Construction of a human immunodeficiency virus type 1 (HIV-1) library containing random combinations of amino acid substitutions in the HIV-1 protease due to resistance by protease inhibitors

Human immunodeficiency virus type 1 (HIV-1) heterogeneity contributes to the emergence of drug-resistant virus, escape from host defense systems, and/or conversion of the cellular tropism. To establish an in vitro system to address a heterogeneous virus population, we constructed a library of HIV-1 molecular clones containing a set of random combinations of zero to 11 amino acid substitutions associated with resistance to protease inhibitors by the HIV-1 protease. The complexity (2.1 x 10(5)) of the HIV-1 library pNG-PRL was large enough to cover all of the possible combinations of zero to 11 amino acid substitutions (a total of 4,096 substitutions possible). The T-cell line MT-2 was infected with the HIV-1 library, and resistant viruses were selected after treatment by the protease inhibitor ritonavir (0.03 to 0.30 microM). The viruses that contained three to eight amino acid substitutions could be selected within 2 weeks. These results demonstrate that this HIV-1 library could serve as an alternative in vitro system to analyze the emergence of drug resistance and to evaluate the antiviral activity of novel compounds against multidrug-resistant viruses.     

Identification of Biased Amino Acid Substitution Patterns in Human Immunodeficiency Virus Type 1 Isolates from Patients Treated with Protease Inhibitors

Human immunodeficiency virus type 1 (HIV-1) amino acid substitutions observed during antiretroviral drug therapy may be caused by drug selection, non-drug-related evolution, or sampling error introduced by the sequencing process. We analyzed HIV-1 sequences from 371 untreated patients and from 178 patients receiving a single protease inhibitor. Amino acid substitution patterns during treatment were compared with inferred substitution patterns arising evolutionarily without treatment. Our results suggest that most treatment-associated amino acid substitutions are caused by selective drug pressure, including substitutions not previously associated with drug resistance.     

Comparative analysis of the sequences and structures of HIV-1 and HIV-2 proteases

The different isolates available for HIV-1 and HIV-2 were compared for the region of the protease (PR) sequence, and the variations in amino acids were analyzed with respect to the crystal structure of HIV-1 PR with inhibitor. Based on the extensive homology (39 identical out of 99 residues), models were built of the HIV-2 PR complexed with two different aspartic protease inhibitors, acetylpepstatin and a renin inhibitor, H-261. Comparison of the HIV-1 PR crystal structure and the HIV-2 PR model structure and the analysis of the changes found in different isolates showed that correlated substitutions occur in the hydrophobic interior of the molecule and at surface residues involved in ionic or hydrogen bond interactions. The substrate binding residues of HIV-1 and HIV-2 PRs show conservative substitutions of four residues. The difference in affinity of HIV-1 and HIV-2 PRs for the two inhibitors appears to be due in part to the change of Val 32 in HIV-1 PR to Ile in HIV-2 PR.     

Amino acid substitutions in Gag protein at non-cleavage sites are indispensable for the development of a high multitude of HIV-1 resistance against protease inhibitors.

Amino acid substitutions in human immunodeficiency virus type 1 (HIV-1) Gag cleavage sites have been identified in HIV-1 isolated from patients with AIDS failing chemotherapy containing protease inhibitors (PIs). However, a number of highly PI-resistant HIV-1 variants lack cleavage site amino acid substitutions. In this study we identified multiple novel amino acid substitutions including L75R, H219Q, V390D/V390A, R409K, and E468K in the Gag protein at non-cleavage sites in common among HIV-1 variants selected against the following four PIs: amprenavir, JE-2147, KNI-272, and UIC-94003. Analyses of replication profiles of various mutant clones including competitive HIV-1 replication assays demonstrated that these mutations were indispensable for HIV-1 replication in the presence of PIs. When some of these mutations were reverted to wild type amino acids, such HIV-1 clones failed to replicate. However, virtually the same Gag cleavage pattern was seen, indicating that the mutations affected Gag protein functions but not their cleavage sensitivity to protease. These data strongly suggest that non-cleavage site amino acid substitutions in the Gag protein recover the reduced replicative fitness of HIV-1 caused by mutations in the viral protease and may open a new avenue for designing PIs that resist the emergence of PI-resistant HIV-1.     

