Integrase Mutations Research Articles

In vitro resistance selections using elvitegravir, raltegravir, and two metabolites of elvitegravir M1 and M4

Elvitegravir is a strand transfer inhibitor of HIV-1 integrase that is currently undergoing phase 3 clinical testing. The two predominant metabolites of elvitegravir, M1 and M4 (elvitegravir hydroxide and elvitegravir glucuronide), have been shown to inhibit HIV-1 integrase in vitro. While they are markedly less potent than elvitegravir and present only at low levels in plasma clinically, we investigated their potential to select for elvitegravir resistance in vitro. Resistance selection experiments using metabolites M1 and M4 led to the development of the previously reported elvitegravir integrase resistance mutations H51Y, T66A, E92G, and S147G, as well as a novel S153F substitution. Additional resistance selection experiments using elvitegravir led to the development of previously reported integrase inhibitor resistance mutations (T66I, F121Y, and S153Y) as well as a novel R263K integrase mutation. Phenotypic analyses of site-directed mutants with these mutations demonstrated broad cross-resistance between elvitegravir and its M1 and M4 metabolites with more limited cross-resistance to the integrase inhibitor raltegravir. Overall, our in vitro studies demonstrate that the resistance profile of the M1 and M4 metabolites of elvitegravir overlaps with that of the parent molecule elvitegravir; as such, their presence at low levels is not considered clinically relevant.

Phenotypic susceptibility of HIV-2 to raltegravir

Raltegravir is the first integrase strand transfer inhibitor approved for treating HIV-1 infection. Although emerging data suggest that raltegravir may also be useful for HIV-2 treatment, studies addressing the in-vitro susceptibility of HIV-2 to raltegravir are scarce, and the genetic pathways leading to raltegravir resistance in HIV-2 have not been adequately characterized. Our objectives were to directly compare the susceptibilities of HIV-1 and HIV-2 to raltegravir and to examine the role of mutations in HIV-2 integrase in emergent raltegravir resistance. Single-cycle and spreading infection assays were used to quantify the sensitivities of wild-type HIV-1 and HIV-2 strains to raltegravir. HIV-2 integrase mutants were constructed by site-directed mutagenesis, and the replication capacities and raltegravir susceptibilities of the resultant variants were analyzed in single-cycle assays. Raltegravir showed comparable activity against wild-type HIV-1 and HIV-2 in both single-cycle and spreading infections, with EC(50) values in the low nanomolar range. Amino acid changes Q148R and N155H individually conferred resistance to raltegravir (14-fold and seven-fold, respectively), whereas the Y143C replacement had no statistically significant effect on raltegravir sensitivity. The combination of Q148R with N155H resulted in high-level raltegravir resistance (>1000-fold). In addition, all HIV-2 integrase variants tested showed impairments in replication capacity. Our data support clinical studies of raltegravir for treating HIV-2 infection and show that the Q148R and N155H changes alone are sufficient for raltegravir resistance in HIV-2. Further efforts are needed to improve access to HIV-2-active antiretrovirals, including raltegravir, in resource-limited areas where HIV-2 is endemic.

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

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. 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. 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. 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.

