Mutation In Protease Research Articles

Detection of A Shared HIV Protease-RT Deletion in Patient Plasma & Cells: A Role For ARV-mediated Selection and Viral Complementation

Background A deletion in HIV pol spanning amino acid residues 59– 99 of protease and 1–204 of reverse transcriptase (RT) was detected in 12/22 plasma specimens from patients receiving antiretovirals (ARVs). It was subsequently detected in cerebrospinal fluid, blood monocytes and T-cells, brain and lymphoid tissues. It was always accompanied by an I54V mutation in protease, and L210W, R211K and L214F mutations in RT. Sequencing and other experiments confirmed that it is not a PCR artifact.

The 2 microm plasmid causes cell death in Saccharomyces cerevisiae with a mutation in Ulp1 protease.

The 2 microm circle plasmid confers no phenotype in wild-type Saccharomyces cerevisiae but in a nib1 mutant, an elevated plasmid copy number is associated with cell death. Complementation was used to identify nib1 as a mutant allele of the ULP1 gene that encodes a protease required for removal of a ubiquitin-like protein, Smt3/SUMO, from protein substrates. The nib1 mutation replaces conserved tryptophan 490 with leucine in the protease domain of Ulp1. Complete deletion of ULP1 is lethal, even in a strain that lacks the 2 microm circle. Partial deletion of ULP1, like the nib1 mutation, results in clonal variations in plasmid copy number. In addition, a subset of these mutant cells produces lineages in which all cells have reduced proliferative capacity, and this phenotype is dependent upon the presence of the 2 microm circle. Segregation of the 2 microm circle requires two plasmid-encoded proteins, Rep1 and Rep2, which were found to colocalize with Ulp1 protein in the nucleus and interact with Smt3 in a two-hybrid assay. These associations and the observation of missegregation of a fluorescently tagged 2 microm circle reporter plasmid in a subset of ulp1 mutant cells suggest that Smt3 modification plays a role in both plasmid copy number control and segregation.

Altered Replicative Capacity of Recombinant HIV Type 1 Containing Mutations at Codons 26 and 29 of the Viral Protease

HIV-1 protease activity is essential for viral replication. In spite of the high rates of HIV mutation, the active site of protease (residues 24–29) is a conserved site, where mutations have not been previously described. To determine the effect of mutations at positions T26 and A29 of the viral protease and its viability in recombinant HIV-1 strains, Sup-t1 cells were transfected by electroporation with PCR products from a protease containing the 26X or 29X mutation. These mutations were constructed by mutagenesis site directed with degenerative primers. Both changes modified the replicative capacity of the virus: viruses containing the 26X mutation have delayed replication as compared to the control virus HTLVIII B; the presence of the 29X mutation in the viral protease results in the absence of HIV-1 replication. These findings confirm that this region of the viral protease appears to be necessary for the viability of HIV-1, and it could be a good target for antiviral therapy.

Altered Replicative Capacity of Recombinant HIV Type 1 Containing Mutations at Codons 26 and 29 of the Viral Protease

HIV-1 protease activity is essential for viral replication. In spite of the high rates of HIV mutation, the active site of protease (residues 24-29) is a conserved site, where mutations have not been previously described. To determine the effect of mutations at positions T26 and A29 of the viral protease and its viability in recombinant HIV-1 strains, Sup-t1 cells were transfected by electroporation with PCR products from a protease containing the 26X or 29X mutation. These mutations were constructed by mutagenesis site directed with degenerative primers. Both changes modified the replicative capacity of the virus: viruses containing the 26X mutation have delayed replication as compared to the control virus HTLVIII B; the presence of the 29X mutation in the viral protease results in the absence of HIV-1 replication. These findings confirm that this region of the viral protease appears to be necessary for the viability of HIV-1, and it could be a good target for antiviral therapy.

Structural basis for coevolution of a human immunodeficiency virus type 1 nucleocapsid-p1 cleavage site with a V82A drug-resistant mutation in viral protease.

