Catalytic Water Co-Existing with a Product Peptide in the Active Site of HIV-1 Protease Revealed by X-Ray Structure Analysis

Background

It is known that HIV-1 protease is an important target for design of antiviral compounds in the treatment of Acquired Immuno Deficiency Syndrome (AIDS). In this context, understanding the catalytic mechanism of the enzyme is of crucial importance as transition state structure directs inhibitor design. Most mechanistic proposals invoke nucleophilic attack on the scissile peptide bond by a water molecule. But such a water molecule coexisting with any ligand in the active site has not been found so far in the crystal structures.

Principal Findings

We report here the first observation of the coexistence in the active site, of a water molecule WAT1, along with the carboxyl terminal product (Q product) peptide. The product peptide has been generated in situ through cleavage of the full-length substrate. The N-terminal product (P product) has diffused out and is replaced by a set of water molecules while the Q product is still held in the active site through hydrogen bonds. The position of WAT1, which hydrogen bonds to both the catalytic aspartates, is different from when there is no substrate bound in the active site. We propose WAT1 to be the position from where catalytic water attacks the scissile peptide bond. Comparison of structures of HIV-1 protease complexed with the same oligopeptide substrate, but at pH 2.0 and at pH 7.0 shows interesting changes in the conformation and hydrogen bonding interactions from the catalytic aspartates.

Conclusions/Significance

The structure is suggestive of the repositioning, during substrate binding, of the catalytic water for activation and subsequent nucleophilic attack. The structure could be a snap shot of the enzyme active site primed for the next round of catalysis. This structure further suggests that to achieve the goal of designing inhibitors mimicking the transition-state, the hydrogen-bonding pattern between WAT1 and the enzyme should be replicated.     

Epistasis among Deleterious Mutations in the HIV-1 Protease

A central goal in molecular evolution is to understand how genetic interactions between protein mutations shape protein function and fitness. While intergenic epistasis has been extensively explored in eukaryotes, bacteria, and viruses, intragenic epistatic interactions have been insufficiently studied. Here, we employ a model system in which lambda phage fitness correlates with the enzymatic activity of human immunodeficiency virus type 1 (HIV-1) protease to systematically determine the epistatic interactions between intragenic pairs of deleterious protein substitutions. We generated 114 genotypes of the HIV-1 protease, each carrying pairs of nucleotide substitution mutations whose separated and combined deleterious effects on fitness were then determined. A high proportion (39%) of pairs displayed lethality. Several pairs exhibited significant interactions for fitness, including positive and negative epistasis. Significant negative epistatic interactions predominated (15%) over positive interactions (2%). However, the average ± SD epistatic effect, ē = 0.0025 ± 0.1334, was not significantly different from zero (p = 0.8368). Notably, epistatic interactions, regardless of epistatic direction, tend to be more frequent in the context of less deleterious mutations. In the present study, the high frequencies of lethality and negative epistasis indicate that the HIV-1 protease is highly sensitive to the effects of deleterious mutations. Therefore, proteins may not be as robust to mutational change as is usually expected.     

Resistance to Protease Inhibitors in a Model of HIV-1 Infection with Impulsive Drug Effects

Background

The emergence of drug resistance is one of the most prevalent reasons for treatment failure in HIV therapy. This has severe implications for the cost of treatment, survival and quality of life.

Methods

We use mathematical modelling to describe the interaction between T cells, HIV-1 and protease inhibitors. We use impulsive differential equations to examine the effects of different levels of protease inhibitors in a T cell. We classify three different regimes according to whether the drug efficacy is low, intermediate or high. The model includes two strains: the wild-type strain, which initially dominates in the absence of drugs, and the mutant strain, which is the less efficient competitor, but has more resistance to the drugs.

Results

Drug regimes may take trajectories through one, two or all three regimes, depending on the dosage and the dosing schedule. Stability analysis shows that resistance does not emerge at low drug levels. At intermediate drug levels, drug resistance is guaranteed to emerge. At high drug levels, either the drug-resistant strain will dominate or, in the absence of longer-lived reservoirs of infected cells, a region exists where viral elimination could theoretically occur. We provide estimates of a range of dosages and dosing schedules where the trajectories lie either solely within a region or cross multiple regions.

