Targeting the Integrated Stress Response Kinase GCN2 to Modulate Retroviral Integration.

Multiple viral targets are now available in the clinic to fight HIV infection. Even if this targeted therapy is highly effective at suppressing viral replication, caregivers are facing growing therapeutic failures in patients due to resistance, with or without treatment-adherence glitches. Accordingly, it is important to better understand how HIV and other retroviruses replicate in order to propose alternative antiviral strategies. Recent studies have shown that multiple cellular factors are implicated during the integration step and, more specifically, that integrase can be regulated through post-translational modifications. We have shown that integrase is phosphorylated by GCN2, a cellular protein kinase of the integrated stress response, leading to a restriction of HIV replication. In addition, we found that this mechanism is conserved among other retroviruses. Accordingly, we developed an in vitro interaction assay, based on the AlphaLISA technology, to monitor the integrase-GCN2 interaction. From an initial library of 133 FDA-approved molecules, we identified nine compounds that either inhibited or stimulated the interaction between GCN2 and HIV integrase. In vitro characterization of these nine hits validated this pilot screen and demonstrated that the GCN2-integrase interaction could be a viable solution for targeting integrase out of its active site.

Evaluation of different analysis pipelines for the detection of HIV-1 minority resistant variants.

OBJECTIVE: Reliable detection of HIV minority resistant variants (MRVs) requires bioinformatics analysis with specific algorithms to obtain good quality alignments. The aim of this study was to analyze ultra-deep sequencing (UDS) data using different analysis pipelines. METHODS: HIV-1 protease, reverse transcriptase (RT) and integrase sequences from antiretroviral-naïve patients were obtained using GS-Junior® (Roche) and MiSeq® (Illumina) platforms. MRVs were defined as variants harbouring resistance-mutation present at a frequency of 1%-20%. Reads were analyzed using different alignment algorithms: Amplicon Variant Analyzer®, Geneious® compared to SmartGene® NGS HIV-1 module. RESULTS: 101 protease and 51 RT MRVs identified in 139 protease and 124 RT sequences generated with a GS-Junior® platform were analyzed using AVA® and SmartGene® software. The correlation coefficients for the MRVs were R2 = 0.974 for protease and R2 = 0.972 for RT. Discordances (n = 13 in protease and n = 15 in RT) mainly concerned low-level MRVs (i.e., with frequencies of 1%-2%, n = 18/28) and they were located in homopolymeric regions (n = 10/15). Geneious® and SmartGene® software were used to analyze 143 protease, 45 RT and 26 integrase MRVs identified in 172 protease, 69 RT, and 72 integrase sequences generated with a MiSeq® platform. The correlation coefficients for the MRVs were R2 = 0.987 for protease, R2 = 0.995 for RT and R2 = 0.993 for integrase. Discordances (n = 9 in protease, n = 3 in RT, and n = 3 in integrase) mainly concerned low-level MRVs (n = 13/15). CONCLUSION: We found an excellent correlation between the various UDS analysis pipelines that we tested. However, our results indicate that specific attention should be paid to low-level MRVs, for which the use of two different analysis pipelines and visual inspection of sequences alignments might be beneficial. Thus, our results argue for use of a 2% threshold for MRV detection, rather than the 1% threshold, to minimize misalignments and time-consuming sight reading steps essential to ensure accurate results for MRV frequencies below 2%.

The S230R Integrase Substitution Associated With Virus Load Rebound During Dolutegravir Monotherapy Confers Low-Level Resistance to Integrase Strand-Transfer Inhibitors.