Maturation of human immunodeficiency virus (HIV) depends on the processing of Gag and Pol polyproteins by the viral protease, making this enzyme a prime target for anti-HIV therapy. Among the protease substrates, the nucleocapsid-p1 (NC-p1) sequence is the least homologous, and its cleavage is the rate-determining step in viral maturation. In the other substrates of HIV-1 protease, P1 is usually either a hydrophobic or an aromatic residue, and P2 is usually a branched residue. NC-p1, however, contains Asn at P1 and Ala at P2. In response to the V82A drug-resistant protease mutation, the P2 alanine of NC-p1 mutates to valine (AP2V). To provide a structural rationale for HIV-1 protease binding to the NC-p1 cleavage site, we solved the crystal structures of inactive (D25N) WT and V82A HIV-1 proteases in complex with their respective WT and AP2V mutant NC-p1 substrates. Overall, the WT NC-p1 peptide binds HIV-1 protease less optimally than the AP2V mutant, as indicated by the presence of fewer hydrogen bonds and fewer van der Waals contacts. AlaP2 does not fill the P2 pocket completely; PheP1' makes van der Waals interactions with Val82 that are lost with the V82A protease mutation. This loss is compensated by the AP2V mutation, which reorients the peptide to a conformation more similar to that observed in other substrate-protease complexes. Thus, the mutant substrate not only binds the mutant protease more optimally but also reveals the interdependency between the P1' and P2 substrate sites. This structural interdependency results from coevolution of the substrate with the viral protease.

Atazanavir: new option for treatment of HIV infection.

Atazanavir is a recently approved human immunodeficiency virus (HIV) protease inhibitor that has an important role in the treatment of both antiretroviral-naive and antiretroviral-experienced individuals. Atazanavir (400 mg) can be administered once per day and requires only 2 capsules. Drug exposure can be safely increased with coadministration of a once-daily regimen of atazanavir (300 mg) and ritonavir (100 mg). Atazanavir is not associated with elevations in serum levels of total cholesterol, low-density lipoprotein cholesterol, or triglycerides, potentially reducing the need for lipid-lowering agents. Atazanavir is associated with elevations in unconjugated bilirubin levels, which are usually not dose limiting. For treatment-naive patients receiving atazanavir who experience virologic rebound, the I50L mutation in HIV protease arises, which does not confer cross-resistance to other protease inhibitors. In treatment-experienced patients with high-level resistance to other protease inhibitors, susceptibility to atazanavir is usually reduced, and optimal effects of atazanavir are seen when it is administered with ritonavir. Similar to other protease inhibitors, careful attention must be paid to drug interactions when administering atazanavir with concomitant medications.

Positive selection detection in 40,000 human immunodeficiency virus (HIV) type 1 sequences automatically identifies drug resistance and positive fitness mutations in HIV protease and reverse transcriptase.

Drug resistance is a major problem in the treatment of AIDS, due to the very high mutation rate of human immunodeficiency virus (HIV) and subsequent rapid development of resistance to new drugs. Identification of mutations associated with drug resistance is critical for both individualized treatment selection and new drug design. We have performed an automated mutation analysis of HIV Type 1 (HIV-1) protease and reverse transcriptase (RT) from approximately 40,000 AIDS patient plasma samples sequenced by Specialty Laboratories Inc. from 1999 to mid-2002. This data set provides a nearly complete mutagenesis of HIV protease and enables the calculation of statistically significant K(a)/K(s) values for each individual amino acid mutation in protease and RT. Positive selection (i.e., a K(a)/K(s) ratio of >1, indicating increased reproductive fitness) detected 19 of 23 known drug-resistant mutation positions in protease and 20 of 34 such positions in RT. We also discovered 163 new amino acid mutations in HIV protease and RT that are strong candidates for drug resistance or fitness. Our results match available independent data on protease mutations associated with specific drug treatments and mutations with positive reproductive fitness, with high statistical significance (the P values for the observed matches to occur by random chance are 10(-5.2) and 10(-16.6), respectively). Our mutation analysis provides a valuable resource for AIDS research and will be available to academic researchers upon publication at http://www.bioinformatics.ucla.edu/HIV. Our data indicate that positive selection mapping is an analysis that can yield powerful insights from high-throughput sequencing of rapidly mutating pathogens.