Conclusion

Under specific circumstances, if the drug level is physiologically tolerable, elimination of free virus is theoretically possible. This forms the basis for theoretical control using combination therapy and for understanding the effects of partial adherence.     

Large Lateral Movement of Transmembrane Helix S5 Is Not Required for Substrate Access to the Active Site of Rhomboid Intramembrane Protease

Rhomboids represent an evolutionarily ancient protease family. Unlike most other proteases, they are polytopic membrane proteins and specialize in cleaving transmembrane protein substrates. The polar active site of rhomboid protease is embedded in the membrane and normally closed. For the bacterial rhomboid GlpG, it has been proposed that one of the transmembrane helices (S5) of the protease can rotate to open a lateral gate, enabling substrate to enter the protease from inside the membrane. Here, we studied the conformational change in GlpG by solving the cocrystal structure of the protease with a mechanism-based inhibitor. We also examined the lateral gating model by cross-linking S5 to a neighboring helix (S2). The crystal structure shows that inhibitor binding displaces a capping loop (L5) from the active site but causes only minor shifts in the transmembrane helices. Cross-linking S5 and S2, which not only restricts the lateral movement of S5 but also prevents substrate from passing between the two helices, does not hinder the ability of the protease to cleave a membrane protein substrate in detergent solution and in reconstituted membrane vesicles. Taken together, these data suggest that a large lateral movement of the S5 helix is not required for substrate access to the active site of rhomboid protease.     

Evidence for Reduced Drug Susceptibility without Emergence of Major Protease Mutations following Protease Inhibitor Monotherapy Failure in the SARA Trial

BackgroundMajor protease mutations are rarely observed following failure with protease inhibitors (PI), and other viral determinants of failure to PI are poorly understood. We therefore characterized Gag-Protease phenotypic susceptibility in subtype A and D viruses circulating in East Africa following viral rebound on PIs.MethodsSamples from baseline and treatment failure in patients enrolled in the second line LPV/r trial SARA underwent phenotypic susceptibility testing. Data were expressed as fold-change in susceptibility relative to a LPV-susceptible reference strain.ResultsWe cloned 48 Gag-Protease containing sequences from seven individuals and performed drug resistance phenotyping from pre-PI and treatment failure timepoints in seven patients. For the six patients where major protease inhibitor resistance mutations did not emerge, mean fold-change EC₅₀ to LPV was 4.07 fold (95% CI, 2.08–6.07) at the pre-PI timepoint. Following viral failure the mean fold-change in EC₅₀ to LPV was 4.25 fold (95% CI, 1.39–7.11, p = 0.91). All viruses remained susceptible to DRV. In our assay system, the major PI resistance mutation I84V, which emerged in one individual, conferred a 10.5-fold reduction in LPV susceptibility. One of the six patients exhibited a significant reduction in susceptibility between pre-PI and failure timepoints (from 4.7 fold to 9.6 fold) in the absence of known major mutations in protease, but associated with changes in Gag: V7I, G49D, R69Q, A120D, Q127K, N375S and I462S. Phylogenetic analysis provided evidence of the emergence of genetically distinct viruses at the time of treatment failure, indicating ongoing viral evolution in Gag-protease under PI pressure.ConclusionsHere we observe in one patient the development of significantly reduced susceptibility conferred by changes in Gag which may have contributed to treatment failure on a protease inhibitor containing regimen. Further phenotype-genotype studies are required to elucidate genetic determinants of protease inhibitor failure in those who fail without traditional resistance mutations whilst PI use is being scaled up globally.     