Background: Dolutegravir (DTG) is an integrase strand-transfer inhibitor (INSTI) used for treatment of human immunodeficiency virus (HIV)-infected individuals. Owing to its high genetic barrier to resistance, DTG has been clinically investigated as maintenance monotherapy to maintain viral suppression and to reduce complication and healthcare costs. Our study aims to explain the underlying mechanism related to the emergence of a S230R substitution in patients who experienced virologic failure while using DTG monotherapy. Methods: We evaluated the effect of the S230R substitution in regard to integrase enzyme activity, viral infectivity, replicative capacity, and susceptibility to different INSTIs by biochemical and cell-based assays. Results: The S230R substitution conferred a 63% reduction in enzyme efficiency. S230R virus was 1.29-fold less infectious than wild-type virus but could replicate in PM1 cells without significant delay. Resistance levels against DTG, cabotegravir, raltegravir, and elvitegravir in tissue culture were 3.85-, 3.72-, 1.52-, and 1.21-fold, respectively, in virus with the S230R substitution. Conclusions: Our data indicate that the S230R substitution is comparable to the previously reported R263K substitution in some respects. Virologic failure during DTG monotherapy can occur through the development of the S230R or R263K mutation, without the need for high-level DTG resistance.

Analyses of HIV-1 integrase sequences prior to South African national HIV-treatment program and available of integrase inhibitors in Cape Town, South Africa.

HIV-Integrase (IN) has proven to be a viable target for highly specific HIV-1 therapy. We aimed to characterize the HIV-1 IN gene in a South African context and identify resistance-associated mutations (RAMs) against available first and second generation Integrase strand-transfer inhibitors (InSTIs). We performed genetic analyses on 91 treatment-naïve HIV-1 infected patients, as well as 314 treatment-naive South African HIV-1 IN-sequences, downloaded from Los Alamos HIV Sequence Database. Genotypic analyses revealed the absence of major RAMs in the cohort collected before the broad availability of combination antiretroviral therapy (cART) and INSTI in South Africa, however, occurred at a rate of 2.85% (9/314) in database derived sequences. RAMs were present at IN-positions 66, 92, 143, 147 and 148, all of which may confer resistance to Raltegravir (RAL) and Elvitegravir (EVG), but are unlikely to affect second-generation Dolutegravir (DTG), except mutations in the Q148 pathway. Furthermore, protein modeling showed, naturally occurring polymorphisms impact the stability of the intasome-complex and therefore may contribute to an overall potency against InSTIs. Our data suggest the prevalence of InSTI RAMs, against InSTIs, is low in South Africa, but natural polymorphisms and subtype-specific differences may influence the effect of individual treatment regimens.

RNA and DNA Sanger sequencing versus next-generation sequencing for HIV-1 drug resistance testing in treatment-naive patients.

Background: Sanger sequencing of plasma RNA is the standard method for HIV-1 drug resistance testing in treatment-naive patients, but is limited by the non-detection of resistance-associated mutations (RAMs) with prevalence below approximately 20%. Objectives: We compared RNA and DNA Sanger sequencing (RSS and DSS) with RNA next-generation sequencing (NGS) for RAM detection in HIV-1 reverse transcriptase (RT), protease (PR) and integrase (IN) genes. Methods: Sanger sequencing was performed on RNA and DNA, following the recommendations of the French Agency for AIDS Research (ANRS). NGS was performed on RNA using the HIV-1 Drug Resistance Assay, v. 3.0 (Roche) on the 454 GS Junior sequencer. The IAS-USA list was used to identify RAMs. ANRS, Rega and Stanford algorithms were used for drug resistance interpretation. Results: The study included 48 ART-naive patients. The number of patients with at least one major RAM was 3, 3, 4 and 8 when using RSS, DSS, NGS 20% and NGS 5%, respectively. Numerous minor mutations were detected in patients, especially in the protease gene. None of the methods detected any major mutation in the integrase gene. Overall, the mutation detection rate was similar between RSS and DSS, and higher with NGS 20%. Differences in drug resistance interpretation were found between algorithms. No impact of the minority RAMs detected by NGS was found on the short-term treatment outcome. Conclusions: DSS does not clearly improve the detection of RAMs in ART-naive patients, as compared with RSS. NGS allows detection of additional minority RAMs; however, their clinical relevance requires further investigation.