Atazanavir: improving the HIV protease inhibitor class

Protease inhibitors are potent agents against HIV but their use is constrained by poor pharmacokinetics, cross-resistance and metabolic toxicities. Atazanavir [Reyataz™] is a new protease inhibitors with once-daily dosing and minimal lipid and glycemic effects. Resistance studies of clinical isolates reveal a mutational pattern distinctive from that of other protease inhibitors. Atazanavir selects for the I50L mutation in HIV protease that confers increased susceptibility to other protease inhibitors in vitro. Clinical trials have shown comparable efficacy to nelfinavir (Viracept®) and efavirenz (Sustiva®) in treatment-naive patients, and in preliminary studies, ritonavir-boosted atazanavir is effective in patients failing previous protease inhibitor-containing regimens. Reversible elevations in bilirubin occur in some patients but are not associated with hepatic injury. Atazanavir improves upon aspects of currently-available protease inhibitors and appears useful for initial and possibly subsequent HIV therapy.

Mutation in a calpain-like protease affects the posttranslational mannosylation of phosphatases in Aspergillus nidulans

In this communication, we show that the palB7 mutation drastically reduced the mannose and N-acetylgalactosamine content of the pacA-encoded acid phosphatase secreted by the fungus Aspergillus nidulans at pH 5.0, compared to a control strain. By using mRNA differential display reverse transcription and polymerase chain reaction, we isolated two cDNAs from the control pabaA1 strain that were not detected in the palB7 mutant strain that encode a mannosyl transferase and a NADH-ubiquinone oxidoreductase. Thus, a defect in the posttranslational mannosylation of proteins could be the consequence of mutations in the palB gene, which encodes for a nuclear calpain-like protease that may have specific functions in the processing of transcription factor(s) similar to its homolog, RIM13, in Saccharomyces cereviseae.

Nelfinavir-resistant, amprenavir-hypersusceptible strains of human immunodeficiency virus type 1 carrying an N88S mutation in protease have reduced infectivity, reduced replication capacity, and reduced fitness and process the Gag polyprotein precursor aberrantly.

The evolution of human immunodeficiency virus type 1 (HIV-1) strains with reduced susceptibility to protease inhibitors (PIs) is a major cause of PI treatment failure. A subset of subjects failing a therapy regimen containing the PI nelfinavir developed mutations at position 88 in the protease region. The N88S mutation occurring in some of these subjects induces amprenavir hypersusceptibility and a reduction of fitness and replication capacity. Here we demonstrate that substitutions L63P and V77I in protease, in combination, partially compensate for the loss of fitness, loss of replication capacity, loss of specific infectivity, and aberrant Gag processing induced by the N88S mutation. In addition, these mutations partially ablate amprenavir hypersusceptibility. Addition of mutation M46L to a strain harboring mutations L63P, V77I, and N88S resulted in a reduction of fitness and infectivity without changing Gag-processing efficiency, while amprenavir hypersusceptibility was further diminished. The ratio of reverse transcriptase activity to p24 protein was reduced in this strain compared to that in the other variants, suggesting that the M46L effect on fitness occurred through a mechanism different from a Gag-processing defect. We utilized these mutant strains to undertake a systematic comparison of indirect, single, cycle-based measures of fitness with direct, replication-based fitness assays and demonstrated that both yield consistent results. However, we observed that the magnitude of the fitness loss for one of the mutants varied depending on the assay used.

Nelfinavir-Resistant, Amprenavir-Hypersusceptible Strains of Human Immunodeficiency Virus Type 1 Carrying an N88S Mutation in Protease Have Reduced Infectivity, Reduced Replication Capacity, and Reduced Fitness and Process the Gag Polyprotein Precursor Aberrantly

Nelfinavir-Resistant, Amprenavir-Hypersusceptible Strains of Human Immunodeficiency Virus Type 1 Carrying an N88S Mutation in Protease Have Reduced Infectivity, Reduced Replication Capacity, and Reduced Fitness and Process the Gag Polyprotein Precursor Aberrantly

An attenuating mutation in the 2A protease of swine vesicular disease virus, a picornavirus, regulates cap- and internal ribosome entry site-dependent protein synthesis.

