Recombinant Integrase Research Articles

HIV-1 subtype F integrase polymorphisms external to the catalytic core domain contribute to severe loss of replication capacity in context of the integrase inhibitor resistance mutation Q148H.

In prior studies, HIV-1 BF recombinants with subtype F integrases failed to develop resistance to raltegravir through the Q148H mutational pathway. We aimed to determine the role of subtype-specific polymorphisms in integrase on drug susceptibility, viral replication and integration. Integrase sequences were retrieved from the Los Alamos Database or obtained from the Garrahan HIV cohort. HIV-1 infectious molecular clones with or without Q148H (+ G140S) resistance mutations were constructed using integrases of subtype B (NL4-3) or F1(BF) ARMA159 and URTR23. Integrase chimeras were generated by reciprocal exchanges of a 200 bp fragment spanning amino acids 85-150 of the catalytic core domain (CCD) of NL4-3-Q148H and either ARMA159-Q148H or URTR23-Q148H. Viral infections were quantified by p24 ELISA and Alu-gag integration PCR assay. At least 18 different polymorphisms distinguish subtype B from F1(BF) recombinant integrases. In phenotypic experiments, p24 at Day 15 post-infection was high (105-106 pg/mL) for WT and NL4-3-Q148H; by contrast, it was low (102-104 pg/mL) for both F1(BF)-Q148H + G140S viruses, and undetectable for the Q148H mutants. Compared with WT viruses, integrated DNA was reduced by 5-fold for NL4-3-Q148H (P = 0.05), 9-fold for URTR23-Q148H (P = 0.01) and 16000-fold for ARMA159-Q148H (P = 0.01). Reciprocal exchange between B and F1(BF) of an integrase CCD region failed to rescue the replicative defect of F1(BF) integrase mutants. The functional impairment of Q148H in the context of subtype F integrases from BF recombinants explains the lack of selection of this pathway in vivo. Non-B polymorphisms external to the integrase CCD may influence the pathway to integrase strand transfer inhibitor resistance.

Cabotegravir, the Long-Acting Integrase Strand Transfer Inhibitor, Potently Inhibits Human T-Cell Lymphotropic Virus Type 1 Transmission in vitro.

Human T-cell lymphotropic virus type 1 (HTLV-1) is a deltaretrovirus most prevalent in southwestern Japan, sub-Saharan Africa, Australia, South America, and the Caribbean. Latest figures approximate 10 million people worldwide to be infected with HTLV-1. This is likely a significant underestimation due to lack of screening in endemic areas and absence of seroconversion symptoms. The two primary diseases associated with HTLV-1 infection are adult T cell leukaemia-lymphoma, a malignant and, sometimes, aggressive cancer; and HTLV-1 associated myelopathy/tropical spastic paraparesis, a debilitating neurological degenerative disease. Unfortunately, despite the poor prognosis, there is currently no effective treatment for HTLV-1 infection. We previously showed that integrase strand transfer inhibitors (INSTIs) clinically used for human immunodeficiency virus type 1 (HIV-1) prophylaxis and treatment are also effective against HTLV-1 transmission in vitro. In 2021 a new INSTI, cabotegravir, was approved by the FDA for HIV-1 treatment. We thus set out to evaluate its efficacy against HTLV-1 infection in vitro. Strand transfer assays performed using recombinant HTLV-1 integrase treated with increasing concentrations of cabotegravir, effectively inhibited strand transfer activity, displaying an IC50 of 77.8 ± 22.4 nM. Furthermore, cabotegravir blocked HTLV-1 transmission in tissue culture; we determined an EC50 of 0.56 ± 0.26 nM, similar to bictegravir. Alu-PCR confirmed the block in integration. Thus, there are four INSTIs and one reverse transcriptase inhibitor approved by the FDA for HIV-1 treatment, that potently block HTLV-1 infection in vitro. This should strongly encourage the establishment of a new standard of HTLV-1 treatment – particularly for pre-exposure prophylaxis and prevention of mother-to-child transmission.

