Impact of frequent natural polymorphisms at the protease gene on the in vitro susceptibility to protease inhibitors in HIV-1 non-B subtypes.

BACKGROUND: Naturally-occurring polymorphisms at the human immunodeficiency virus type 1 (HIV-1) protease which have been associated to resistance to protease inhibitors (PIs) in clade B viruses are frequently found in non-B subtypes, with unknown clinical significance. OBJECTIVE: To assess the susceptibility of non-B viruses to different PIs. STUDY DESIGN: Plasma samples from 58 drug-naive individuals infected with HIV-1 non-B subtypes (2A, 22C, 2D, 1F, 29G and 2J) defined by phylogenetic analyses of the protease gene were tested using a phenotypic assay (PhenoSense, ViroLogic, South San Francisco, CA, USA). Twenty of them were further analyzed with another assay (Antivirogram, Virco, Mechelen, Belgium). All 58 non-B viruses harbored amino acid substitutions associated with reduced PI susceptibility in clade B (positions 10, 20, 36, 63, 70, 77 and 82). RESULTS: Using PhenoSense-HIV assay, all but two individuals harbored viruses completely susceptible to all six PIs tested (indinavir (IDV), ritonavir (RTV), saquinavir (SQV), nelfinavir (NFV), amprenavir (APV), lopinavir (LPV)). The two viruses with reduced susceptibility belonged to clade G. The first virus, which had K20I, M36I and V82I, showed 2.9-fold decreased susceptibility to APV, while the second virus showed 3.9-fold decreased susceptibility to both NFV and RTV, with amino acid substitutions K20I, M36I, L63P and V82I. Of note, several other viruses displayed the same constellation of mutations but without showing any reduced susceptibility, suggesting that these polymorphisms per se do not affect PI susceptibility. CONCLUSION: PI susceptibility in HIV-1 non-B viruses seems to be preserved despite the presence of polymorphic changes which have been associated to PI resistance in clade B viruses.     

Changes in the Vif protein of HIV-1 associated with the development of resistance to inhibitors of viral protease

The protease (PR) and virus infectivity factor (vif) gene sequences of a cohort of HIV-1 infected patients showing evidence of developing protease inhibitor (PI) resistance whilst undergoing highly active antiretroviral therapy (HAART) have been determined. The PR sequences showed the presence of the classical mutations associated with resistance to PIs. The sequence of the Vif protein showed less variation in samples from PI treated patients than in specimens prepared from treatment-naive patients. In addition a number of amino acid positions within Vif showed highly significant preferences for a particular amino acid in the PI-treated cohort compared to the untreated control cohort.     

Genetic heterogeneity of the NS3 protease gene in hepatitis C virus genotype 1 from untreated infected patients

NS3 protease is essential for hepatitis C Virus (HCV) replication, and is one of the most promising targets for specific anti-HCV therapy. Its natural polymorphism has not been studied at the quasispecies level. In the present work, the genetic heterogeneity of the NS3 protease gene was analyzed in 17 HCV genotype 1 (5 subtypes 1a and 12 subtypes 1b) samples collected from infected patients before anti-viral therapy. A total of 294 clones were sequenced. Although the protease NS3 is considered to be one of the less variable genes in the HCV genome, variability of both nucleotide and amino acid sequences was found. In variants belonging to 1a and 1b subtypes, 224 and 267 of 543 positions showed one or more nucleotide substitutions, respectively. Forty and 74 of the 181 NS3 amino acid positions showed at least one mutation in HCV-1a and HCV-1b isolates, respectively. Most substitutions were conservative. This substantial polymorphism of the NS3 protease produced by HCV-1a and HCV-1b suggests that, despite the numerous functional and structural constraints, the enzyme is sufficiently flexible to tolerate substitutions.     