Virulent and avirulent strains of swine vesicular disease virus (SVDV), a picornavirus, have been characterized previously. The major determinants for attenuation have been mapped to specific residues in the 1D-2A-coding region. The properties of the 2A proteases from the virulent and avirulent strains of SVDV have now been examined. Both proteases efficiently cleaved the 1D/2A junction in vitro and in vivo. However, the 2A protease of the avirulent strain of SVDV was much less effective than the virulent-virus 2A protease at inducing cleavage of translation initiation factor eIF4GI within transfected cells. Hence the virulent-virus 2A protease is much more effective at inhibiting cap-dependent protein synthesis. Furthermore, the virulent-virus 2A protease strongly stimulated the internal ribosome entry sites (IRESs) from coxsackievirus B4 and from SVDV, while the avirulent-virus 2A protease was significantly less active in these assays. Thus, the different properties of the 2A proteases from the virulent and avirulent strains of SVDV in regulating protein synthesis initiation reflect the distinct pathogenic properties of the viruses from which they are derived. A single amino acid substitution, adjacent to His21 of the catalytic triad, is sufficient to confer the characteristics of the virulent-strain 2A protease on the avirulent-strain protease. It is concluded that the efficiency of picornavirus protein synthesis, controlled directly by the IRES or indirectly by the 2A protease, can determine virus virulence.

Altered Viral Fitness of HIV-1 Following Failure of Protease Inhibitor-Based Therapy

HIV-1 isolated from patients with improved CD4+ T-cell counts despite virologic failure on a nucleoside reverse transcriptase inhibitor (NRTI) and protease inhibitor (PI)-containing regimen were characterized. Five paired virus isolates from patients before and after zidovudine, lamivudine, and ritonavir treatment were tested. Human peripheral blood leukocyte-reconstituted severe combined immunodeficient (hu-PBL-SCID) mice were infected with pre-or posttreatment isolates and plasma HIV-1 RNA levels and CD4+ T cells were measured. Two of five post-treatment isolates exhibited decreased replication in hu-PBL-SCID mice compared with the paired pretreatment isolate, and both had the V82A mutation in protease associated with resistance to PI. One additional posttreatment isolate with the M184V mutation in reverse transcriptase showed diminished replication. CD4+ T-cell depletion was similar following infection with either the pre-or posttreatment isolates. Subtle losses in the replication capacity of PI-or NRTI-resistant viruses may contribute to relative preservation of CD4+ T-cell counts in persons who experience virologic failure. Cytopathic effects of viral infection for target T cells vary from patient to patient but appear not to be influenced by mutations associated with failure of therapy in this system.

Altered Viral Fitness of HIV-1 Following Failure of Protease Inhibitor-Based Therapy

HIV-1 isolated from patients with improved CD4+ T-cell counts despite virologic failure on a nucleoside reverse transcriptase inhibitor (NRTI) and protease inhibitor (PI)-containing regimen were characterized. Five paired virus isolates from patients before and after zidovudine, lamivudine, and ritonavir treatment were tested. Human peripheral blood leukocyte-reconstituted severe combined immunodeficient (hu-PBL-SCID) mice were infected with pre- or posttreatment isolates and plasma HIV-1 RNA levels and CD4+ T cells were measured. Two of five post-treatment isolates exhibited decreased replication in hu-PBL-SCID mice compared with the paired pretreatment isolate, and both had the V82A mutation in protease associated with resistance to PI. One additional posttreatment isolate with the M184V mutation in reverse transcriptase showed diminished replication. CD4+ T-cell depletion was similar following infection with either the pre- or posttreatment isolates. Subtle losses in the replication capacity of PI- or NRTI-resistant viruses may contribute to relative preservation of CD4+ T-cell counts in persons who experience virologic failure. Cytopathic effects of viral infection for target T cells vary from patient to patient but appear not to be influenced by mutations associated with failure of therapy in this system.

A mutation in human immunodeficiency virus type 1 protease, N88S, that causes in vitro hypersensitivity to amprenavir.