Ty1 integrase is composed of an active N-terminal domain and a large disordered C-terminal module dispensable for its activity in vitro

Long-terminal repeat (LTR) retrotransposons are genetic elements that, like retroviruses, replicate by reverse transcription of an RNA intermediate into a complementary DNA (cDNA) that is next integrated into the host genome by their own integrase. The Ty1 LTR retrotransposon has proven to be a reliable working model to investigate retroelement integration site preference. However, the low yield of recombinant Ty1 integrase production reported so far has been a major obstacle for structural studies. Here we analyze the biophysical and biochemical properties of a stable and functional recombinant Ty1 integrase highly expressed in E.coli. The recombinant protein is monomeric and has an elongated shape harboring the three-domain structure common to all retroviral integrases at the N-terminal half, an extra folded region, and a large intrinsically disordered region at the C-terminal half. Recombinant Ty1 integrase efficiently catalyzes concerted integration in vitro, and the N-terminal domain displays similar activity. These studies that will facilitate structural analyses may allow elucidating the molecular mechanisms governing Ty1 specific integration into safe places in the genome.

Construction and validation of a plasmid for HIV-1 phenotyping assays using Luminescence

Objective: The objective of these studies was the construction of a recombination vector for HIV-1 Integrase containing luciferase reporter gene, for the generation of recombinant viruses that can be used in phenotyping assays with integrase inhibitors. Methods: In this work, the vector pNL4-3Luc was molecularly manipulated in order to delete the integrase gene and build a vector for recombination of integrases from different virus subtypes. Results: As a result, viruses with recombinant integrases were generated in HEK293T cells transfected with plasmids, showing significant levels of luminescence. After the purification of the produced viral particles, susceptible lymphocytes were infected with the viruses containing the recombinant integrases and luminescence was detected in both integrases from HIV-1 subtype B and subtype C. Conclusion: The observation of light emission from cells infected by viruses with different integrases can be an efficient method to assess the susceptibility of these viruses in the presence of specific inhibitors for HIV-1 Integrase.

Progressive emergence of an S153F plus R263K combination of integrase mutations in the proviral DNA of one individual successfully treated with dolutegravir.

The development of HIV drug resistance against the integrase strand transfer inhibitor dolutegravir is rare. We report here the transient detection, by near full-genome ultradeep sequencing, of minority HIV-1 subtype B variants bearing the S153F and R263K integrase substitutions in the proviral DNA from blood cells of one patient who successfully initiated dolutegravir-based ART, over 24 weeks. Our objective was to study the effects of these substitutions. Strand transfer and DNA-binding activities of recombinant integrase proteins were measured in cell-free assays. Cell-based resistance, infectivity and replicative capacities were measured using molecular clones. Structural modelling was performed to understand experimental results. R263K emerged first, followed by the addition of S153F at Week 12. By Week 24, both mutations remained present, but at lower prevalence. We confirmed the coexistence of S153F and R263K on single viral genomes. Combining S153F or S153Y with R263K decreased integration and viral replicative capacity and conferred high levels of drug resistance against all integrase inhibitors. Alone, S153Y and S153F did little to infectivity or dolutegravir resistance. We identified altered DNA binding as a mechanism of resistance. The patient remained with undetectable viral loads at all timepoints. Drug-resistant minority variants have often been reported under suppressive ART. Our study adds to these observations by unravelling a progression towards higher levels of resistance through a novel pathway despite continuous undetectable viral loads. Poorly replicative HIV drug-resistant minority proviral variants did not compromise viral suppression in one individual treated with dolutegravir.

Two-long terminal repeat (LTR) DNA circles are a substrate for HIV-1 integrase

Integration of the HIV-1 DNA into the host genome is essential for viral replication and is catalyzed by the retroviral integrase. To date, the only substrate described to be involved in this critical reaction is the linear viral DNA produced in reverse transcription. However, during HIV-1 infection, two-long terminal repeat DNA circles (2-LTRcs) are also generated through the ligation of the viral DNA ends by the host cell's nonhomologous DNA end-joining pathway. These DNAs contain all the genetic information required for viral replication, but their role in HIV-1's life cycle remains unknown. We previously showed that both linear and circular DNA fragments containing the 2-LTR palindrome junction can be efficiently cleaved in vitro by recombinant integrases, leading to the formation of linear 3'-processed-like DNA. In this report, using in vitro experiments with purified proteins and DNAs along with DNA endonuclease and in vivo integration assays, we show that this circularized genome can also be efficiently used as a substrate in HIV-1 integrase-mediated integration both in vitro and in eukaryotic cells. Notably, we demonstrate that the palindrome cleavage occurs via a two-step mechanism leading to a blunt-ended DNA product, followed by a classical 3'-processing reaction; this cleavage leads to integrase-dependent integration, highlighted by a 5-bp duplication of the host genome. Our results suggest that 2-LTRc may constitute a reserve supply of HIV-1 genomes for proviral integration.