HIV-1蛋白酶的表达、纯化及其抑制剂体外筛选方法的建立

从HIV-1IIIB病毒RNA经RT-PCR得到HIV-1蛋白酶编码序列,克隆到pet28a质粒中构建HIV-1蛋白酶表达载体。阳性克隆转染E.coliBL21DE3,经IPTG诱导,蛋白酶以包涵体的形式表达,表达量占菌体总蛋白量的40%。包涵体经TritonX-100洗涤后溶解于8M尿素,溶解后的蛋白溶液经sephacyls-200H.R分子筛柱纯化后纯度达到90%以上,收集蛋白酶峰稀释复性并通过超滤进行浓缩。经检测,纯化的蛋白酶具有较高的活性。用荧光标记的蛋白酶底物检测不同浓度indinavir对蛋白酶活性的影响,表明该方法可以用于蛋白酶抑制剂的筛选。     

HIV-1蛋白酶的表达、纯化及其抑制剂体外筛选方法的建立

从HIV-1 ⅢB病毒RNA经RT-PCR得到HIV-1蛋白酶编码序列,克隆到pet28a质粒中构建HIV-1蛋白酶表达载体.阳性克隆转染E.coli BL21 DE3,经IPTG诱导,蛋白酶以包涵体的形式表达,表达量占菌体总蛋白量的40%.包涵体经Triton X-100洗涤后溶解于8M尿素,溶解后的蛋白溶液经sephacyl s-200 H.R分子筛柱纯化后纯度达到90%以上,收集蛋白酶峰稀释复性并通过超滤进行浓缩.经检测,纯化的蛋白酶具有较高的活性.用荧光标记的蛋白酶底物检测不同浓度indinavir对蛋白酶活性的影响,表明该方法可以用于蛋白酶抑制剂的筛选.     

Mutations in Multiple Domains of Gag Drive the Emergence of In Vitro Resistance to the Phosphonate-Containing HIV-1 Protease Inhibitor GS-8374

GS-8374 is a potent HIV protease inhibitor (PI) with a unique diethyl-phosphonate moiety. Due to a balanced contribution of enthalpic and entropic components to its interaction with the protease (PR) active site, the compound retains activity against HIV mutants with high-level multi-PI resistance. We report here the in vitro selection and characterization of HIV variants resistant to GS-8374. While highly resistant viruses with multiple mutations in PR were isolated in the presence of control PIs, an HIV variant displaying moderate (14-fold) resistance to GS-8374 was generated only after prolonged passaging for >300 days. The isolate showed low-level cross-resistance to darunavir, atazanavir, lopinavir, and saquinavir, but not other PIs, and contained a single R41K mutation in PR combined with multiple genotypic changes in the Gag matrix, capsid, nucleocapsid, and SP2 domains. Mutations also occurred in the transframe peptide and p6* domain of the Gag-Pol polyprotein. Analysis of recombinant HIV variants indicated that mutations in Gag, but not the R41K in PR, conferred reduced susceptibility to GS-8374. The Gag mutations acted in concert, since they did not affect susceptibility when introduced individually. Analysis of viral particles revealed that the mutations rendered Gag more susceptible to PR-mediated cleavage in the presence of GS-8374. In summary, the emergence of resistance to GS-8374 involved a combination of substrate mutations without typical resistance mutations in PR. These substrate changes were distributed throughout Gag and acted in an additive manner. Thus, they are classified as primary resistance mutations indicating a unique mechanism and pathway of resistance development for GS-8374.     

用两步MS-PCR结合ELISA检测HIV-1逆转录酶基因的耐药性突变

高效抗逆转录病毒治疗法（HAART）的推广使用有效地抑制了HIV-1病毒的传播和艾滋病（AIDS）的发病率、死亡率。近年来,HIV-1逆转录酶基因突变所导致的耐药性成为了HAART的治疗失败的主要原因,耐药性突变的检测对于指导病人用药以及新药开发具有重要意义。本工作发展了一种检测HIV-1逆转录酶基因耐药性突变的新方法。采用两步MS-PCR方法检测HIV-1 B亚型野生型和耐药突变型逆转录酶基因突变,检测点包括M41L、K70R、K103N、Y181C和T215,并在两步MS-PCR基础上,设计比野生型引物长的突变型引物,结合微孔板杂交ELISA呈色技术检测各点突变。结果显示,简化的两步MS-PCR能保持灵敏度和特异性高的优点,ELISA的阳性结果与阴性结果的比值（P/N）达到要求,两步MS-PCR结合ELISA方法灵敏度高,操作简单、结果直观、成本低、相对耗时短且能进行高通量检测,其无论在HIV-1耐药性突变及其它的点突变检测中具有较好的临床应用前景。     