Molecular and functional analysis of a conserved CTL epitope in HIV-1 p24 recognized from a long-term nonprogressor: constraints on immune escape associated with targeting a sequence essential for viral replication

It has been hypothesized that sequence variation within CTL epitopes leading to immune escape plays a role in the progression of HIV-1 infection. Only very limited data exist that address the influence of biologic characteristics of CTL epitopes on the emergence of immune escape variants and the efficiency of suppression HIV-1 by CTL. In this report, we studied the effects of HIV-1 CTL epitope sequence variation on HIV-1 replication. The highly conserved HLA-B14-restricted CTL epitope DRFYKTLRAE in HIV-1 p24 was examined, which had been defined as the immunodominant CTL epitope in a long-term nonprogressing individual. We generated a set of viral mutants on an HX10 background differing by a single conservative or nonconservative amino acid substitution at each of the P1 to P9 amino acid residues of the epitope. All of the nonconservative amino acid substitutions abolished viral infectivity and only 5 of 10 conservative changes yielded replication-competent virus. Recognition of these epitope sequence variants by CTL was tested using synthetic peptides. All mutations that abrogated CTL recognition strongly impaired viral replication, and all replication-competent viral variants were recognized by CTL, although some variants with a lower efficiency. Our data indicate that this CTL epitope is located within a viral sequence essential for viral replication. Targeting CTL epitopes within functionally important regions of the HIV-1 genome could limit the chance of immune evasion.     

Altered HIV-1 Gag protein interactions with cyclophilin A (CypA) on the acquisition of H219Q and H219P substitutions in the CypA binding loop

HIV-1 Gag protein interaction with cyclophilin A (CypA) is critical for viral fitness. Among the amino acid substitutions identified in Gag noncleavage sites in HIV-1 variants resistant to protease inhibitors, H219Q (Gatanaga, H., Suzuki, Y., Tsang, H., Yoshimura, K., Kavlick, M. F., Nagashima, K., Gorelick, R. J., Mardy, S., Tang, C., Summers, M. F., and Mitsuya, H. (2002) J. Biol. Chem. 277, 5952-5961) and H219P substitutions in the viral CypA binding loop confer the greatest replication advantage to HIV-1. These substitutions represent polymorphic amino acid residues. We found that the replication advantage conferred by these substitutions was far greater in CypA-rich MT-2 and H9 cells than in Jurkat cells and peripheral blood mononuclear cells (PBM), both of which contained less CypA. High intracellular CypA content in H9 and MT-2 cells, resulting in excessive CypA levels in virions, limited wild-type HIV-1 (HIV-1(WT)) replication and H219Q introduction into HIV-1 (HIV-1(H219Q)), reduced CypA incorporation of HIV-1, and potentiated viral replication. H219Q introduction also restored the otherwise compromised replication of HIV-1(P222A) in PBM, although the CypA content in HIV-1(H219Q/P222A) was comparable with that in HIV-1(P222A), suggesting that H219Q affected the conformation of the CypA-binding motif, rendering HIV-1 replicative in a low CypA environment. Structural modeling analyses revealed that although hydrogen bonds are lost with H219Q and H219P substitutions, no significant distortion of the CypA binding loop of Gag occurred. The loop conformation of HIV-1(P222A) was found highly distorted, although H219Q introduction to HIV-1 restored the conformation of the loop close to that of HIV-1 (P222A). The present data suggested that the effect of CypA on HIV-1 replicative (WT) ability is bimodal (both high and low CypA content limits HIV-1 replication), that the conformation of the CypA binding region of Gag is important for viral fitness, and that the function of CypA is to maintain the conformation.     