Consensus Integrase of a New HIV-1 Genetic Variant CRF63_02A1

The high genetic variability of the human immunodeficiency virus (HIV-1) leads to a constant emergence of new genetic variants, including the recombinant virus CRF63_02A1, which is widespread in the Siberian Federal District of Russia. We studied HIV-1 CRF63_02A1 integrase (IN_CRF) catalyzing the incorporation of viral DNA into the genome of an infected cell. The consensus sequence was designed, recombinant integrase was obtained, and its DNA-binding and catalytic activities were characterized. The stability of the IN_CRF complex with the DNA substrate did not differ from the complex stability for subtype A and B integrases; however, the rate of complex formation was significantly higher. The rates and efficiencies of 3'-processing and strand transfer reactions catalyzed by IN_CRF were found to be higher, too. Apparently, all these distinctive features of IN_CRF may result from specific amino acid substitutions in its N-terminal domain, which plays an important role in enzyme multimerization and binding to the DNA substrate. It was also found that the drug resistance mutations Q148K/G140S and G118R/E138K significantly reduce the catalytic activity of IN_CRF and its sensitivity to the strand transfer inhibitor raltegravir. Reduction in sensitivity to raltegravir was found to be much stronger in the case of double-mutation Q148K/G140S.

Characterization of HIV-1 integrase interaction with human Ku70 protein and initial implications for drug targeting

Human Ku70/Ku80 protein is known to influence HIV-1 replication. One of the possible reasons may be the protection of integrase from proteasomal degradation by Ku70 subunit. We demonstrated that recombinant HIV-1 integrase and Ku70 form a stable complex, while no interaction of Ku70 with integrase from prototype foamy virus was observed. By analyzing protein subdomains we determined two binding sites in the structure of both Ku70 and integrase: the 51–160 a.a. region of integrase interacts with residues 251–438 of Ku70, whereas Ku70 N-terminal domain (1–250 a.a.) contacts an α6-helix in the 200–220 a.a. integrase region. Single substitutions within integrase (E212A or L213A) block the interaction with Ku70 thus indicating that the binding site formed by the 200–220 a.a. integrase region is crucial for complex formation. E212A/L213A substitutions decreased the integrase capacity to bind Ku70 in HEK293T cells. A conjugate of 2′-ОMe-GGUUUUUGUGU oligonucleotide with eosin is shown by molecular modeling to shield integrase residues E212/L213 and is effective in blocking complex formation of Ku70 with integrase what makes the complex between α6-helix and Ku70(1–250) a possible target for drug development.

Development of a phenotypic susceptibility assay for HIV-1 integrase inhibitors

Phenotypic resistance analysis is an indispensable method for determination of HIV-1 resistance and cross-resistance to novel drug compounds. Since integrase inhibitors are essential components of recent antiretroviral combination therapies, phenotypic resistance data, in conjunction with the corresponding genotypes, are needed for improving rules-based and data-driven tools for resistance prediction, such as HIV-Grade and geno2pheno[integrase]. For generation of phenotypic resistance data to recent integrase inhibitors, a recombinant phenotypic integrase susceptibility assay was established. For validation purposes, the phenotypic resistance to raltegravir, elvitegravir and dolutegravir of nine subtype-B virus strains, isolated from integrase inhibitor-naïve and raltegravir-treated patients was determined. Genotypic resistance analysis identified four virus strains harbouring RAL resistance-associated mutations. Phenotypic resistance analysis was performed as follows. The HIV-1 integrase genes were cloned into a modified pNL4-3 vector and transfected into 293T cells for the generation of recombinant virus. The integrase-inhibitor susceptibility of the recombinant viruses was determined via an indicator cell line. While raltegravir resistance profiles presented a high cross-resistance to elvitegravir, dolutegravir maintained in-vitro activity in spite of the Y143R and N155H mutations, confirming the strong activity of dolutegravir against raltegravir-resistant viruses. Solely a Q148H+G140S variant presented reduced susceptibility to dolutegravir. In conclusion, our phenotypic susceptibility assay permits resistance analysis of the integrase gene of patient-derived viruses for integrase inhibitors by replication-competent recombinants. Thus, this assay can be used to analyze phenotypic drug resistance of integrase inhibitors in vitro. It provides the possibility to determine the impact of newly appearing mutational patterns to drug resistance of recent integrase inhibitors.