Polymorphism and drug-selected mutations in the protease gene of human immunodeficiency virus type 2 from patients living in Southern France

The susceptibility of human immunodeficiency virus type 2 (HIV-2) to protease inhibitors (PI) is largely unknown. We studied HIV-2 protease genes from 21 HIV-2-infected patients who were exposed or not exposed to PI. The aim of this study was (i). to characterize the polymorphism of HIV-2 protease in the absence of drug, (ii). to know whether the HIV-2 protease gene naturally harbors HIV-1 drug resistance codons, and (iii). to identify mutations emerging under PI-selective pressure. Sixty-five HIV-2 RNA or proviral DNA samples were directly sequenced from the plasma or peripheral blood mononuclear cells of 8 patients who had received PI and 13 patients who had never received any antiretroviral. In untreated patients, the highest amino acid variability in HIV-2 protease was observed at positions 14, 40, 43, 46, 65 and 70, and seven codons (10V, 32I, 36I, 46I, 47V, 71V, and 73A) associated with drug resistance in HIV-1 were highly prevalent. In addition, at six positions (positions 7, 46, 62, 71, 90, and 99), the amino acid variability or the amino acid frequencies or both differed significantly in PI-treated and untreated patients, suggesting that mutations 7K-->R, 46V-->I, 62V-->A/T, 71V-->I, 90L-->M and 99L-->F were occurring under PI-selective pressure. At these positions, at least one sample simultaneously harbored both wild-type and mutated codons, while substitutions at positions 62, 71, 90, and 99 were confirmed in a longitudinal analysis. Moreover, the presence of codons 46I and 99F in the absence of drug in HIV-2 subtype B proteases may reflect natural resistance to PI. In conclusion, the present study revealed that HIV-2 strains harbor specific patterns of natural polymorphism and resistance.     

Genetic mechanisms of resistance to protease inhibitors and entry inhibitors

Resistance to protease inhibitors (PIs) often appears as a result of changes at codons within the protease gene, which differ from one drug to another. When PIs are boosted with low doses of ritonavir, multiple mutations at the protease are required to reduce significantly the antiviral activity of these compounds. Moreover, mutations at gag cleavage sites may restore the loss of viral fitness caused by the selection of specific PI resistance mutations. In respect to entry inhibitors, a new class of compounds already in phase II/III clinical trials, resistance may emerge as a result of mutations outside the pol gene. Loss of susceptibility to agents that block virus attachment or the interaction with co-receptors often select HIV strains with mutations in the gp120 envelope-coding region. In contrast, agents that target gp41-dependent fusion select for HIV variants with mutations in the gp41 envelope gene.     

Impact of highly active antiretroviral therapy on hepatitis C virus protease quasispecies diversity in HIV co‐infected patients

Many hepatitis C virus (HCV)‐infected patients are also infected with HIV, and undergo antiretroviral (ARV) treatment for their human immunodeficiency virus (HIV) infection. Due to changes in HIV burden and immunologic status, HIV ARV treatment may have indirect effects on the HCV population, which could impact the effectiveness of subsequent HCV protease inhibitor (PI) treatment. The genetic variability of the protease‐encoding HCV NS3 gene was evaluated in 10 co‐infected patients initiating ARVs (both before and after ARV initiation), and compared to the genetic variability in 10 patients on stable ARV therapy. After RT‐PCR of plasma‐derived HCV RNA, a mean of 20 clones per patient time‐point were sequenced and analyzed for changes in the HCV quasispecies population. No significant differences in sequence diversity or complexity at the nucleic acid or amino acid levels were seen at baseline between groups or between the two time points in either group. HCV protease diversity in the pre‐ and post‐ARV treatment samples was not significantly different than samples from patients on stable ARV therapy. There was no significant development of amino acid substitutions known to confer HCV PI resistance in either group. Initiation of ARV for HIV infection does not significantly alter the genetic diversity or complexity of the HCV NS3 gene or result in increased number of HCV PI‐associated amino acid changes. These results suggest ARV treatment for HIV would not affect the efficacy of HCV PI treatment. J. Med. Virol. 82: 791–798, 2010. © 2010 Wiley‐Liss, Inc.     

Genetic mechanisms of resistance to protease inhibitors and entry inhibitors

Abstract

Resistance to protease inhibitors (PIs) often appears as a result of changes at codons within the protease gene, which differ from one drug to another. When PIs are boosted with low doses of ritonavir, multiple mutations at the protease are required to reduce significantly the antiviral activity of these compounds. Moreover, mutations at gag cleavage sites may restore the loss of viral fitness caused by the selection of specific PI resistance mutations. In respect to entry inhibitors, a new class of compounds already in phase II/III clinical trials, resistance may emerge as a result of mutations outside the pol gene. Loss of susceptibility to agents that block virus attachment or the interaction with co-receptors often select HIV strains with mutations in the gp120 envelope-coding region. In contrast, agents that target gp41-dependent fusion select for HIV variants with mutations in the gp41 envelope gene.     