HIV-1 integrase SUMOylation and viral replication

Background HIV-1 integrase catalyzes the integration of the reverse transcribed viral cDNA into the cellular genome, a key event of retroviral replication that is targeted by novel anti-HIV therapeutic agents. Numerous studies have contributed to understand of the molecular basis of integrase catalytic functions. Besides integration, HIV-1 integrase participates in other steps of viral replication such as reverse transcription and/or uncoating, PIC nuclear import and virion morphology. However, the underlying mechanisms are still not fully elucidated. Cellular and viral factors assist integrase in performing its multiple activities. Moreover, post-translational modifications (i.e. acetylation, ubiquitination and phosphorylation), which represent a common, rapid and generally reversible mechanism for fine tuning of protein activities, have also been shown to regulate integrase functions. Materials and methods and results By in vitro SUMOylation assay and purification on NiNTA beads in denaturing conditions, we show that HIV1 integrase is covalently modified by the three SUMO paralogues. By mutating SUMO-acceptor lysine residues within phylogenetically conserved SUMOylation consensus motifs identified in silico, we demonstrate that they represent major sites of SUMO conjugation. We introduced the same changes in the integrase sequence of a plasmid encoding the viral genome, which was used to generate mutant viral particles, and we compare their infectivity to that of WT HIV-1. We find that viruses harboring SUMOylation-defective integrase mutants are less infectious that the WT counterpart. Real-time PCR analysis of viral cDNA synthesis reveals that cells infected with mutant viruses display similar content of traverse transcripts, but a lower number of integrated proviruses, than WT HIV-1-infected cells. This integration defect at the integration step is not due to impairment of integrase binding to LEDGF/p7S, a key chromatin-tethering factor of HIV-1 pre-integration complex. Indeed, both WT and SUMOylation-defective integrase proteins coprecipitated with similar efficiency with LEDGF/p75. Finally, we establish that SUMOylation-defective integrase mutants retained WT catalytic activity as determined by Vprfusion protein complementation assay. Conclusions Here, we show that HIV-1 IN is modified by SUMO proteins and that phylogenetically conserved SUMOylation consensus motifs represent major SUMO-acceptor sites. Viruses harboring SUMOylation-site IN mutants displayed a replication defect that was mapped during the early stages of infection, before integration but after reverse transcription. Since SUMOylation-defective IN mutants retained WT catalytic activity as well as LEDGF-binding, we hypothesize that SUMOylation might regulate the affinity of IN for co-factors, contributing to efficient HIV-1 replication.

The dynamics of appearance and disappearance of HIV-1 integrase mutations during and after withdrawal of raltegravir therapy

The dynamics of appearance and disappearance of HIV-1 integrase mutations during and after withdrawal of raltegravir therapy

Clinical efficacy of raltegravir against B and non-B subtype HIV-1 in phase III clinical studies

We evaluated the long-term efficacy of raltegravir according to HIV-1 subtype (B and non-B) using data from three phase III studies in treatment-experienced (BENCHMRK-1 and 2) and treatment-naive (STARTMRK) HIV-infected patients. HIV-1 subtypes were identified from baseline plasma specimens using genotypic data of the PhenoSense GT test (Monogram Biosciences, South San Francisco, California, USA). Non-B subtypes were combined for the current analyses due to small numbers of each specific subtype. An observed failure approach was used (only discontinuations due to lack of efficacy were treated as failures). Resistance evaluation was performed in patients with documented virologic failure. Seven hundred and forty-three patients received raltegravir and 519 received comparator (efavirenz in STARTMRK; optimized background therapy in BENCHMRK). Non-B subtype virus (A, A/C, A/D, A/G, A1, AE, AG, B/G, BF, C, D, D/F, F, F1, G, and complex) was isolated at baseline in 98 (13%) raltegravir recipients and 62 (12%) comparator recipients. Subtypes AE and C were most common, isolated in 41 and 43 patients, respectively. The proportion of raltegravir recipients achieving HIV RNA less than 50 copies/ml was similar between non-B and B subtypes (STARTMRK: 94.5 vs. 88.7%; BENCHMRK-1 and 2: 66.7 vs. 60.7%); change in CD4 cell count also was similar between non-B and B subtypes (STARTMRK: 243 vs. 221 cells/μl; BENCHMRK-1 and 2: 121 vs. 144 cells/μl). Phenotypic resistance to raltegravir in non-B virus was associated with integrase mutations observed previously in subtype B virus. In phase III studies in treatment-naive and treatment-experienced patients, raltegravir showed comparable and potent clinical efficacy against B and non-B HIV-1 subtypes.