Membrane Potential Depolarization as a Triggering Mechanism for Vpu-Mediated HIV-1 Release

Vpu, a component unique to HIV-1, greatly enhances the efficiency of viral particle release by unclear mechanisms. This Vpu function is intrinsically linked to its channel-like structure, which enables it to interfere with homologous transmembrane structures in infected cells. Because Vpu interacts destructively with host background K⁺ channels that set the cell resting potential, we hypothesized that Vpu might trigger viral release by destabilizing the electric field across a budding membrane. Here, we found that the efficiency of Vpu-mediated viral release is inversely correlated with membrane potential polarization. By inhibiting the background K⁺ currents, Vpu dissipates the voltage constraint on viral particle discharge. As a proof of concept, we show that HIV-1 release can be accelerated by externally imposed depolarization alone. Our findings identify the trigger of Vpu-mediated release as a manifestation of the general principle of depolarization-stimulated exocytosis.     

Analysis of Non-Typeable Haemophilous influenzae VapC1 Mutations Reveals Structural Features Required for Toxicity and Flexibility in the Active Site

Bacteria have evolved mechanisms that allow them to survive in the face of a variety of stresses including nutrient deprivation, antibiotic challenge and engulfment by predator cells. A switch to dormancy represents one strategy that reduces energy utilization and can render cells resistant to compounds that kill growing bacteria. These persister cells pose a problem during treatment of infections with antibiotics, and dormancy mechanisms may contribute to latent infections. Many bacteria encode toxin-antitoxin (TA) gene pairs that play an important role in dormancy and the formation of persisters. VapBC gene pairs comprise the largest of the Type II TA systems in bacteria and they produce a VapC ribonuclease toxin whose activity is inhibited by the VapB antitoxin. Despite the importance of VapBC TA pairs in dormancy and persister formation, little information exists on the structural features of VapC proteins required for their toxic function in vivo. Studies reported here identified 17 single mutations that disrupt the function of VapC1 from non-typeable H. influenzae in vivo. 3-D modeling suggests that side chains affected by many of these mutations sit near the active site of the toxin protein. Phylogenetic comparisons and secondary mutagenesis indicate that VapC1 toxicity requires an alternative active site motif found in many proteobacteria. Expression of the antitoxin VapB1 counteracts the activity of VapC1 mutants partially defective for toxicity, indicating that the antitoxin binds these mutant proteins in vivo. These findings identify critical chemical features required for the biological function of VapC toxins and PIN-domain proteins.     

An Assay to Monitor HIV-1 Protease Activity for the Identification of Novel Inhibitors in T-Cells

The emergence of resistant HIV strains, together with the severe side-effects of existing drugs and lack of development of effective anti-HIV vaccines highlight the need for novel antivirals, as well as innovative methods to facilitate their discovery. Here, we have developed an assay in T-cells to monitor the proteolytic activity of the HIV-1 protease (PR). The assay is based on the inducible expression of HIV-1 PR fused within the Gal4 DNA-binding and transactivation domains. The fusion protein binds to the Gal4 responsive element and activates the downstream reporter, enhanced green fluorescent protein (eGFP) gene only in the presence of an effective PR Inhibitor (PI). Thus, in this assay, eGFP acts as a biosensor of PR activity, making it ideal for flow cytometry based screening. Furthermore, the assay was developed using retroviral technology in T-cells, thus providing an ideal environment for the screening of potential novel PIs in a cell-type that represents the natural milieu of HIV infection. Clones with the highest sensitivity, and robust, reliable and reproducible reporter activity, were selected. The assay is easily adaptable to other PR variants, a multiplex platform, as well as to high-throughput plate reader based assays and will greatly facilitate the search for novel peptide and chemical compound based PIs in T-cells.     