Removal of nuclease contamination during purification of recombinant prototype foamy virus integrase

Retroviral infection requires integration of the viral genome into the host genome. Recombinant integrase proteins may be purified following bacterial expression. A bulk biochemical assay of integrase function relies on the conversion of supercoiled plasmids to linear or relaxed circles. Single molecule molecular tweezer assays of integrase also evaluate the conversion of supercoiled DNA to nicked and broken species. A bacterial nuclease that co-purifies with retroviral integrase may affect the quantitation of integration activity in bulk or single molecule assays. During purification of retroviral integrase from bacteria, fractions may be screened for contaminating nuclease activity. In order to efficiently separate the nuclease from integrase, the binding affinities of each protein must differ. We find that a co-purifying nuclease may be efficiently separated from integrase based on heparin affinity, but not ionic affinity.

Characterization of the Drug Resistance Profiles of Integrase Strand Transfer Inhibitors in Simian Immunodeficiency Virus SIVmac239.

We previously showed that the simian immunodeficiency virus SIVmac239 is susceptible to human immunodeficiency virus (HIV) integrase (IN) strand transfer inhibitors (INSTIs) and that the same IN drug resistance mutations result in similar phenotypes in both viruses. Now we wished to determine whether tissue culture drug selection studies with SIV would yield the same resistance mutations as in HIV. Tissue culture selection experiments were performed using rhesus macaque peripheral blood mononuclear cells (PBMCs) infected with SIVmac239 viruses in the presence of increasing concentrations of dolutegravir (DTG), elvitegravir (EVG), and raltegravir (RAL). We now show that 22 weeks of selection pressure with DTG yielded a mutation at position R263K in SIV, similar to what has been observed in HIV, and that selections with EVG led to emergence of the E92Q substitution, which is a primary INSTI resistance mutation in HIV associated with EVG treatment failure. To study this at a biochemical level, purified recombinant SIVmac239 wild-type (WT) and E92Q, T97A, G118R, Y143R, Q148R, N155H, R263K, E92Q T97A, E92Q Y143R, R263K H51Y, and G140S Q148R recombinant substitution-containing IN enzymes were produced, and each of the characteristics strand transfer, 3'-processing activity, and INSTI inhibitory constants was assessed in cell-free assays. The results show that the G118R and G140S Q148R substitutions decreased Km' and Vmax'/Km' for strand transfer compared to those of the WT. RAL and EVG showed reduced activity against both viruses and against enzymes containing Q148R, E92Q Y143R, and G140S Q148R. Both viruses and enzymes containing Q148R and G140S Q148R showed moderate levels of resistance against DTG. This study further confirms that the same mutations associated with drug resistance in HIV display similar profiles in SIV. Our goal was to definitively establish whether HIV and simian immunodeficiency virus (SIV) share similar resistance pathways under tissue culture drug selection pressure with integrase strand transfer inhibitors and to test the effect of HIV-1 integrase resistance-associated mutations on SIV integrase catalytic activity and resistance to integrase strand transfer inhibitors. Clinically relevant HIV integrase resistance-associated mutations were selected in SIV in our tissue culture experiments. Not only do we report on the characterization of SIV recombinant integrase enzyme catalytic activities, we also provide the first research anywhere on the effect of mutations within recombinant integrase SIV enzymes on drug resistance.

Dolutegravir-Selected HIV-1 Containing the N155H and R263K Resistance Substitutions Does Not Acquire Additional Compensatory Mutations under Drug Pressure That Lead to Higher-Level Resistance and Increased Replicative Capacity.