Comparison of transgene expression in Aedes aegypti generated by mariner Mos1 transposition and ΦC31 site‐directed recombination

Transgenic mosquitoes generated by transposable elements (TEs) often poorly express transgenes owing to position effects. To avoid these effects, the ΦC31 site-directed recombination system was used to insert transgenes into a locus favourable for gene expression in Aedes aegypti. We describe phenotypes of mariner Mos1 TE and ΦC31 transgenic mosquitoes expressing the enhanced green fluorescent protein (EGFP) reporter in midguts of blood-fed females. Mosquitoes of nine TE-generated lines [estimated transformation frequency (TF): 9.3%] clearly expressed the eye-specific selection marker but only 2/9 lines robustly expressed the EGFP reporter. The piggyBac TE-generated ΦC31 docking strain, attP26, supported recombination with attB site containing donors at an estimated TF of 1.7-4.9%. Using a codon-optimized ΦC31 integrase mutant instead of the 'wild-type' enzyme did not affect TF. Site-directed recombination of line attP26 with an attB-containing donor expressing EGFP from the Ae. aegypti carboxypeptidase promoter produced one transgenic line with blood-fed females expressing the reporter in midgut tissue. Docking strain attP26 also supported robust expression of Flock House virus B2 from the Ae. aegypti polyubiquitin promoter. Our data confirm that eye-specific selection marker expression alone is not a reliable indicator for robust gene-of-interest expression in Ae. aegypti and that the ΦC31 system can ensure predictable transgene expression in this mosquito species.

Resistance to raltegravir highlights integrase mutations at codon 148 in conferring cross-resistance to a second-generation HIV-1 integrase inhibitor

Raltegravir is the first integrase strand-transfer inhibitor (INSTI) approved for use in highly active antiretroviral therapy (HAART) for the management of HIV infection. Resistance to antiretrovirals can compromise the efficacy of HAART regimens. Therefore it is important to understand the emergence of resistance to RAL and cross-resistance to other INSTIs including potential second-generation INSTIs such as MK-2048. We have now studied the question of whether in vitro resistance selection (IVRS) with RAL initiated with viruses derived from clinical isolates would result in selection of resistance mutations consistent with those arising during treatment regimens with HAART containing RAL. Some correlation was observed between the primary mutations selected in vitro and during therapy, initiated with viruses with identical IN sequences. Additionally, phenotypic cross-resistance conferred by specific mutations to RAL and MK-2048 was quantified. N155H, a RAL-associated primary resistance mutation, was selected after IVRS with MK-2048, suggesting similar mechanisms of resistance to RAL and MK-2048. This was confirmed by phenotypic analysis of 766 clonal viruses harboring IN sequences isolated at the point of virological failure from 106 patients on HAART (including RAL), where mutation Q148H/K/R together with additional secondary mutations conferred reduced susceptibility to both RAL and MK-2048. A homology model of full length HIV-1 integrase complexed with viral DNA and RAL or MK-2048, based on an X-ray structure of the prototype foamy virus integrase–DNA complex, was used to explain resistance to RAL and cross-resistance to MK-2048. These findings will be important for the further discovery and profiling of next-generation INSTIs.

Elvitegravir overcomes resistance to raltegravir induced by integrase mutation Y143

In this study, we characterized elvitegravir activity in the context of raltegravir resistance mutations. Using site-directed mutagenesis, we generated recombinant integrase proteins and viruses harboring raltegravir resistance mutation to assess the biochemical and cellular activity of elvitegravir in the presence of such mutants. Recombinant proteins were used in gel-based assays. Antiviral data were obtained with reporter viruses in a single-round infection using a luciferase-based assay. Although main raltegravir resistance pathways involving mutations at integrase position 148 and 155 confer cross-resistance to elvitegravir, elvitegravir remains fully active against the Y143R mutant integrase and virus particles. In addition to favorable pharmacokinetics compared to raltegravir, our findings provide the rationale for using elvitegravir in patients failing raltegravir because of the integrase mutation Y143.