Mutations in a Conserved Residue in the Influenza Virus Neuraminidase Active Site Decreases Sensitivity to Neu5Ac2en-Derived Inhibitors

The influenza virus neuraminidase (NA)-specific inhibitor zanamivir (4-guanidino-Neu5Ac2en) is effective in humans when administered topically within the respiratory tract. The search for compounds with altered pharmacological properties has led to the identification of a novel series of influenza virus NA inhibitors in which the triol group of zanamivir has been replaced by a hydrophobic group linked by a carboxamide at the 6 position (6-carboxamide). NWS/G70C variants generated in vitro, with decreased sensitivity to 6-carboxamide, contained hemagglutinin (HA) and/or NA mutations. HA mutants bound with a decreased efficiency to the cellular receptor and were cross-resistant to all the NA inhibitors tested. The NA mutation, an Arg-to-Lys mutation, was in a previously conserved site, Arg292, which forms part of a triarginyl cluster in the catalytic site. In enzyme assays, the NA was equally resistant to zanamivir and 4-amino-Neu5Ac2en but showed greater resistance to 6-carboxamide and was most resistant to a new carbocyclic NA inhibitor, GS4071, which also has a hydrophobic side chain at the 6 position. Consistent with enzyme assays, the lowest resistance in cell culture was seen to zanamivir, more resistance was seen to 6-carboxamide, and the greatest resistance was seen to GS4071. Substrate binding and enzyme activity were also decreased in the mutant, and consequently, virus replication in both plaque assays and liquid culture was compromised. Altered binding of the hydrophobic side chain at the 6 position or the triol group could account for the decreased binding of both the NA inhibitors and substrate.Influenza virus possesses two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). HA is responsible for recognition of the cell surface receptor, and NA is thought to be responsible for the elution of progeny virions from infected cells, and from each other by cleavage of terminal sialic acid residues (Neu5Ac). The potential of NA as a target for antiviral therapy was investigated many years ago, when Meindl and Tuppy (13) first synthesized the unsaturated sialic acid analog Neu5Ac2en, which inhibited influenza virus replication in vitro but not in vivo (16, 17). Based on the knowledge of the three-dimensional structure of NA complexed with Neu5Ac (23), a derivative of Neu5Ac2en with a substitution of a guanidinium group at the 4 position, 4-guanidino-Neu5Ac2en (zanamivir), has been synthesized and has been shown to have potent antiviral activity both in vitro and in vivo when administered topically within the respiratory tract (7, 25, 27). The search for compounds with altered pharmacological properties has led to the identification of a novel series of influenza virus NA inhibitors in which the triol group of zanamivir was replaced with a hydrophobic group linked by a carboxamide at the 6 position (21). An essential aspect of drug development is determining if and how resistant variants may arise after prolonged exposure to the inhibitor. We and others have reported the generation of variants with decreased sensitivity to zanamivir as a result of mutations in either NA (1, 3, 4, 12, 22) or HA (3, 11). We were interested in determining whether we could also isolate variants to the 6-carboxamide derivative of zanamivir by in vitro passaging in the presence of the inhibitor.     

Potential Elucidation of a Novel CTL Epitope in HIV-1 Protease by the Protease Inhibitor Resistance Mutation L90M

The combination of host immune responses and use of antiretrovirals facilitate partial control of human immunodeficiency virus type 1 (HIV-1) infection and result in delayed progression to Acquired Immunodeficiency Syndrome (AIDS). Both treatment and host immunity impose selection pressures on the highly mutable HIV-1 genome resulting in antiretroviral resistance and immune escape. Researchers have shown that antiretroviral resistance mutations can shape cytotoxic T-lymphocyte immunity by altering the epitope repertoire of HIV infected cells. Here it was discovered that an important antiretroviral resistance mutation, L90M in HIV protease, occurs at lower frequencies in hosts that harbor the B*15, B*48 or A*32 human leukocyte antigen subtypes. A likely reason is the elucidation of novel epitopes by L90M. NetMHCPan predictions reveal increased affinity of the peptide spanning the HIV protease region, PR 89–97 and PR 90–99 to HLA-B*15/B*48 and HLA-A*32 respectively due to the L90M substitution. The higher affinity could increase the chance of the epitope being presented and recognized by Cytotoxic T-lymphocytes and perhaps provide additional immunological pressures in the presence of antiretroviral attenuating mutations. This evidence supports the notion that knowledge of HLA allotypes in HIV infected individuals could augment antiretroviral treatment by the elucidation of epitopes due to antiretroviral resistance mutations in HIV protease.     