Development of a G118R mutation in HIV-1 integrase following a switch to dolutegravir monotherapy leading to cross-resistance to integrase inhibitors.

OBJECTIVES: Dolutegravir shows a high barrier to resistance with no previously reported cases of acquired integrase mutations during first-line therapy. In this study, rapid development of the G118R mutation arose following a switch from first-line elvitegravir/cobicistat/tenofovir disoproxil fumarate/emtricitabine to dolutegravir monotherapy. The G118R mutation also arose in a treatment-experienced patient switched to dolutegravir monotherapy. The genetic basis for G118R selection and potential phenotypic outcome was ascertained. PATIENT AND METHODS: Genotypic analysis was performed on patients with virological failure (<1000 copies/mL) on dolutegravir-containing regimens. The Los Alamos database was queried for glycine codon 118 polymorphisms. Cell culture selections and phenotypic drug susceptibility assays assessed resistance via the G118R pathway. RESULTS: We report on two patients who developed viral failure while on dolutegravir monotherapy. Both patients had been on a current or previous regimen containing integrase inhibitors. Virological failure (<1000 copies/mL) emerged early within 2 months following the dolutegravir switch. The appearance of G118R in these two cases and subtype C and CRF02_AG in vitro selections were related to a rare GGA natural polymorphism at codon 118 (1.5% prevalence), facilitating a GGA to AGA transition. Cell culture selections were used to assess the in vitro progression of the G118R pathway leading to cross-resistance to all integrase inhibitors. CONCLUSIONS: Although resistance to dolutegravir is typically rare, genetic polymorphisms and monotherapy can facilitate the acquisition of G118R.

Mutagenesis of lysines 156 and 159 in human immunodeficiency virus type 1 integrase (IN) reveals differential interactions between these residues and different IN inhibitors.

Human immunodeficiency virus (HIV) type I integrase (IN) active site, and viral DNA-binding residues K156 and K159 are predicted to interact both with strand transfer-selective IN inhibitors (STI), e.g. L-731,988, Elvitegravir (EVG), and the FDA-approved IN inhibitor, Raltegravir (RGV), and strand transfer non-selective inhibitors, e.g. dicaffeoyltartaric acids (DCTAs), e.g. L-chicoric acid (L-CA). To test posited roles for these two lysine residues in inhibitor action we assayed the potency of L-CA and several STI against a panel of K156 and K159 mutants. Mutagenesis of K156 conferred resistance to L-CA and mutagenesis of either K156 or K159 conferred resistance to STI indicating that the cationic charge at these two viral DNA-binding residues is important for inhibitor potency. IN K156N, a reported polymorphism associated with resistance to RGV, conferred resistance to L-CA and STI as well. To investigate the apparent preference L-CA exhibits for interactions with K156, we assayed the potency of several hybrid inhibitors containing combinations of DCTA and STI pharmacophores against recombinant IN K156A or K159A. Although K156A conferred resistance to diketo acid-branched bis-catechol hybrid inhibitors, neither K156A nor K159A conferred resistance to their monocatechol counterparts, suggesting that bis-catechol moieties direct DCTAs toward K156. In contrast, STI were more promiscuous in their interaction with K156 and K159. Taken together, the results of this study indicate that DCTAs interact with IN in a manner different than that of STI and suggest that DCTAs are an attractive candidate chemotype for development into drugs potent against STI-resistant IN.

Evolution of a novel pathway leading to dolutegravir resistance in a patient harbouring N155H and multiclass drug resistance.