Amprenavir (Agenerase, 141-W94, VX-478) is a human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PRI) recently approved for the treatment of HIV-1 infection in the United States. A major cause of treatment failure is the development of resistance to PRIs. One potential use for amprenavir is as salvage therapy for patients for whom treatment that includes one (or more) of the other four currently approved PRIs-saquinavir, indinavir, ritonavir, and nelfinavir-has failed. We evaluated the cross-resistance to amprenavir of viruses that evolved during treatment with the two most commonly prescribed PRIs, nelfinavir and indinavir. Unexpectedly, a dramatic increase in susceptibility (2.5- to 12. 5-fold) was observed with 20 of 312 (6.4%) patient viruses analyzed. The most pronounced increases in susceptibility were strongly associated with an N88S mutation in protease. All viruses that carried the N88S mutation were hypersensitive to amprenavir. Site-directed mutagenesis studies confirmed the causal role of N88S in determining amprenavir hypersensitivity. The presence of the N88S mutation and associated amprenavir hypersensitivity may be useful in predicting an improved clinical response to amprenavir salvage therapy.

Reduced antiretroviral drug susceptibility among patients with primary HIV infection.

The transmission of drug-resistant human immunodeficiency virus (HIV) has been documented, but the prevalence of such transmission is unknown. To assess the spectrum and frequency of antiretroviral susceptibility among subjects with primary HIV infection. Retrospective analysis of 141 subjects identified from clinical research centers in 5 major metropolitan areas, enrolled from 1989 to 1998, with HIV seroconversion within the preceding 12 months and no more than 7 days' prior antiretroviral (ARV) therapy. Phenotypic and genotypic ARV susceptibility of HIV from plasma samples. The transmission of drug-resistant HIV as assessed by a greater than 10-fold reduction in ARV susceptibility to 1 or more drugs was observed in 3 (2%) of 141 subjects, including to a nonnucleoside reverse transcriptase inhibitor in 1 patient and to a nucleoside reverse transcriptase inhibitor and a protease inhibitor in 2 patients. Population-based sequence analysis of these 3 samples identified multidrug-resistance mutations in reverse transcriptase (M184V, T215Y, K219K/R) and protease (L101/V, K20R, M361, M46I, G48V, L63P, A71T, V771, V82T, 184V, L90M) in the 2 latter patient samples, along with numerous polymorphisms. A reduction in susceptibility of greater than 2.5- to 10-fold to 1 or more drugs was observed in viral isolates from 36 patients (26%). Sequence analysis of these 36 samples identified well-characterized drug resistance mutation in reverse transcriptase and protease in only 1 of these patients. Reductions in drug susceptibility of more than 10-fold were rare among this cohort of recently HIV-infected subjects and were distributed among each of the 3 major classes of ARV drugs tested. Reductions in susceptibility of more than 2.5- to 10-fold to certain ARV drugs of unknown clinical significance were highly prevalent among newly infected patients. Resistance testing may be warranted to monitor the frequency of drug resistance over time and to assess the potential for geographic variability.

Characterization of novel cathepsin K mutations in the pro and mature polypeptide regions causing pycnodysostosis.

Cathepsin K, a lysosomal cysteine protease critical for bone remodeling by osteoclasts, was recently identified as the deficient enzyme causing pycnodysostosis, an autosomal recessive osteosclerotic skeletal dysplasia. To investigate the nature of molecular lesions causing this disease, mutations in the cathepsin K gene from eight families were determined, identifying seven novel mutations (K52X, G79E, Q190X, Y212C, A277E, A277V, and R312G). Expression of the first pro region missense mutation in a cysteine protease, G79E, in Pichia pastoris resulted in an unstable precursor protein, consistent with misfolding of the proenzyme. Expression of five mature region missense defects revealed that G146R, A277E, A277V, and R312G precursors were unstable, and no mature proteins or protease activity were detected. The Y212C precursor was activated to its mature form in a manner similar to that of the wild-type cathepsin K. The mature Y212C enzyme retained its dipeptide substrate specificity and gelatinolytic activity, but it had markedly decreased activity toward type I collagen and a cathepsin K-specific tripeptide substrate, indicating that it was unable to bind collagen triple helix. These studies demonstrated the molecular heterogeneity of mutations causing pycnodysostosis, indicated that pro region conformation directs proper folding of the proenzyme, and suggested that the cathepsin K active site contains a critical collagen-binding domain.

Template-dependent initiation of Sindbis virus RNA replication in vitro.