Glycan Microheterogeneity at the PGT135 Antibody Recognition Site on HIV-1 gp120 Reveals a Molecular Mechanism for Neutralization Resistance

Broadly neutralizing antibodies have been isolated that bind the glycan shield of the HIV-1 envelope spike. One such antibody, PGT135, contacts the intrinsic mannose patch of gp120 at the Asn332, Asn392, and Asn386 glycosylation sites. Here, site-specific glycosylation analysis of recombinant gp120 revealed glycan microheterogeneity sufficient to explain the existence of a minor population of virions resistant to PGT135 neutralization. Target microheterogeneity and antibody glycan specificity are therefore important parameters in HIV-1 vaccine design.     

Molecular Characterization of Ambiguous Mutations in HIV-1 Polymerase Gene: Implications for Monitoring HIV Infection Status and Drug Resistance

Detection of recent HIV infections is a prerequisite for reliable estimations of transmitted HIV drug resistance (t-HIVDR) and incidence. However, accurately identifying recent HIV infection is challenging due partially to the limitations of current serological tests. Ambiguous nucleotides are newly emerged mutations in quasispecies, and accumulate by time of viral infection. We utilized ambiguous mutations to establish a measurement for detecting recent HIV infection and monitoring early HIVDR development. Ambiguous nucleotides were extracted from HIV-1 pol-gene sequences in the datasets of recent (HIVDR threshold surveys [HIVDR-TS] in 7 countries; n=416) and established infections (1 HIVDR monitoring survey at baseline; n=271). An ambiguous mutation index of 2.04×10^-3 nts/site was detected in HIV-1 recent infections which is equivalent to the HIV-1 substitution rate (2×10^-3 nts/site/year) reported before. However, significantly higher index (14.41×10^-3 nts/site) was revealed with established infections. Using this substitution rate, 75.2% subjects in HIVDR-TS with the exception of the Vietnam dataset and 3.3% those in HIVDR-baseline were classified as recent infection within one year. We also calculated mutation scores at amino acid level at HIVDR sites based on ambiguous or fitted mutations. The overall mutation scores caused by ambiguous mutations increased (0.54×10^-23.48×10^-2/DR-site) whereas those caused by fitted mutations remained stable (7.50-7.89×10^-2/DR-site) in both recent and established infections, indicating that t-HIVDR exists in drug-naïve populations regardless of infection status in which new HIVDR continues to emerge. Our findings suggest that characterization of ambiguous mutations in HIV may serve as an additional tool to differentiate recent from established infections and to monitor HIVDR emergence.     

Identification of Novel Mutations Responsible for Resistance to MK-2048, a Second-Generation HIV-1 Integrase Inhibitor