Single Mutations in HIV Integrase Confer High-Level Resistance to Raltegravir in Primary Human Macrophages

CD4(+) T cells and macrophages are the primary target cells for HIV in vivo, and antiretroviral drugs can vary in their ability to inhibit the infection of these different cell types. Resistance pathways to the HIV integrase inhibitor raltegravir have previously been investigated in T cells. Primary raltegravir resistance mutations, most often at integrase amino acid position 148 or 155, afford some resistance to the drug. The acquisition of pathway-specific secondary mutations then provides higher-level resistance to viruses infecting T cells. We show here that during macrophage infection, the presence of a single primary raltegravir resistance mutation (Q148H, Q148R, N155H, or N155S) is sufficient to provide resistance to raltegravir comparable to that seen in viruses expressing both primary and secondary mutations in costimulated CD4(+) T cells. These data implicate macrophages as a potential in vivo reservoir that may facilitate the development of resistance to raltegravir. Notably, the newer integrase inhibitor MK-2048 effectively suppressed the infection of all raltegravir-resistant viruses in both T cells and macrophages, indicating that more recently developed integrase inhibitors are capable of inhibiting infection in both major HIV cellular reservoirs, even in patients harboring raltegravir-resistant viruses.

Structure-Analysis of the HIV-1 Integrase Y143C/R Raltegravir Resistance Mutation in Association with the Secondary Mutation T97A

The HIV-1 integrase (IN) mutations Y143C/R are known as raltegravir (RAL) primary resistance mutations. In a previous study (S. Reigadas et al., PLoS One 5:e10311, 2010), we investigated the genetic pathway and the dynamics of emergence of the Y143C/R mutations in three patients failing RAL-containing regimens. In these patients, the Y143C/R mutation was associated with the T97A mutation. The aim of the present biochemical and molecular studies in vitro was to evaluate whether the secondary mutation, T97A, associated with the Y143C/R mutation could increase the level of resistance to RAL and impact IN activities. Site-directed mutagenesis experiments were performed with expression vectors harboring the region of the pol gene coding for IN. With a 3'-end processing assay, the 50% inhibitory concentrations (IC(50)) were 1.2 μM, 1.2 μM, 2.4 μM (fold change [FC], 2), and 20 μM (FC, 16.7) for IN wild type (WT), the IN T97A mutation, the IN Y143C/T97A mutation, and the IN Y143R/T97A mutation, respectively. FCs of 18 and 100 were observed with the strand transfer assay for IN Y143C/T97A and Y143R/T97A mutations, with IC(50) of 0.625 μM and 2.5 μM, respectively. In the strand transfer assay, the IN Y143C or R mutation combined with the secondary mutation T97A severely impaired susceptibility to RAL compared to results with the IN Y143C or R mutation alone. Assays without RAL suggested that the T97A mutation could rescue the catalytic activity which was impaired by the presence of the Y143C/R mutation. The combination of the T97A mutation with the primary RAL resistance mutations Y143C/R strongly reduces the susceptibility to RAL and rescues the catalytic defect due to the Y143C/R mutation. This result indicates that the emergence of the Y143C/R/T97A double-mutation pattern in patients is a signature of a high resistance level.

Plasma Raltegravir Exposure Influences the Antiviral Activity and Selection of Resistance Mutations

Raltegravir (RAL) resistance is associated with the selection of integrase mutations at positions 92, 143, 148, and/or 155. A substantial proportion of RAL failures, however, occurs in the absence of these changes. An examination of RAL plasma concentrations may help in interpreting this observation. All early RAL virological failures seen at 22 clinics in Spain during 2009 were identified. HIV integrase sequences and RAL plasma trough concentrations (C(t)) were examined. A total of 106 patients experiencing virological failure on RAL were identified. Only the earliest sample on failure was examined. Integrase sequences could be obtained for 89 (84%), of whom 30 (33.7%) depicted primary RAL resistance mutations (15 N155H, eight Q148H/R, three Y143R, one E92Q, and three more than one of them). Another nine (10.1%) patients showed only secondary changes. The remaining 50 RAL early failures (56.2%) did not select any integrase change. RAL C(t) could be measured in 66 patients at failure and in 21 of them before failure. In a control group of 37 patients with viral suppression on RAL, detectable plasma levels were seen in all cases, with greater median RAL C(t) than in failures, either at the time of viral rebound (p<0.001) or before it (p=0.055). Moreover, median C(t) at the time of failure was greater in patients selecting primary RAL resistance mutations than in the rest of the failures (p<0.001). Undetectable RAL C(t) was seen only in patients failing RAL without integrase resistance mutations (64.1% of them). RAL failures in the absence of integrase resistance mutations mainly reflect poor drug compliance.