Mutation patterns and structural correlates in human immunodeficiency virus type 1 protease following different protease inhibitor treatments

Although many human immunodeficiency virus type 1 (HIV-1)-infected persons are treated with multiple protease inhibitors in combination or in succession, mutation patterns of protease isolates from these persons have not been characterized. We collected and analyzed 2,244 subtype B HIV-1 isolates from 1,919 persons with different protease inhibitor experiences: 1,004 isolates from untreated persons, 637 isolates from persons who received one protease inhibitor, and 603 isolates from persons receiving two or more protease inhibitors. The median number of protease mutations per isolate increased from 4 in untreated persons to 12 in persons who had received four or more protease inhibitors. Mutations at 45 of the 99 amino acid positions in the protease-including 22 not previously associated with drug resistance-were significantly associated with protease inhibitor treatment. Mutations at 17 of the remaining 99 positions were polymorphic but not associated with drug treatment. Pairs and clusters of correlated (covarying) mutations were significantly more likely to occur in treated than in untreated persons: 115 versus 23 pairs and 30 versus 2 clusters, respectively. Of the 115 statistically significant pairs of covarying residues in the treated isolates, 59 were within 8 A of each other-many more than would be expected by chance. In summary, nearly one-half of HIV-1 protease positions are under selective drug pressure, including many residues not previously associated with drug resistance. Structural factors appear to be responsible for the high frequency of covariation among many of the protease residues. The presence of mutational clusters provides insight into the complex mutational patterns required for HIV-1 protease inhibitor resistance.     

Amino acid insertions near Gag cleavage sites restore the otherwise compromised replication of human immunodeficiency virus type 1 variants resistant to protease inhibitors

A variety of amino acid substitutions in the protease and Gag proteins have been reported to contribute to the development of human immunodeficiency virus type 1 (HIV-1) resistance to protease inhibitors. In the present study, full-length molecular infectious HIV-1 clones were generated by using HIV-1 variants isolated from heavily drug-experienced and therapy-failed AIDS patients. Of six full-length infectious clones generated, four were found to have unique insertions (TGNS, SQVN, AQQA, SRPE, APP, and/or PTAPPA) near the p17/p24 and p1/p6 Gag cleavage sites, in addition to the known resistance-related multiple amino acid substitutions within the protease. The addition of such Gag inserts mostly compromised the replication of wild-type HIV-1, whereas the primary multidrug-resistant HIV infectious clones containing inserts replicated significantly better than those modified to lack the inserts. Western blot analyses revealed that the processing of Gag proteins by wild-type protease was impaired by the presence of the inserts, whereas that by mutant protease was substantially improved. The present study represents the first report clearly demonstrating that the inserts seen in the proximity of the Gag cleavage sites in highly multi-PI resistant HIV-1 variants restore the otherwise compromised enzymatic activity of mutant protease, enabling the multi-PI-resistant HIV-1 variants to remain replication competent.     

A Symmetric Region of the HIV-1 Integrase Dimerization Interface Is Essential for Viral Replication

HIV-1 integrase (IN) is an important target for contemporary antiretroviral drug design research. Historically, efforts at inactivating the enzyme have focused upon blocking its active site. However, it has become apparent that new classes of allosteric inhibitors will be necessary to advance the antiretroviral field in light of the emergence of viral strains resistant to contemporary clinically used IN drugs. In this study we have characterized the importance of a close network of IN residues, distant from the active site, as important for the obligatory multimerization of the enzyme and viral replication as a whole. Specifically, we have determined that the configuration of six residues within a highly symmetrical region at the IN dimerization interface, composed of a four-tiered aromatic interaction flanked by two salt bridges, significantly contributes to proper HIV-1 replication. Additionally, we have utilized a quantitative luminescence assay to examine IN oligomerization and have determined that there is a very low tolerance for amino acid substitutions along this region. Even conservative residue substitutions negatively impacted IN multimerization, resulting in an inactive viral enzyme and a non-replicative virus. We have shown that there is a very low tolerance for amino acid variation at the symmetrical dimeric interface region characterized in this study, and therefore drugs designed to target the amino acid network detailed here could be expected to yield a significantly reduced number of drug-resistant escape mutations compared to contemporary clinically-evaluated antiretrovirals.     