We have previously shown that the addition of the raltegravir/elvitegavir (RAL/EVG) primary resistance mutation N155H to the R263K dolutegravir (DTG) resistance mutation partially compensated for the fitness cost imposed by R263K while also slightly increasing DTG resistance in vitro (K. Anstett, T. Mesplede, M. Oliveira, V. Cutillas, and M. A. Wainberg, J Virol 89:4681-4684, 2015, doi:10.1128/JVI.03485-14). Since many patients failing RAL/EVG are given DTG as part of rescue therapy, and given that the N155H substitution often is found in combination with other compensatory resistance mutations in such individuals, we investigated the effects of multiple such substitutions within integrase (IN) on each of integrase function, HIV-1 infectivity, and levels of drug resistance. To this end, each of the L74M, E92Q, T97A, E157Q, and G163R substitutions were introduced into NL4.3 subtype B HIV-1 vectors harboring N155H and R263K in tandem [termed NL4.3IN(N155H/R263K)]. Relevant recombinant integrase enzymes also were expressed, and purified and biochemical assays of strand transfer efficiency as well as viral infectivity and drug resistance studies were performed. We found that the addition of T97A, E157Q, or G163R somewhat improved the affinity of INN155H/R263K for its target DNA substrate, while the presence of L74M or E92Q had a negative effect on this process. However, viral infectivity was significantly decreased from that of NL4.3IN(N155H/R263K) after the addition of each tertiary mutation, and no increases in levels of DTG resistance were observed. This work shows that the compensatory mutations that evolve after N155H under continued DTG or RAL/EVG pressure in patients are unable to improve either enzyme efficiency or viral infectivity in an N155H/R263K background. In contrast to other drugs, dolutegravir has not selected for resistance in HIV-positive individuals when used in first-line therapy. We had previously shown that HIV containing the primary raltegravir/elvitegravir resistance substitution N155H could select for R263K under dolutegravir pressure and that this virus was fit and displayed low-level resistance to dolutegravir (Anstett et al., J Virol 89: 4681-4684). Therefore, the current study aimed to uncover whether accessory mutations that appear after N155H in response to raltegravir/elvitegravir were compatible with N155H and R263K. We found, however, that the addition of a third mutation negatively impacted both the enzyme and the virus in terms of activity and infectivity without large shifts in integrase inhibitor resistance. Thus, it is unlikely that these substitutions would be selected under dolutegravir pressure. These data support the hypothesis that primary resistance against DTG cannot evolve through RAL/EVG resistance pathways and that the selection of R263K leads HIV into an evolutionary dead-end.

Development of an integrase-based ELISA for specific diagnosis of individuals infected with HIV

Currently, enzyme immunoassays (EIAs) are the most common immunological diagnostic methods that are used as the screening tool in HIV detection. Among all three major genes of HIV, the products of gag and env are usually used in EIAs (ELISAs and rapid tests). Hence, the presence of cross reacting antibodies against these antigens leads to the appearance of repetitive false positive results in screening tests. Re-testing the primary reactive samples with EIAs using other HIV antigens can considerably reduce the rate of false positive results. The products of pol gene may act as an appropriate candidate in this context. Integrase is a conserved and immunogenic product of HIV, encoded by the pol gene. The aim of this research was to determine the sensitivity and specificity of an ELISA detecting integrase antibodies. Recombinant integrase was produced in Escherichia coli to develop the integrase-based ELISA. Assay performance was evaluated by HIV positive and negative sera and an HIV panel of BBI (PRB-601). The sensitivity and specificity of assay was determined as 96.7 [95% confidence interval: 91.3–98.9%] and 100% [95% CI: 96.1–100%], respectively. High specificity of this assay may suggest its possible use in the detection of HIV.

The R263K mutation in HIV integrase that is selected by dolutegravir may actually prevent clinically relevant resistance to this compound.

IntroductionDrug resistance against dolutegravir (DTG) or the nucleosides with which it has been co-administered has never been observed in patients receiving this drug in first-line therapy. In contrast, a R263K mutation that confers low-level resistance (3–4 fold) to DTG has been selected by DTG in culture. Our group has ascribed the absence of resistance to DTG to the high fitness cost exacted by the R263K mutation and an inability of HIV to generate compensatory mutations.Materials and MethodsWe generated recombinant integrase enzymes and viruses containing various combinations of mutations and studied these enzymatically and in culture. We also selected for resistance against raltegravir (RAL) using viruses containing the R263K mutation.ResultsThe R263K mutation alone conferred an approximate 3-fold level of resistance to DTG and a 40% loss in viral replicative capacity and recombinant integrase activity. Secondary mutations selected at positions H51Y or E138K did not individually affect either enzyme activity or DTG resistance, but the combination of R263K together with H51Y or E138K increased DTG resistance to about 7-fold accompanied by a ≈75% loss in each of viral replication capacity, and both in vitro and in vivo integrase activity. Conversely, combinations of R263K together with multiple resistance mutations for RAL and/or EVG at positions 92,143, 148 and 155 resulted in even further diminished enzymatic activity that may be incompatible with viral survival. Modelling of the 3-dimensional structure of integrase suggests that R263K is located in a region that may not permit further mutagenesis if secondary mutations at H51Y or E138K are also present. Moreover, integrase that contains R263K together with substitutions at positions 92, 143, 148 and 155 may be enzymatically inactive. The use of the R263K-containing virus to select for resistance to RAL led to the appearance of RAL-containing mutations but the loss of R263K.ConclusionsSecondary mutations to R263K following selection with DTG have all led to diminished viral and enzymatic fitness, helping to explain why resistance to DTG in previously drug-naïve subjects has never been observed. The use of DTG in first-line therapy may prevent the facile development of drug resistance and help to forestall ongoing HIV transmission.