Catalytic efficiency and phenotype of HIV-1 proteases encoding single critical resistance substitutions

We have shown that a bacteriophage lambda genetic screen system may be useful in predicting the activity and phenotype of HIV-1 protease in the course of viral infection and antiretroviral therapy. This simple and rapid genetic screening system has been used here to characterize HIV-1 proteases encoding single primary resistance substitutions. Except for proteases with amino acid changes at positions 46 and 84, proteases containing single-resistance substitutions displayed a lower catalytic efficiency than the WT enzyme. Single mutants could be identified by their efficiency, demonstrating that modest differences in protease activity can be monitored with this simple assay. Overall, drug susceptibility could be reduced by introduction of single mutations. However, high-level protease inhibitor (PI) resistance was only achieved by multiple mutated proteases. The small but reproducible increase in resistance displayed by single mutants also demonstrated the ability of this genetic screen system for detecting minor reductions in drug susceptibility. These results show that the bacteriophage lambda genetic screen system used here is a useful tool in the analysis of specific contribution of mutations in the HIV protease-coding region or in specific cleavage sites that affect the process of PI resistance.     

The HIV-1 protease resistance mutation I50L is associated with resistance to atazanavir and susceptibility to other protease inhibitors in multiple mutational contexts

BACKGROUND: The HIV-1 protease mutation I50 L causes atazanavir resistance but increases susceptibility to other PIs. Predicted phenotypic FC values were obtained from viral genotypes, using the virtual Phenotype-LM bioinformatics tool (powering vircoTYPE). OBJECTIVE: To evaluate I50 L's effect on susceptibility to 8 PIs, in a large genotype database. STUDY DESIGN: I50 L containing routine clinical isolate samples in Virco's genotype database were paired with samples having like patterns (or profiles) of IAS-USA-defined primary PI mutations, but lacking I50 L. Using vircoTYPE (version 4.1), the median predicted FC for each mutational profile was determined. I50 L-associated shifts in FC were evaluated using drug-specific CCOs. RESULTS: We selected 307 and 37098 samples with and without I50 L. These corresponded to 31 mutation patterns of > or =3 samples each. I50 L caused resistance to atazanavir in all 31 mutation contexts, but was associated with higher susceptibility for other PIs. The largest I50 L-associated shifts in median predicted FC were: 1.2 to 42.4 (atazanavir), 10.2 to 3.2 (amprenavir), 3.3 to 0.5 (darunavir), 13 to 0.5 (indinavir), 34.9 to 1.3 (lopinavir), 22.3 to 1.3 (nelfinavir), 5.2 to 0.3 (saquinavir) and 29.9 to 5.2 (tipranavir). CONCLUSIONS: The PI mutation I50 L causes clinically relevant resistance and increased susceptibility to atazanavir and other PIs respectively.     

Emergence of drug resistance-associated mutations in HIV-1 subtype C protease gene in north India

A major cause of anti-retroviral therapy (ART) failure is the drug resistance-associated mutations in polymerase gene of HIV-1. Paucity of data regarding potential drug resistance to protease inhibitors (PIs) prompted us to carry out this study. Drug resistance (DR) genotyping of the entire protease gene was performed in 104 HIV-1 ART-naive and first-line ART-experienced patients. The DR pattern was analyzed using the Stanford HIV-DR database, International AIDS Society-USA mutation list and REGA algorithm version 8.0. Subtyping was done using Mega 4 and REGA HIV-1 subtyping tool-v2.01. Majority of our sequences 98 (96 %) were subtype C and remaining four (3.92 %) were subtype A1. In three (2.9 %) DE patients, major DR-associated mutation at D30 N and M46I positions were detected. Approximately 70 % polymorphisms as minor mutations were observed in protease gene, of which 14 distinct amino acids changes were linked to partial DR such as G16E, K20R, M36I, D60E, I62V, L63P, I64M, H69K, T74A/S, V77I, V82I, I85V, L89M, and I93L. The two major and several minor mutations detected in this study confer low/intermediate levels of resistance to most PIs independently or together. Our results conclude that resistance testing in HIV-1-infected patients should be performed before the initiation of PI therapy for better therapeutic outcome in this region. This information not only will shed light on the extent of current DR in HIV strains but also will aid in patient treatment and drug designing.     