Sequence homology required by human immunodeficiency virus type 1 to escape from short interfering RNAs

Short interfering RNAs (siRNAs) targeting viral or cellular genes can efficiently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Nevertheless, the emergence of mutations in the gene being targeted could lead to the rapid escape from the siRNA. Here, we simulate viral escape by systematically introducing single-nucleotide substitutions in all 19 HIV-1 residues targeted by an effective siRNA. We found that all mutant viruses that were tested replicated better in the presence of the siRNA than in the presence of the wild-type virus. The antiviral activity of the siRNA was completely abolished by single substitutions in 10 (positions 4 to 11, 14, and 15) out of 16 positions tested (substitution at 3 of the 19 positions explored rendered nonviable viruses). With the exception of the substitution observed at position 12, substitutions at either the 5' end or the 3' end (positions 1 to 3, 16, and 18) were better tolerated by the RNA interference machinery and only in part affected siRNA inhibition. Our results show that optimal HIV-1 gene silencing by siRNA requires a complete homology within most of the target sequence and that substitutions at only a few positions at the 5' and 3' ends are partially tolerated.     

In vivo sequence diversity of the protease of human immunodeficiency virus type 1: presence of protease inhibitor-resistant variants in untreated subjects.

We have evaluated the sequence diversity of the protease human immunodeficiency virus type 1 in vivo. Our analysis of 246 protease coding domain sequences obtained from 12 subjects indicates that amino acid substitutions predicted to give rise to protease inhibitor resistance may be present in patients who have not received protease inhibitors. In addition, we demonstrated that amino acid residues directly involved in enzyme-substrate interactions may be varied in infected individuals. Several of these substitutions occurred in combination either more or less frequently than would be expected if their appearance was independent, suggesting that one substitution may compensate for the effects of another. Taken together, our analysis indicates that the human immunodeficiency virus type 1 protease has flexibility sufficient to vary critical subsites in vivo, thereby retaining enzyme function and viral pathogenicity.     

Role of minority populations of human immunodeficiency virus type 1 in the evolution of viral resistance to protease inhibitors

Human immunodeficiency virus type 1 (HIV-1) drug resistance results from the accumulation of mutations in the viral genes targeted by the drugs. These genetic changes, however, are commonly detected and monitored by techniques that only take into account the dominant population of plasma virus. Because HIV-1-infected patients harbor a complex and diverse mixture of virus populations, the mechanisms underlying the emergence and the evolution of resistance are not fully elucidated. Using techniques that allow the quantification of resistance mutations in minority virus species, we have monitored the evolution of resistance in plasma virus populations from patients failing protease inhibitor treatment. Minority populations with distinct resistance genotypes were detected in all patients throughout the evolution of resistance. The emergence of new dominant genotypes followed two possible mechanisms: (i) emergence of a new mutation in a currently dominant genotype and (ii) emergence of a new genotype derived from a minority virus species. In most cases, these population changes were associated with an increase in resistance at the expense of a reduction in replication capacity. Our findings provide a preliminary indication that minority viral species, which evolve independently of the majority virus population, can eventually become dominant populations, thereby serving as a reservoir of diversity and possibly accelerating the development of drug resistance.     