The R262K Substitution Combined with H51Y in HIV-1 Subtype B Integrase Confers Low-Level Resistance against Dolutegravir

Clinical studies have shown that integrase strand transfer inhibitors (INSTIs) can be used effectively against HIV-1 infection. To date, no resistance substitution has been found in INSTI-naive patients treated with the new integrase inhibitor dolutegravir (DTG). In a recent selection study with DTG, using a virus bearing the H51Y substitution in integrase, the emergence of an R to K substitution at position 262 (R262K) was observed. We characterized this double mutant with respect to integrase strand transfer activity and susceptibility to DTG both biochemically and in tissue culture. We showed that the addition of R262K to H51Y decreased recombinant integrase strand transfer activity but improved integrase DNA-binding affinity, compared to wild-type or H51Y-containing enzymes. The defect in strand transfer activity did not translate into a decrease in HIV-1 infectivity. The combination of H51Y and R262K substitutions slightly decreased susceptibility to DTG (fold change = 1.87) in cell-based resistance assays. Although viral replication was not affected and enzyme efficiency was impaired by the addition of R262K to H51Y, there was an overall increase in the level of biochemical drug resistance against DTG. Our findings suggest that the R at position 262 plays an important role in DNA binding.

6-(1-Benzyl-1H-pyrrol-2-yl)-2,4-dioxo-5-hexenoic Acids as Dual Inhibitors of Recombinant HIV-1 Integrase and Ribonuclease H, Synthesized by a Parallel Synthesis Approach

The increasing efficiency of HAART has helped to transform HIV/AIDS into a chronic disease. Still, resistance and drug-drug interactions warrant the development of new anti-HIV agents. We previously discovered hit 6, active against HIV-1 replication and targeting RNase H in vitro. Because of its diketo-acid moiety, we speculated that this chemotype could serve to develop dual inhibitors of both RNase H and integrase. Here, we describe a new series of 1-benzyl-pyrrolyl diketohexenoic derivatives, 7a-y and 8a-y, synthesized following a parallel solution-phase approach. Those 50 analogues have been tested on recombinant enzymes (RNase H and integrase) and in cell-based assays. Approximately half (22) exibited inhibition of HIV replication. Compounds 7b, 7u, and 8g were the most active against the RNase H activity of reverse-transcriptase, with IC50 values of 3, 3, and 2.5 μM, respectively. Compound 8g was also the most potent integrase inhibitor with an IC50 value of 26 nM.

Dialysis Purification of Integrase-DNA Complexes Provides High-Resolution Atomic Force Microscopy Images: Dimeric Recombinant HIV-1 Integrase Binding and Specific Looping on DNA

It remains difficult to obtain high-resolution atomic force microscopy images of HIV-1 integrase bound to DNA in a dimeric or tetrameric fashion. We therefore constructed specific target DNAs to assess HIV-1 integrase binding and purified the complex by dialysis prior to analysis. Our resulting atomic force microscopy analyses indicated precise size of binding human immunodeficiency virus type 1 (HIV-1) recombinant integrase in a tetrameric manner, inducing formation of a loop-like or figure-eight-like secondary structure in the target DNA. Our findings regarding the target DNA secondary structure provide new insights into the intermediate states of retroviral integration.