HIV-1 integrase modulates the interaction of the HIV-1 cellular cofactor LEDGF/p75 with chromatin

BackgroundChromatin binding plays a central role in the molecular mechanism of LEDGF/p75 in HIV-1 DNA integration. Conflicting results have been reported in regards to the relevance of the LEDGF/p75 chromatin binding element PWWP domain in its HIV-1 cofactor activity.ResultsHere we present evidence that re-expression of a LEDGF/p75 mutant lacking the PWWP domain (ΔPWWP) rescued HIV-1 infection in cells verified to express background levels of endogenous LEDGF/p75 that do not support efficient HIV-1 infection. The HIV-1 cofactor activity of LEDGF/p75 ΔPWWP was similar to that of LEDGF/p75 wild type (WT). A possible molecular explanation for the nonessential role of PWWP domain in the HIV-1 cofactor activity of LEDGF/p75 comes from the fact that coexpression of HIV-1 integrase significantly restored the impaired chromatin binding activity of LEDGF/p75 ΔPWWP. However, integrase failed to promote chromatin binding of a non-chromatin bound LEDGF/p75 mutant that lacks both the PWWP domain and the AT hook motifs (ΔPWWP/AT) and that exhibits negligible HIV-1 cofactor activity. The effect of integrase on the chromatin binding of LEDGF/p75 requires the direct interaction of these two proteins. An HIV-1 integrase mutant, unable to interact with LEDGF/p75, failed to enhance chromatin binding, whereas integrase wild type did not increase the chromatin binding strength of a LEDGF/p75 mutant lacking the integrase binding domain (ΔIBD).ConclusionsOur data reveal that the PWWP domain of LEDGF/p75 is not essential for its HIV-1 cofactor activity, possibly due to an integrase-mediated increase of the chromatin binding strength of this LEDGF/p75 mutant.

Prevalence of resistance mutations related to integrase inhibitor S/GSK1349572 in HIV-1 subtype B raltegravir-naive and -treated patients

To compare the frequency of previously in vitro-selected integrase mutations (T124A, T124A/S153F, S153Y, T124A/S153Y and L101I/T124A/S153Y) conferring resistance to S/GSK1349572 between HIV-1 subtype B integrase inhibitor (INI)-naive and raltegravir-treated patients. Integrase sequences from 650 INI-naive patients and 84 raltegravir-treated patients were analysed. The T124A mutation alone and the combination T124A/L101I were more frequent in raltegravir-failing patients than in INI-naive patients (39.3% versus 24.5%, respectively, P = 0.005 for T124A and 20.2% versus 10.0%, respectively, P = 0.008 for T124A/L101I). The S153Y/F mutations were not detected in any integrase sequence (except for S153F alone, only detected in one INI-naive patient). T124A and T124A/L101I, more frequent in raltegravir-treated patients, could have some effect on raltegravir response and their presence could play a role in the selection of other mutations conferring S/GSK1349572 resistance. The impact of raltegravir-mediated changes such as these on the virological response to S/GSK1349572 should be studied further.