OBJECTIVES: Dolutegravir has been recently approved for treatment-naive and -experienced HIV-infected subjects, including integrase inhibitor (INI)-experienced patients. Dolutegravir is a second-generation INI that can overcome many prior raltegravir and elvitegravir failures. Here, we report the evolution of resistance to dolutegravir in a highly treatment-experienced patient harbouring the major N155H mutation consequent to raltegravir treatment failure. METHODS: Genotypic and phenotypic analyses were done on longitudinal samples to determine viral resistance to INIs. Integrase amino acid sequence interactions with raltegravir and dolutegravir were assessed by molecular modelling and docking simulations. RESULTS: Five mutations (A49P, L68FL, T97A, E138K and L234V) were implicated in emergent dolutegravir resistance, with a concomitant severe compromise in viral replicative capacity. Molecular modelling and docking simulations revealed that dolutegravir binding to integrase was affected by these acquired dolutegravir mutations. CONCLUSIONS: Our findings identify a novel mutational pathway involving integrase mutations A49P and L234V, leading to dolutegravir resistance in a patient with the N155H raltegravir mutation.

The evaluation of statins as potential inhibitors of the LEDGF/p75-HIV-1 integrase interaction.

Lovastatin was identified through virtual screening as a potential inhibitor of the LEDGF/p75-HIV-1 integrase interaction. In an AlphaScreen assay, lovastatin inhibited the purified recombinant protein-protein interaction (IC50 = 1.97 ± 0.45 µm) more effectively than seven other tested statins. None of the eight statins, however, yielded antiviral activity in vitro, while only pravastatin lactone yielded detectable inhibition of HIV-1 integrase strand transfer activity (31.65% at 100 µm). A correlation between lipophilicity and increased cellular toxicity of the statins was observed.

[Isolation, idetification and anti-HIV-1 integrase activity of culturable endophytic fungi from Tibetan medicinal plant Phlomis younghusbandii Mukerjee].

A total of 52 endophytic fungi were isolated from roots and stems of Tibetan medicinal plant Phlomis younghusbandii Mukerjee. These fungal isolates were molecularly identified based on ITS sequnces and 28S sequences distributed to 12 genera, including Phoma, Chaetosphaeronema, Fusarium and Leptosphaeria, etc. Among them, the dominant genus was Phoma. Extracts of all strains were evaluated for anti-HIV-1 integrase activity by using soluable integrase expressed in E. coli BL21 (DE3). The results showed that seven samples from five fungal endophytes PHY-24, PHY-38, PHY-40, PHY-51, PHY-53, which belonged to genus Chaetosphaeronema, inhibited strand transfer reaction catalyzed by HIV-1 integrase with IC50 values, of 6.60, 5.20, 2.86, 7.86, 4.47, 4.56 and 3.23 microg x mL(-1) respectively. In conclusion, the endophytic fungi of Phlomis younghusbandii Mukerjee are valuable for further screening anti-HIV-1 integrase agents.

A novel anti-HIV active integrase inhibitor with a favorable in vitro cytochrome P450 and uridine 5'-diphospho-glucuronosyltransferase metabolism profile.

Research efforts on the human immunodeficiency virus (HIV) integrase have resulted in two approved drugs. However, co-infection of HIV with Mycobacterium tuberculosis and other microbial and viral agents has introduced added complications to this pandemic, requiring favorable drug-drug interaction profiles for antiviral therapeutics targeting HIV. Cytochrome P450 (CYP) and uridine 5'-diphospho-glucuronosyltransferase (UGT) are pivotal determining factors in the occurrence of adverse drug-drug interactions. For this reason, it is important that anti-HIV agents, such as integrase inhibitors, possess favorable profiles with respect to CYP and UGT. We have discovered a novel HIV integrase inhibitor (compound 1) that exhibits low nM antiviral activity against a diverse set of HIV-1 isolates, and against HIV-2 and the simian immunodeficiency virus (SIV). Compound 1 displays low in vitro cytotoxicity and its resistance and related drug susceptibility profiles are favorable. Data from in vitro studies revealed that compound 1 was not a substrate for UGT isoforms and that it was not an inhibitor or activator of key CYP isozymes.

A new chimeric protein represses HIV-1 LTR-mediated expression by DNA methylase.