Recent insights into the early events in Sindbis virus RNA replication suggest a requirement for either the P123 or P23 polyprotein, as well as mature nsP4, the RNA-dependent RNA polymerase, for initiation of minus-strand RNA synthesis. Based on this observation, we have succeeded in reconstituting an in vitro system for template-dependent initiation of SIN RNA replication. Extracts were isolated from cells infected with vaccinia virus recombinants expressing various SIN proteins and assayed by the addition of exogenous template RNAs. Extracts from cells expressing P123C>S, a protease-defective P123 polyprotein, and nsP4 synthesized a genome-length minus-sense RNA product. Replicase activity was dependent upon addition of exogenous RNA and was specific for alphavirus plus-strand RNA templates. RNA synthesis was also obtained by coexpression of nsP1, P23C>S, and nsP4. However, extracts from cells expressing nsP4 and P123, a cleavage-competent P123 polyprotein, had much less replicase activity. In addition, a P123 polyprotein containing a mutation in the nsP2 protease which increased the efficiency of processing exhibited very little, if any, replicase activity. These results provide further evidence that processing of the polyprotein inactivates the minus-strand initiation complex. Finally, RNA synthesis was detected when soluble nsP4 was added to a membrane fraction containing P123C>S, thus providing a functional assay for purification of the nsP4 RNA polymerase.

A mutation in human immunodeficiency virus type 1 protease at position 88, located outside the active site, confers resistance to the hydroxyethylurea inhibitor SC-55389A.

The hydroxyethylurea human immunodeficiency virus type 1 (HIV-1) protease inhibitors SC-55389A and SC-52151 were used to select drug-resistant variants in vitro. One clinical HIV-1 strain (89-959) and one laboratory HIV-1 strain (LAI) were passaged in peripheral blood mononuclear cells or CEMT4 cells in the presence of SC-55389A. Resistant isolates from both strains consistently had a mutation to serine for asparagine at amino acid 88 (N88S) in the protease gene either alone or in combination with a change to phenylalanine at position 10. The N88S mutation, recreated by oligonucleotide-mediated site-directed mutagenesis in HXB2, was sufficient to confer resistance to SC-55389A. In contrast, SC-52151-resistant variants selected from the monocytotropic strain SF162 had multiple substitutions in the protease gene (I11V, M461, F53L, A71V, and N88D), and the N88D mutation, re-created by oligonucleotide-mediated site-directed mutagenesis in HXB2, did not confer resistance to SC-52151. The potencies of L735,524 and Ro31-8959 were not reduced when these compounds were assayed against variants with either the N88S or N88D substitution. Position 88 is in a helix that lies behind the substrate binding pocket and may indirectly influence inhibitor binding through interactions with the amino acid at position 31. The selected mutations were persistent in the viral populations after more than 20 passages in the absence of drugs. Passaging of virus first in SC-55389A alone and then in combination with SC-52151 resulted in the accumulation of more mutations in the protease gene (L10F, D35E, D37M, I47V, 154L, A71V, V82I, and S88D) and in the selection of a variant that was cross-resistant to multiple protease inhibitors. These results indicate that a mutation in the HIV-1 protease at a position that is located outside of the substrate binding pocket confers resistance to a protease inhibitor and that mutations in the protease gene accumulate with increasing selection pressure and can persist in the absence of selection pressure.

Lack of integrase can markedly affect human immunodeficiency virus type 1 particle production in the presence of an active viral protease.

The Gag-Pol polyprotein of human immunodeficiency virus type 1 is not required for efficient viral particle assembly or release. However, in this report we demonstrate that the synthesis of a truncated Gag-Pol precursor due to a premature termination codon in pol can reduce the ability of a full-length provirus to direct the formation of viral particles. Marked effects on particle production were seen when premature termination codons were introduced into the integrase (IN)-coding region. By contrast, a mutant which lacked both IN and reverse transcriptase (RT) formed particles with normal efficiency. Particle production by IN mutants was restored to wild-type levels when a second premature termination codon was introduced at the 5' end of the RT-coding sequence. Particle formation was similarly restored by a second site mutation in the viral protease (PR) gene which prevented proteolytic processing of the Gag polyprotein. Finally particle formation was restored in the presence of A77003, a specific inhibitor of human immunodeficiency virus type 1 PR. These results suggest that the effects of a lack of IN sequences on particle formation require the synthesis of a Gag-Pol precursor which contains RT sequences and are due to inappropriate PR activity.