Human immunodeficiency virus type 1 group m protease in cameroon: genetic diversity and protease inhibitor mutational features

To establish a baseline for monitoring resistance to protease inhibitors (PIs) and examining the efficacy of their use among persons in Cameroon infected with human immunodeficiency virus type 1 (HIV-1), we analyzed genetic variability and PI resistance-associated substitutions in PCR-amplified protease (PR) sequences in strains isolated from 110 HIV-1-infected, drug-na?ve Cameroonians. Of the 110 strains, 85 were classified into six HIV-1 PR subtypes, A (n = 1), B (n = 1), F (n = 4), G (n = 7), H (n = 1), and J (n = 7), and a circulating recombinant form, CRF02-AG (n = 64). PR genes from the remaining 25 (23%) specimens were unclassifiable, whereas 2% (7 of 301) unclassifiable PR sequences were reported for a global collection. Two major PI resistance-associated mutations, 20M and 24I, were detected in strains from only two specimens, whereas secondary mutations were found in strains from all samples except one strain of subtype B and two strains of CRF02-AG. The secondary mutations showed the typical PI resistance-associated pattern for non-subtype B viruses in both classifiable and unclassifiable PR genes, with 36I being the predominant (99%) mutation, followed by 63P (18%), 20R (15%), 77I (13%), and 10I or 10V (11%). Of these mutations, dual and triple PI resistance-associated substitutions were found in 38% of all the Cameroonian strains. Compared with classifiable PR sequences, unclassifiable sequences had significantly more dual and triple substitutions (64% versus 30%; P = 0.004). Phenotypic and clinical evaluations are needed to estimate whether PI resistance during antiretroviral drug treatment occurs more rapidly in individuals infected with HIV-1 strains harboring multiple PI resistance-associated substitutions. This information may be important for determination of appropriate drug therapies for HIV-1-infected persons in Cameroon, where more than one-third of HIV-1 strains were found to carry dual and triple minor PI resistance-associated mutations.     

Natural polymorphisms in the protease gene modulate the replicative capacity of non-B HIV-1 variants in the absence of drug pressure.

BACKGROUND: Genetic variation in the HIV-1 pol gene, which encodes the main targets for anti-HIV drugs, may favors different susceptibility and resistance pathways to antiretroviral agents. Several amino acid substitutions occur frequently in some non-B viruses at positions associated with drug resistance in clade B viruses. The clinical relevance of those polymorphisms is unclear. OBJECTIVE: To evaluate the effect of two natural protease (PR) polymorphisms, K20I and M36I, which are frequently found in non-B subtypes, on the virus replicative capacity in the presence and absence of protease inhibitors (PI). STUDY DESIGN: Infectious HIV-1 clones carrying K20I, M36I or K20I/M36I were designed. Their replication kinetics were analyzed by viral competition in the absence of PI. Susceptibility to six different PI was phenotypically assessed in clones and in recombinant viruses carrying non-B proteases from 16 drug-naive individuals. RESULTS: In the absence of drug, the M36I clone replicated more rapidly than wt (wild type) or the double mutant K20I/M36I. Natural polymorphisms 20I and/or 36I improved the virus replicative capacity under drug pressure, reducing the susceptibility to saquinavir and indinavir, with IC(50) values 2-3.5-fold higher than wt. All but one drug-naive individual carrying non-B viruses were fully susceptibility to all tested PI, suggesting that additional substitutions within the PR might compensate the reduced PI susceptibility caused by K20I and/or M36I. CONCLUSION: Natural PR polymorphisms in non-B HIV-1 variants can influence in vitro the virus replication capacity in the presence and/or absence or certain PI. Hypothetically, the improved viral replication of mutant 36I might favor a more rapid spreading of non-B subtypes of HIV-1.     