Selection in culture of HIV resistance to dolutegravir by mutations at integrase positions R263K and H51Y that diminish viral replication fitness

Purpose of the studyWe selected for resistance in tissue culture against dolutegravir (DTG), a second‐generation HIV integrase strand transfer inhibitor, which is now in phase 2/3 clinical trials, in order to try to characterize the resistance profile of this compound.MethodsHIV‐1 of different subtypes was grown in both MT‐2 cells and peripheral blood mononuclear cells over protracted periods, with the concentration of DTG being incrementally increased from an initial concentration of 0.05 nM, i.e. 4 times less than the EC50. After a total of 6 months of growth, a final drug concentration of 50–100 nM was achieved, beyond which virus could no longer be grown. Viral DNA was then sequenced to reveal the presence of mutations that might confer resistance to DTG. The biological relevance of these mutations was confirmed through site‐directed mutagenesis experiments in which individual mutations or combinations of mutations were studied in comparison with wild‐type (wt) virus in tissue culture and with recombinant HIV integrase enzyme in biochemical assays.Summary of resultsThe most common integrase resistance mutation to arise in subtype B and recombinant A/G viruses was R263K followed by H51Y. In the case of subtype C viruses, the most common mutation was G118R followed by H51Y. The presence of R263K alone conferred an approximate 3–6‐fold level of resistance to DTG in culture, a 30% drop in levels of recombinant integrase strand transfer activity, as well as an approximate 20–30% loss in viral replicative capacity. Biochemical experiments indicated that the t½ residency times of DTG for subtype B and C viruses for wt integrase were 26 h and 38 h, respectively, and for R263K, 16h and 22h, respectively. In contrast, H51Y by itself did not significantly affect either strand transfer activity or resistance to DTG. However, the combination of R263K together with H51Y led to an increase in levels of DTG resistance to about 15‐fold accompanied by an ~50% loss in both viral replication capacity and integrase strand transfer activity.ConclusionsR263K and H51Y can combine to augment levels of resistance to DTG yet result in a more severe attenuation of viral replication capacity and integrase strand transfer activity than R263K alone. These data suggest that viruses containing both mutations may be at a severe replicative disadvantage and help to explain why primary resistance to DTG is so rare to arise in clinical studies performed to date.

Correlation of Recombinant Integrase Activity and Functional Preintegration Complex Formation during Acute Infection by Replication-Defective Integrase Mutant Human Immunodeficiency Virus

Previous studies characterized two types of replication-defective human immunodeficiency virus type 1 (HIV-1) integrase mutants: class I, which are specifically blocked at the integration step, and class II, which harbor additional virion production and/or reverse transcription defects. Class I mutant enzymes supported little if any metal ion-dependent 3'-processing and DNA strand transfer activities in vitro, whereas class II enzymes displayed partial or full catalytic function in studies with simplified assay designs, suggesting that defective interaction(s) with heterologous integrase binding proteins might underlie the class II mutant viral phenotype. To address this hypothesis, class I and II mutant enzymes were interrogated under expanded sets of in vitro conditions. The majority failed to catalyze the concerted integration of two viral DNA ends into target DNA, highlighting defective integrase function as the root cause of most class II in addition to all class I mutant virus infection defects. One mutant protein, K264E, in contrast, could support the wild-type level of concerted integration activity. After accounting for its inherent reverse transcription defect, HIV-1(K264E) moreover formed preintegration complexes that supported the efficient integration of endogenous viral DNA in vitro and normal levels and sequences of 2-long terminal repeat-containing circle junctions during acute infection. K264E integrase furthermore efficiently interacted in vitro with two heterologous binding partners, LEDGF/p75 and reverse transcriptase. Our results underscore the physiological relevance of concerted integration assays for tests of integrase mutant function and suggest that the K264E mutation disrupts an interaction with an intranuclear integrase binding partner that is important for HIV-1 integration.

Study of the activity of integrase of retrotransposone Gypsy Drosophila melanogaster in vitro

Introduction of retrotransposone gypsy in the genome of Drosophila melanogaster occurs specifically and is performed by integrase of this mobile element. Gypsy integrase is capable not only of introduction but also of precise excision of retrotransposon DNA from the target site. In the present work, we study the endonuclease activity of recombinant integrase gypsy in vitro. It is indicated that the enzyme hydrolyzes the substrate, forming single-chain and double-chain breaks. Conformation of the substrate greatly influences hydrolysis: integrase conducts double-chain breaks only in a supertwisted circular DNA molecule. The data make it possible to understanding the integration mechanism of retroviruses and transposition of mobile elements.