The impact of the nelfinavir resistance-conferring mutation D30N on the susceptibility of HIV-1 subtype B to other protease inhibitors

The human immunodeficiency virus type 1 (HIV-1) protease mutation D30N is exclusively selected by the protease inhibitor (PI) nelfinavir and confers resistance to this drug. We demonstrate that D30N increases the susceptibility to saquinavir (SQV) and amprenavir in HIV-1 subtype B isolates and that the N88D mutation in a D30N background neutralizes this effect. D30N also suppresses indinavir (IDV) resistance caused by the M46I mutation. Interestingly, in patients with viruses originally containing the D30N mutation who were treated with IDV or SQV, the virus either reversed this mutation or acquired N88D, suggesting an antagonistic effect of D30N upon exposure to these PIs. These findings can improve direct salvage drug treatment in resource limited countries where subtype B is epidemiologically important and extend the value of first and second line PIs in these populations.     

Efficiency of a second-generation HIV-1 protease inhibitor studied by molecular dynamics and absolute binding free energy calculations

A subnanomolar inhibitor of human immunodeficiency virus type 1 (HIV-1) protease, designated QF34, potently inhibits the wild-type and drug-resistant enzyme. To explain its broad activity, the binding of QF34 to the wild-type HIV-1 protease is investigated by molecular dynamics simulations and compared to the binding of two inhibitors that are used clinically, saquinavir (SQV) and indinavir (IDV). Analysis of the flexibility of protease residues and inhibitor segments in the complex reveals that segments of QF34 were more mobile during the dynamics studies than the segments of SQV and IDV. The dynamics of hydrogen bonding show that QF34 forms a larger number of stable hydrogen bonds than the two inhibitors that are used clinically. Absolute binding free energies were calculated with molecular mechanics-generalized Born surface area (MM-GBSA) methodology using three protocols. The most consistent results were obtained using the single-trajectory approach, due to cancellation of errors and inadequate sampling in the separate-trajectory protocols. For all three inhibitors, energy components in favor of binding include van der Waals and electrostatic terms, whereas polar solvation and entropy terms oppose binding. Decomposition of binding energies reveals that more protease residues contribute significantly to the binding of QF34 than to the binding of SQV and IDV. Moreover, contributions from protease main chains and side chains are balanced in the case of QF34 (52:48 ratio, respectively), whereas side chain contributions prevail in both SQV and IDV (main-chain:side-chain ratios of 41:59 and 45:55, respectively). The presented results help explain the ability of QF34 to inhibit multiple resistant mutants and should be considered in the design of broad-specificity second-generation HIV-1 protease inhibitors.     

Population Genetic Analysis of the Protease Locus of Human Immunodeficiency Virus Type 1 Quasispecies Undergoing Drug Selection, Using a Denaturing Gradient-Heteroduplex Tracking Assay

Monitoring the evolution of human immunodeficiency virus type 1 (HIV-1) drug resistance requires measuring the frequency of closely related genetic variants making up the complex viral quasispecies found in vivo. In order to resolve both major and minor (>/=2%) protease gene variants differing by one or more nucleotide substitutions, we analyzed PCR products derived from plasma viral quasispecies by using a combination of denaturing gradient gel electrophoresis and DNA heteroduplex tracking assays. Correct population sampling of the high level of genetic diversity present within viral quasispecies could be documented by parallel analysis of duplicate, independently generated PCR products. The composition of genetically complex protease gene quasispecies remained constant over short periods of time in the absence of treatment and while plasma viremia fell >100-fold following the initiation of protease inhibitor ritonavir monotherapy. Within a month of initiating therapy, a strong reduction in the genetic diversity of plasma viral populations at the selected protease locus was associated with rising plasma viremia and the emergence of drug resistance. The high levels of protease genetic diversity seen before treatment reemerged only months later. In one patient, reduction in genetic diversity at the protease gene was observed concomitantly with an increase in diversity at the envelope gene (E. L. Delwart, P. Heng, A. Neumann, and M. Markowitz, J. Virol. 72:2416-2421, 1998), indicating that opposite population genetic changes can take place in different HIV-1 loci. The rapid emergence of drug-resistant HIV-1 was therefore associated with a strong, although only transient, reduction in genetic diversity at the selected locus. The denaturing gradient-heteroduplex tracking assay is a simple method for the separation and quantitation of very closely related, low-frequency, genetic variants within complex viral populations.