y/R263K combination may explain the absence of resistance mutations in integrase inhibitor-naïve patients treated to date with DTG [6, 12, 15, 16], since viruses that possess these two mutations in tandem may replicate so poorly as to be undetectable by the conventional assays that were employed for detection of drug resistance in the above-referenced clinical studies.
A more important issue may be the potential value of using a drug to select for mutations that severely compromise both viral replication capacity and the ability of viral DNA to integrate into host cells. In addition, we have continued our DTG tissue culture selection efforts for over one year and have not yet identified any potential compensatory mutations that might restore integrase function while increasing levels of drug resistance above those seen with the combination of R263K and H51Y. This may be related to either the low replication capacity of viruses containing the H51Y/R263K combination or the poor integrase strand transfer capacity of enzymes containing these substitutions, or both. The findings presented here may be related to the fact that DTG posesses a very long residency time on the HIV integrase enzyme . Of course, it is possible as well that tissue culture drug selection protocols are inadequate to select for all mutations of relevance.
We agree that definitive results that further define drug resistance to DTG, including additional DTG-related mutations, may arise in first-line treatment, especially from the clinical use of DTG after its approval in settings that may not always emphasize the importance of adherence to antiretroviral treatment regimens in the same way as do registrational clinical trials. However, if the results reported here are further clinically validated, consideration should be given to exploring the possibility that the H51Y/R263K combination of mutations, perhaps in concert with other strategies, might reduce or prevent new cycles of HIV DNA integration into host cells.
We are currently in the process of studying whether the R263K and H51Y mutations in simian immunodeficiency virus (SIV) have similar effects as those described here. If so, this would serve to justify further studies in macaque monkeys that employ our mutated viruses, both in the presence and absence of DTG, aimed at achieving retroviral eradication from infected hosts. The use of mutated HIV in humanized mouse models could also be tested.
One caveat of the above arguments is that viruses that fail to achieve integration will not help us to deal with the problem of the HIV reservoir in individuals previously infected by HIV, even if such subjects have been treated with drugs such as DTG. However, it is possible that the withholding of all anti-HIV drugs from treated subjects might result in an activation of latent viruses from reservoirs. In this context, viruses that fail to achieve efficient integration, such as those decribed here, might fail to effectively repopulate reservoirs and multiple cycles of on/off therapy might diminish the size of the latent reservoir over time. While awaiting the results of further clinical studies involving DTG, these are concepts that could be studied in animal models.
Our findings suggest that DTG may be intrinsically resistant to the emergence of resistance and that this drug should be used in first-line therapy to minimize the emergence of possible drug resistance. The finding that a secondary mutation, i.e. H51Y, may simultaneously reduce viral replication and enzymatic activity, while augmenting levels of drug resistance in the presence of a primary resistance mutation, i.e. R263K, could potentially be advantageous not only for HIV treatment but for strategies aimed at HIV eradication as well.
Cells and antiviral compounds
TZM-bl, 293T, and PM1 cells were used as described . Human primary peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of healthy donors using Ficoll-Hypaque and stimulated with 10 μg/ml phytohemagglutinin A (PHA) and 20 U/ml human interleukin-2 (IL-2) for 72 h. Merck & Co., Inc. and ViiV Healthcare Ltd. kindly provided raltegravir (RAL) and dolutegravir (DTG), respectively. Efavirenz (EFV) was obtained from the NIH AIDS Research and Reference Reagent Program.
Integrase strand-transfer activity assay
Integrase strand transfer reactions with recombinant purified proteins were carried out as published , with the major difference being the use of pre-processed LTR DNA (sense amino-group-5′-ACCCTTTTAGTCAGTGTGGAAAATCTCTAGCA-3′ and antisense 5′-ACTGCTAGAGATTTTCCACACTGACTAAAAG-3′). LTR DNA was covalently linked to Costar DNA Bind 96-well plates (Corning) and the plates were blocked and washed as described . Purified integrase proteins were incubated for 30 minutes before the biotinylated target DNA (sense 5′-TGACCAAGGGCTAATTCACT-3Bio and antisense 5′-AGTGAATTAGCCCTTGGTCA-3Bio) was added, followed by an additional incubation of 1 h at 37°C for the strand-transfer reaction to occur. After washes, strand-transfer was quantified through the use of Eu-labelled streptavidin (Perkin Elmer), as described previously .
Generation of replication-competent genetically homogenous HIV-1
pNL4.3IN(R263K) has been described previously . To study the H51Y mutation and the H51Y/R263K combination, pNL4.3IN(H51Y) and pNL4.3IN(H51Y/R263K) were generated by site-directed mutagenesis using H51Y primers (sense: 5′-CTAAAAGGGGAAGCCATGTATGGACAAGTAGACTGTA-3′ and antisense: 5′-TACAGTCTACTTGTCCATACATGGCTTCCCCTTTTAG-3′), and the QuickChange II XL Site-Directed mutagenesis kit (Stratagene). The presence of the mutations was confirmed by sequencing. Genetically homogenous viruses were produced by transfecting wild-type and mutated pNL4.3 plasmids into 293T cells as described previously .
HIV susceptibility to antiretroviral compounds
HIV susceptibilities to DTG, RAL, and EFV were measured in TZM-bl cell at 48 h after infection as described previously . Fifty percent inhibitory concentrations (IC50s) and 95% confidence intervals were calculated on the basis of at least three experiments by using GraphPad Prism 4.0 Software.
Monogram biosciences PhenoSense replication capacity and phenotyping assays
HIV replication capacity and susceptibilities to DTG, RAL, and EFV were measured as previously described . Briefly, murine leukemia virus envelope-pseudotyped viruses bearing the integrase H51Y and R263K mutations and a luciferase reporter gene were used to inoculate human embryonic kidney HEK-293 cells. The resultant luciferase activity was used to calculate changes in HIV replication capacity relative to a wild-type reference strain. Drug susceptibility was expressed as a fold-change in IC50.
HIV infectivity and replication capacity
HIV infectivity was evaluated using a noncompetitive short-term infectivitiy assay in TZM-bl cells as previously described . HIV replication capacity was measured over time in PM1 cells by quantifiying RT activity (in counts per minute) in culture fluids as described previously .
Determination of HIV integration in PBMCs
A quantitative PCR (qPCR) assay for integrated DNA in primary human PBMCs was performed as previously described [17, 24]. Briefly, cellular DNA was extracted using the DNeasy blood and tissue extraction kit from Qiagen and amplified in a two-step PCR. The second step was performed with Platinum qPCR SuperMix-UDG (Invitrogen) on a Corbett Rotor-Gene 6000 (Corbett) by using the following conditions: 50°C for 2 min, 95°C for 2 min, and 50 repeats of 95°C for 10 s, 60°C for 20 s, and 72°C for 45 s. The samples were normalized for their β-globin gene content. Primers and probes have been described previously .
In silicostudies of HIV integrase
This project was supported by the Canadian Institutes for Health Research (CIHR), the Canadian Foundation for AIDS Research (CANFAR) and ISTP Canada. TM is a BMS/CTN Postdoctoral Fellow. PKQ is the recipient of a CIHR pre-doctoral fellowship. DNS is the recipient of a CIHR doctoral scholarship. We thank Drs. John Coffin, Eric Cohen, and Joseph Eron for their review of our manuscript and suggestions for its improvement.
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