Once the human immunodeficiency virus (HIV) genome is inserted into the host genome, the virus cannot be removed, which results in latency periods and makes it difficult to eradicate. The majority of strategies to eradicate HIV have been based on preventing virus latency, thereby enabling antiretroviral drugs to act against HIV replication. Another innovative strategy is permanently silencing the integrated virus to prevent the spread of infection. Epigenetic processes are natural mechanisms that can silence viral replication. We describe a new chimeric protein (IN3b) that consists of a HIV-1 integrase domain, which recognises the HIV long terminal repeat (LTR) and the catalytic domain of DNA methyltransferase DNMT3b. Our objective was to silence HIV replication by the specific delivery of the catalytic methyltransferase domain to the LTR promoter to induce its methylation. We found that our IN3b chimeric protein was expressed in the nucleus and decreased LTR-associated HIV genome expression and HIV replication. Therefore, the IN3b chimeric protein may be an effective tool against HIV replication and maybe used in a new line of research to induce or maintain HIV latency.

Discovery of novel inhibitors of LEDGF/p75-IN protein-protein interactions.

Human lens epithelium-derived growth factor (LEDGF)/p75 plays an important role in the HIV life cycle by stimulating integrase (IN)-led viral DNA integration into cellular chromosomes. Mechanistic studies show the majority of IN inhibitors chelate magnesium ions in the catalytic active site, a region topologically distant from the LEDGF/p75 binding site. Compounds disrupting the formation of LEDGF/p75 and IN complexes serve as a novel mechanistic approach different from current antiretroviral therapies. We previously built pharmacophore models mimicking LEDGF/p75 residues and identified four classes of LEDGF/p75-IN inhibitors. Substructure and similarity searches yielded additional LEDGF/p75-IN inhibitors containing an acylhydrazone moiety. The most potent of the acylhydrazones inhibited LEDGF/p75-IN interaction with an IC(50) value of 400nM. We explored structure-activity relationships (SAR) and identified new acylhydrazones, hydrazines, and diazenes as lead molecules for further optimization. Two lead LEDGF/p75-IN inhibitors showed antiviral activity.

d(GGGT) 4 and r(GGGU) 4 are both HIV-1 inhibitors and interleukin-6 receptor aptamers.

Aptamers are oligonucleotides that bind targets with high specificity and affinity. They have become important tools for biosensing, target detection, drug delivery and therapy. We selected the quadruplex-forming 16-mer DNA aptamer AID-1 [d(GGGT) 4] with affinity for the interleukin-6 receptor (IL-6R) and identified single nucleotide variants that showed no significant loss of binding ability. The RNA counterpart of AID-1 [r(GGGU) 4] also bound IL-6R as quadruplex structure. AID-1 is identical to the well-known HIV inhibitor T30923, which inhibits both HIV infection and HIV-1 integrase. We also demonstrated that IL-6R specific RNA aptamers not only bind HIV-1 integrase and inhibit its 3' processing activity in vitro, but also are capable of preventing HIV de novo infection with the same efficacy as the established inhibitor T30175. All these aptamer target interactions are highly dependent on formation of quadruplex structure.

Parallel screening of low molecular weight fragment libraries: do differences in methodology affect hit identification?

Fragment screening is becoming widely accepted as a technique to identify hit compounds for the development of novel lead compounds. In neighboring laboratories, we have recently, and independently, performed a fragment screening campaign on the HIV-1 integrase core domain (IN) using similar commercially purchased fragment libraries. The two campaigns used different screening methods for the preliminary identification of fragment hits; one used saturation transfer difference nuclear magnetic resonance spectroscopy (STD-NMR), and the other used surface plasmon resonance (SPR) spectroscopy. Both initial screens were followed by X-ray crystallography. Using the STD-NMR/X-ray approach, 15 IN/fragment complexes were identified, whereas the SPR/X-ray approach found 6 complexes. In this article, we compare the approaches that were taken by each group and the results obtained, and we look at what factors could potentially influence the final results. We find that despite using different approaches with little overlap of initial hits, both approaches identified binding sites on IN that provided a basis for fragment-based lead discovery and further lead development. Comparison of hits identified in the two studies highlights a key role for both the conditions under which fragment binding is measured and the criteria selected to classify hits.