Hyperlipidemia and insulin resistance are induced by protease inhibitors independent of changes in body composition in patients with HIV infection

Although protease inhibitor (PI) therapy has improved the clinical status of patients with HIV infection, concerns have arisen that such treatment may have deleterious effects on glucose control, lipid metabolism, and body fat distribution. To determine whether initiation of PI therapy uniquely affects glucose and lipid metabolism, we analyzed paired data in HIV-infected patients before and after beginning antiretroviral therapy that included a PI (PI; N = 20) or lamivudine (3TC) but no PI (3TC; N = 9); and a control group on stable regimens that included neither of these agents (CONT: N = 12). Measurements included fasting glucose; insulin; triglycerides; total, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol; HIV RNA; CD4+ lymphocytes; weight; and total and regional body composition. Neither weight nor total or regional fat content changed significantly in any group during the period of observation. Nonetheless, in patients beginning PI therapy, there were significant increases in glucose (+9+/-3 mg/dl; p = .0136), insulin (+12.2+/-4.9 U/ml; p = .023), triglycerides (+53+/-17 mg/dl; p = .0069), and total and LDL cholesterol (+32+/-11 and +18+/-5 mg/dl; p = .0082 and .0026, respectively). None of these parameters changed significantly in the 3TC or CONT groups. The PI and 3TC groups experienced comparable increases in CD4+ lymphocytes, suggesting that the observed metabolic effects may be associated with PIs uniquely, rather than improvement in clinical status. However, it is also possible that the metabolic effects of PIs are associated with more effective viral suppression, because a greater proportion of patients in the PI group achieved undetectable levels of virus. We conclude that changes in glucose and lipid metabolism are induced by PI therapy in the absence of significant changes in weight or fat distribution. Longer periods of follow-up will be required to determine the clinical significance of these changes. However, at the moment, the risks associated with these metabolic effects do not appear to outweigh improvements in survival seen with PI therapy.     

Mutagenesis of amino acids at two tomato ringspot nepovirus cleavage sites: effect on proteolytic processing in cis and in trans by the 3C-like protease.

Tomato ringspot nepovirus (ToRSV) encodes two polyproteins that are processed by a 3C-like protease at specific cleavage sites. Analysis of ToRSV cleavage sites identified previously and in this study revealed that cleavage occurs at conserved Q/(G or S) dipeptides. In addition, a Cys or Val is found in the -2 position. Amino acid substitutions were introduced in the -6 to +1 positions of two ToRSV cleavage sites: the cleavage site between the protease and putative RNA-dependent RNA polymerase, which is processed in cis, and the cleavage site at the N-terminus of the movement protein, which is cleaved in trans. The effect of the mutations on proteolytic processing at these sites was tested using in vitro translation systems. Substitution of conserved amino acids at the -2, -1, and +1 positions resulted in a significant reduction in proteolytic processing at both cleavage sites. The effects of individual substitutions were stronger on the cleavage site processed in trans than on the one processed in cis. The cleavage site specificity of the ToRSV protease is discussed in comparison to that of related proteases.     

Direct comparison of the acute in vivo effects of HIV protease inhibitors on peripheral glucose disposal

The clinical use of HIV protease inhibitors (PIs) is associated with the development of peripheral insulin resistance. The incidence and degree of impaired glucose tolerance observed in treated patients vary considerably between drugs, however. To compare the ability of HIV PIs to alter peripheral glucose disposal acutely in a genetically identical model system at therapeutically relevant drug levels, healthy lean male rats previously naive to PI exposure were given ritonavir, amprenavir, lopinavir/ritonavir (4:1), or atazanavir by continuous intravenous infusion to achieve steady state drug levels of 10 or 25 muM rapidly. Under euglycemic hyperinsulinemic clamp conditions, a dose-dependent reduction in the peripheral glucose disposal rate (Rd) was observed with all the PIs except atazanavir. The rank order of sensitivity was ritonavir, lopinavir, and then amprenavir. Changes in skeletal muscle and heart 2-deoxyglucose (2-DOG) uptake correlated with reductions in Rd. All 3 of these PIs also produced significant reductions in 2-DOG uptake into primary rat adipocytes in vitro. Atazanavir had no effect on glucose uptake in vitro or in vivo. The in vivo potency of PIs to impair peripheral glucose disposal acutely correlates with the degree of insulin resistance observed in HIV-infected patients receiving these drugs. Preclinical testing of novel candidate PIs in a rodent model system may be useful in identifying the future risk of altering glucose homeostasis.