MK-2048 represents a prototype second-generation integrase strand transfer inhibitor (INSTI) developed with the goal of retaining activity against viruses containing mutations associated with resistance to first-generation INSTIs, raltegravir (RAL) and elvitegravir (EVG). Here, we report the identification of mutations (G118R and E138K) which confer resistance to MK-2048 and not to RAL or EVG. These mutations were selected in vitro and confirmed by site-specific mutagenesis. G118R, which appeared first in cell culture, conferred low levels of resistance to MK-2048. G118R also reduced viral replication capacity to approximately 1% that of the isogenic wild-type (wt) virus. The subsequent selection of E138K partially restored replication capacity to ≈13% of wt levels and increased resistance to MK-2048 to ≈8-fold. Viruses containing G118R and E138K remained largely susceptible to both RAL and EVG, suggesting a unique interaction between this second-generation INSTI and the enzyme may be defined by these residues as a potential basis for the increased intrinsic affinity and longer “off” rate of MK-2048. In silico structural analysis suggests that the introduction of a positively charged arginine at position 118, near the catalytic amino acid 116, might decrease Mg²⁺ binding, compromising enzyme function and thus leading to the significant reduction in both integration and viral replication capacity observed with these mutations.Selective pressure exerted by antiretroviral drugs, in conjunction with high viral mutation rates, promotes the inevitable emergence of drug-resistant HIV-1 variants. This necessitates an ongoing search for novel antiretroviral compounds that either have novel mechanisms and inhibit different stages of viral replication or inhibit targets that have acquired resistance to existing drugs. In the latter case, such newer next-generation agents should ideally display resistance profiles which are distinct and nonoverlapping with those of the first-generation drugs.Integration of viral cDNA into the host cell genome is a distinct feature of retroviral replication, and inhibitors of HIV-1 integrase have recently been added to the arsenal of clinically approved antiretroviral drugs. Raltegravir (RAL) was the first integrase strand transfer inhibitor (INSTI) to be approved by the U.S. Food and Drug Administration (FDA) after clinical trials showed that this drug promoted a rapid and sustained antiviral effect (13). Elvitegravir (EVG), another integrase inhibitor, is currently in phase III clinical trials (27). Resistance mutations common to both of these first-generation integrase inhibitors have been reported and can result in high levels of drug resistance (26). Mutations which engender cross-resistance between RAL and EVG have been reported in clinical trials, cell culture studies, and biochemical assays (9, 26). This has prompted the search for second-generation integrase inhibitors that might display novel patterns of resistance, allowing their use in patients who have failed therapy with RAL or EVG. MK-2048 (28) is a prototype second-generation INSTI that retains potency against viruses containing common single and double mutations observed in the clinic with first-generation agents with a 95% inhibitory concentration (IC₉₅) in the nM range. MK-2048 has been previously reported to be active against viruses resistant to RAL and EVG (28, 29). Given common mechanisms of action among INSTIs and a lack of structural information on integrase inhibitor complexes with resistance mutations, an understanding of resistance to second-generation agents such as MK-2048 is important.This article describes the selection of resistance to MK-2048 in tissue culture and the characterization of mutations associated with such resistance, G118R and E138K. The identification of distinct mutations which appear to confer resistance to MK-2048 and not to either RAL or EVG has potential implications for understanding the structural basis for the second-generation profile of this compound as well as future drug discovery and development efforts focused on this mechanism.     

Dominant Mutations in S. cerevisiae PMS1 Identify the Mlh1-Pms1 Endonuclease Active Site and an Exonuclease 1-Independent Mismatch Repair Pathway

Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A) that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway.     

Effects of Mutations in Residues near the Active Site of Human Immunodeficiency Virus Type 1 Integrase on Specific Enzyme-Substrate Interactions

The phylogenetically conserved catalytic core domain of human immunodeficiency virus type 1 (HIV-1) integrase contains elements necessary for specific recognition of viral and target DNA features. In order to identify specific amino acids that determine substrate specificity, we mutagenized phylogenetically conserved residues that were located in close proximity to the active-site residues in the crystal structure of the isolated catalytic core domain of HIV-1 integrase. Residues composing the phylogenetically conserved DD(35)E active-site motif were also mutagenized. Purified mutant proteins were evaluated for their ability to recognize the phylogenetically conserved CA/TG base pairs near the viral DNA ends and the unpaired dinucleotide at the 5′ end of the viral DNA, using disintegration substrates. Our findings suggest that specificity for the conserved A/T base pair depends on the active-site residue E152. The phenotype of IN(Q148L) suggested that Q148 may be involved in interactions with the 5′ dinucleotide of the viral DNA end. The activities of some of the proteins with mutations in residues in close proximity to the active-site aspartic and glutamic acids were salt sensitive, suggesting that these mutations disrupted interactions with DNA.