Study of genotypic and phenotypic HIV-1 dynamics of integrase mutations during raltegravir treatment: a refined analysis by ultra-deep 454 pyrosequencing.

BACKGROUND: The dynamics of raltegravir-resistant variants and their impact on virologic response in 23 HIV-1-infected patients, who started a salvage raltegravir-containing regimen, were investigated. METHODS: Integrase population sequencing and Ultra-Deep-454 Pyrosequencing (UDPS) were performed on plasma samples at baseline and at raltegravir failure. All integrase mutations detected at a frequency ≥1% were considered to be reliable for the UDPS analyses. Phylogenetic and phenotypic resistance analyses were also performed. RESULTS: At baseline, primary resistance mutations were not detected by both population and UDPS genotypic assays; few secondary mutations (T97A-V151I-G163R) were rarely detected and did not show any statistically association either with virologic response at 24-weeks or with the development of resistant variants at failure. At UDPS, not all resistant variants appearing early during treatment evolved as major populations during failure; only specific resistance pathways (Y143R-Q148H/R-N155H) associated with an increased rate of fitness and phenotypic resistance were selected. CONCLUSIONS: Resistance to raltegravir in integrase strand transfer inhibitor-naive patients remains today a rare event, which might be changed by future extensive use of such drugs. In our study, pathways of resistance at failure were not predicted by baseline mutations, suggesting that evolution plus stochastic selection plays a major role in the appearance of integrase-resistance mutations, whereas fitness and resistance are dominant factors acting for the late selection of resistant quasispecies.

Evaluation of a system to screen for stimulators of non-specific DNA nicking by HIV-1 integrase: application to a library of 50,000 compounds.

BACKGROUND: In addition to activities needed to catalyse integration, retroviral integrases exhibit non-specific endonuclease activity that is enhanced by certain small compounds, suggesting that integrase could be stimulated to damage viral DNA before integration occurs. METHODS: A non-radioactive, plate-based, solution phase, fluorescence assay was used to screen a library of 50,080 drug-like chemicals for stimulation of non-specific DNA nicking by HIV-1 integrase. RESULTS: A semi-automated workflow was established and primary hits were readily identified from a graphic output. Overall, 0.6% of the chemicals caused a large increase in fluorescence (the primary hit rate) without also having visible colour that could have artifactually caused this result. None of the potential stimulators from this moderate-size library, however, passed a secondary test that included an inactive integrase mutant that assessed whether the increased fluorescence depended on the endonuclease activity of integrase. CONCLUSIONS: This first attempt at identifying integrase stimulator compounds establishes the necessary logistics and workflow required. The results from this study should encourage larger scale high-throughput screening to advance the novel antiviral strategy of stimulating integrase to damage retroviral DNA.

Dolutegravir (S/GSK1349572) exhibits significantly slower dissociation than raltegravir and elvitegravir from wild-type and integrase inhibitor-resistant HIV-1 integrase-DNA complexes.