Analysis of Low-Frequency Mutations Associated with Drug Resistance to Raltegravir before Antiretroviral Treatment

Raltegravir is highly efficacious in the treatment of HIV-1 infection. The prevalence and impact on virologic outcome of low-frequency resistant mutations among HIV-1-infected patients not previously treated with raltegravir have not been fully established. Samples from HIV treatment-experienced patients entering a clinical trial of raltegravir treatment were analyzed using a parallel allele-specific sequencing (PASS) assay that assessed six primary and six secondary integrase mutations. Patients who achieved and sustained virologic suppression (success patients, n = 36) and those who experienced virologic rebound (failure patients, n = 35) were compared. Patients who experienced treatment failure had twice as many raltegravir-associated resistance mutations prior to initiating treatment as those who achieved sustained virologic success, but the difference was not statistically significant. The frequency of nearly all detected resistance mutations was less than 1% of viral population, and the frequencies of mutations between the success and failure groups were similar. Expansion of pre-existing mutations (one primary and five secondary) was observed in 16 treatment failure patients in whom minority resistant mutations were detected at baseline, suggesting that they might play a role in the development of drug resistance. Two or more mutations were found in 13 patients (18.3%), but multiple mutations were not present in any single viral genome by linkage analysis. Our study demonstrates that low-frequency primary RAL-resistant mutations were uncommon, while minority secondary RAL-resistant mutations were more frequently detected in patients naïve to raltegravir. Additional studies in larger populations are warranted to fully understand the clinical implications of these mutations.

Effect of Raltegravir Resistance Mutations in HIV-1 Integrase on Viral Fitness

Raltegravir resistance is conferred by mutations at integrase codons 143, 148, and 155 together with associated secondary mutations. The N155H mutants emerge first, and are eventually replaced by Q148H mutants, usually in combination with G140S. These mutations have different effects on susceptibility and replication capacity, but data on the relative fitness of RAL-resistant viruses are limited. To understand the impact of the different RAL resistance pathways on viral fitness, mutations at integrase codons 74, 92, 138, 140, 148, 155, and/or 163 were introduced into HIV-1NL4-3 by site-directed mutagenesis and expressed in recombinant viruses. Relative fitness and drug susceptibility were determined in the absence or presence of RAL. In the absence of drug, RAL-resistant mutants were less fit than wild type, and the Q148H mutant was significantly less fit than the N155H mutant. Fitness was partially restored by the presence of additional RAL resistance mutations at positions G140S and E92Q or E138K, respectively. In the presence of RAL, the N155H mutant remained fitter than the Q148H mutant, but the G140S/Q148H double mutant was fitter than single mutants or the E92Q/N155H double mutant. These findings correspond well with the clinical trials data and help explain the temporal pattern of RAL resistance evolution.

Critical Amino Acid Residues Within the ɸC31 Integrase DNA-Binding Domain Affect Recombination Activities in Mammalian Cells

The bacteriophage-derived ϕC31 integrase system represents an attractive tool for site-directed recombination in mammalian cells. Its integration reaction is based on recombination between the attachment site attB within an episomal substrate plasmid and either the bacteriophage-derived wild-type attachment site attP or pseudo-attP attachment sites (attP') present in the mammalian genome. In the present study we aimed at increasing the safety and efficiency of ϕC31 integrase-mediated recombination by mutating the DNA-binding domain located at the C terminus. Using an alanine mutagenesis approach, we generated 22 ϕC31 point mutants that were screened for activities in mammalian cells. Intramolecular excision assays based on recombination between attB and attP revealed five mutants with 2-fold enhanced excision activity. Importantly, we also identified mutants showing enhanced recombination activities between attB and three previously described attP' sites detected in the mammalian genome, indicating that there may be enhanced specificity for these hot spots. Several mutants showed, in mammalian cells, integration activities that increased in a cell line-dependent manner. The combination of beneficial mutations in addition to optimization of the integrase plasmid dose enhanced integration efficiencies up to 5.5-fold. We also identified three ϕC31 integrase mutants that were recombination defective in all applied assays, suggesting that these amino acid residues are essential for the functionality of ϕC31 integrase in mammalian cells. In summary, we identified critical amino acid residues within the ϕC31 DNA-binding domain. With respect to site-directed recombination and genome engineering these findings have important implications for improved ϕC31 protein design.