The integrase inhibitor (INI) dolutegravir (DTG; S/GSK1349572) has significant activity against HIV-1 isolates with raltegravir (RAL)- and elvitegravir (ELV)-associated resistance mutations. As an initial step in characterizing the different resistance profiles of DTG, RAL, and ELV, we determined the dissociation rates of these INIs with integrase (IN)-DNA complexes containing a broad panel of IN proteins, including IN substitutions corresponding to signature RAL and ELV resistance mutations. DTG dissociates slowly from a wild-type IN-DNA complex at 37°C with an off-rate of 2.7 × 10(-6) s(-1) and a dissociative half-life (t(1/2)) of 71 h, significantly longer than the half-lives for RAL (8.8 h) and ELV (2.7 h). Prolonged binding (t(1/2), at least 5 h) was observed for DTG with IN-DNA complexes containing E92, Y143, Q148, and N155 substitutions. The addition of a second substitution to either Q148 or N155 typically resulted in an increase in the off-rate compared to that with the single substitution. For all of the IN substitutions tested, the off-rate of DTG from IN-DNA complexes was significantly slower (from 5 to 40 times slower) than the off-rate of RAL or ELV. These data are consistent with the potential for DTG to have a higher genetic barrier to resistance, provide evidence that the INI off-rate may be an important component of the mechanism of INI resistance, and suggest that the slow dissociation of DTG may contribute to its distinctive resistance profile.

Evaluation of HIV-1 integrase inhibitors on human primary macrophages using a luciferase-based single-cycle phenotypic assay.

Macrophages represent an important site for productive infection of HIV-1 and the evaluation of integrase (IN) inhibitors on this cell subset is of fundamental importance. In this report, preclinical evaluation of IN inhibitors on primary human macrophages was attempted successfully using a 96-well microtiter phenotypic assay developed recently for the evaluation of IN inhibitors in a cell-based system by taking advantage of HIV-derived lentiviral vectors expressing luciferase. IN inhibitors were also tested using a lentiviral vector containing an IN with introduced T66I/S153Y mutations, known to affect the activity of azido-group-containing diketo acid (DKA) IN inhibitors. Utilizing different classes of HIV integrase inhibitors against the wild-type IN and the mutant mentioned above, some of the IN inhibitors used were also active on this particular mutant, suggesting that should HIV-1 develop additional or different mutations to become resistant to such anti-IN drugs, new drugs can be developed with a better resistance profile. This assay provides a standardized method for the preclinical evaluation of the efficacy of IN inhibitors on wild-type and mutated IN that can be adapted easily for the evaluation of anti-IN activity on IN sequences derived from patients.

HIV-1 resistance patterns to integrase inhibitors in antiretroviral-experienced patients with virological failure on raltegravir-containing regimens.

BACKGROUND: Our aim was to study the in vivo viral genetic pathways for resistance to raltegravir, in antiretroviral-experienced patients with virological failure (VF) on raltegravir-containing regimens. METHODS: We set up a prospective study including antiretroviral-experienced patients receiving raltegravir-based regimens. Integrase (IN) genotypic resistance analysis was performed at baseline. IN was also sequenced at follow-up points in the case of VF, i.e. plasma HIV-1 RNA>400 copies/mL at month 3 and/or >50 copies/mL at month 6. For phenotyping, the IN region was recombined with an IN-deleted HXB2-based HIV-1 backbone. A titrated amount of IN recombinant viruses was used for antiviral testing against raltegravir and elvitegravir. RESULTS: Among 51 patients, 11 (21.6%) had VF. Four different patterns of IN mutations were observed: (i) emergence of Q148H/R with secondary mutations (n=5 patients); (ii) emergence of N155H, then replaced by a pattern including Y143C/H/R (n=3); (iii) selection of S230N (n=1); and (iv) no evidence of selection of IN mutations (n=2). The median raltegravir and elvitegravir fold changes (FCs) were 244 (154-647) and 793 (339-892), respectively, for the Q148H/R pattern, while the median raltegravir and elvitegravir FCs were 21 (6-52) and 3 (2-3), respectively, with Y143C/H/R. The median plasma raltegravir Cmin was lower in patients with selection of the N155H mutation followed by Y143C/H/R compared with patients with Q148H/R and with patients without emerging mutations or without VF. CONCLUSIONS: Diverse genetic profiles can be associated with VF on raltegravir-containing regimens, including the dynamics of replacement of mutational profiles. Pharmacokinetic parameters could be involved in this genetic evolution.