Mutation Q95K enhances N155H-mediated integrase inhibitor resistance and improves viral replication capacity

The genetic barrier to development of raltegravir resistance is considered to be low, requiring at least one primary integrase mutation: Y143C, Q148H/K/R or N155H to confer raltegravir therapy failure. However, during continued raltegravir treatment failure, additional mutations may be selected. In a patient failing raltegravir therapy, we investigated the impact of multiple integrase mutations on resistance and viral replication. Furthermore, in vivo fitness was investigated during failure of raltegravir-containing highly active antiretroviral therapy and after raltegravir was discontinued from the regimen. Patient-derived viral integrase genes were cloned into a reference strain. These recombinant viruses were used to determine the contribution of individual integrase mutations to raltegravir resistance and replication capacity in vitro. To determine in vivo fitness, the relative proportion of specific integrase mutations was monitored over time by in-depth clonal analysis of the viral integrase at baseline, during and after raltegravir treatment. Raltegravir therapy failure was associated with the initial selection of primary resistance mutation N155H. This mutation conferred a 3.8-fold reduction in raltegravir susceptibility and a severe reduction in viral replication. Acquisition of integrase mutation Q95K increased resistance (6.2-fold) and partly restored viral replication. Selection of a third mutation, V151I, further increased raltegravir resistance (20-fold), but decreased viral replication. After prolonged raltegravir interruption, raltegravir resistance mutations were lost, demonstrating the reduced replication capacity of the resistant virus. We describe selection of Q95K as a secondary resistance mutation during raltegravir therapy failure. In the background of N155H, Q95K enhances raltegravir and elvitegravir resistance and improves the impaired replication of the virus.

Resistance to new antiretrovirals (ETV, anti-integrases, entry inhibitors)

HIV is extremely variable and newly arisen variants may be rapidly selected upon drug pressure. New antivirals are available, new therapeutic options are at hand for all patients, either naive or multiexperienced. Individualization is the hallmark of new approaches to antiretroviral therapies. Knowledge of drug resistance is important to guide clinicians in devising treatment regimens and to optimally preserve later treatment options for patients. This presentation will focus on the new technologies, methodologies, models and research tools to enhance detection and interpretation of HIV-1 drug-resistance. Particular attention will be dedicated to the resistance to the new antivirals. The development of the new second-generation NNRTI etravirine, which has a higher genetic barrier than the first-generation drugs, has led to new understanding of the complexity of NNRTI resistance. To date, 17 mutations have been identified as being associated with etravirine resistance and reduced virological response. Integrase inhibitors are very potent drugs providing a great opportunity for clinicians, to construct new regimens for individuals with HIV infection resistant to all three traditional classes of antivirals. However, these drugs have a relatively low genetic barrier to resistance and thus require both the selection of an optimised backbone to ensure durable viral suppression and a clear understanding of their residual activity in a failing integrase regimen. Resistance tests able to sequence and detect mutations in integrase are crucial for their proper use in clinical practice. Viral escape of entry inhibitors may occur through 'classic' viral resistance to the compounds, but also by usage of alternative cell co-receptors. To date the main focus of research on co-receptor antagonists has been on determining and quantifying the CCR5, CXCR4, dual/mixed coreceptor tropism. Recent data begun to emerge on how resistance develops with the accumulation of mutations in gp120 to maraviroc, the first-in-class R5 co-receptor antagonist.

301. Expression of the LEDGF Integrase Binding Domain (IBD) Severely Restricts HIV-1 Replication and Drives Evolution of Phenotypically Distinctive Integrase Mutants

301. Expression of the LEDGF Integrase Binding Domain (IBD) Severely Restricts HIV-1 Replication and Drives Evolution of Phenotypically Distinctive Integrase Mutants