Variability at Human Immunodeficiency Virus Type 1 Subtype C Protease Cleavage Sites: an Indication of Viral Fitness?

Oliveira, Túlio de; Engelbrecht, Susan; Rensburg, Estrelita Janse van; Gordon, Michelle; Bishop, Karen; Megede, Jan zur; Barnett, Susan W.; Cassol, Sharon

doi:10.1128/jvi.77.17.9422-9430.2003

Cited by 58 publications

(54 citation statements)

References 47 publications

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“…de Oliveira et al (22) also listed several variations on cleavage sites in their paper. It was possible to generate 160 octamers from those lists, of which 140 were not in the 746-peptide set.…”

Section: Resultsmentioning

confidence: 99%

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Comprehensive Bioinformatic Analysis of the Specificity of Human Immunodeficiency Virus Type 1 Protease

You

Garwicz

Rögnvaldsson

2005

J Virol

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Rapidly developing viral resistance to licensed human immunodeficiency virus type 1 (HIV-1) protease inhibitors is an increasing problem in the treatment of HIV-infected individuals and AIDS patients. A rational design of more effective protease inhibitors and discovery of potential biological substrates for the HIV-1 protease require accurate models for protease cleavage specificity. In this study, several popular bioinformatic machine learning methods, including support vector machines and artificial neural networks, were used to analyze the specificity of the HIV-1 protease. A new, extensive data set (746 peptides that have been experimentally tested for cleavage by the HIV-1 protease) was compiled, and the data were used to construct different classifiers that predicted whether the protease would cleave a given peptide substrate or not. The best predictor was a nonlinear predictor using two physicochemical parameters (hydrophobicity, or alternatively polarity, and size) for the amino acids, indicating that these properties are the key features recognized by the HIV-1 protease. The present in silico study provides new and important insights into the workings of the HIV-1 protease at the molecular level, supporting the recent hypothesis that the protease primarily recognizes a conformation rather than a specific amino acid sequence. Furthermore, we demonstrate that the presence of 1 to 2 lysine residues near the cleavage site of octameric peptide substrates seems to prevent cleavage efficiently, suggesting that this positively charged amino acid plays an important role in hindering the activity of the HIV-1 protease.In less than a quarter of a century, over 20 million people have succumbed to AIDS, and at the end of 2003, an estimated 38 million people were living with a human immunodeficiency virus (HIV) infection. With an increase of almost 5 million new cases per year, more than 40 million people are likely to be infected with HIV today, with over 2 million of those afflicted being children under the age of 15 years (see the UNAIDS Report on the Global AIDS Epidemic and the AIDS Epidemic Update December 2004 from UNAIDS/WHO [48a, 48b]).Drugs that inhibit the HIV-1 protease, so-called protease inhibitors, are an important part of AIDS therapy today (20), since the HIV-1 protease cleaves viral Gag and Gag-Pol polyproteins into structure and replication proteins that are necessary for the virus to become infectious (28). Currently licensed protease inhibitors are all peptidomimetic; they mimic a peptide that the HIV-1 protease normally cleaves but are chemically modified such that the scissile bond cannot be cleaved (21, 37). Hence, rational design of an efficient inhibitor requires a good understanding of the HIV-1 protease specificity, i.e., knowing which amino acid sequences are cleaved by the protease and which are not. This is, however, difficult since it cleaves at several different sites that have little or no sequence similarity.A problem with the clinical use of protease inhibitors is the fact that th...

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Section: Resultsmentioning

confidence: 99%

“…The website http://www.bioafrica.net (22) lists several HIV-1 cleavage sites, of which about half are not in our data set. Table 3 lists some of these cleavage sites together with the SP with sparse orthogonal encoding and GSVM with property-coding classifier predictions.…”

Section: Resultsmentioning

confidence: 99%

Comprehensive Bioinformatic Analysis of the Specificity of Human Immunodeficiency Virus Type 1 Protease

You

Garwicz

Rögnvaldsson

2005

J Virol

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“…The amino acid changes of Leu to Pro at the P1Ј position (LP1ЈP) and Pro to Leu at the P5Ј position (PP5ЈL) in p1/p6 were observed. These changes have been reported as polymorphisms (2,5,8). The polymorphisms were preserved during the course of therapy without changing.…”

Section: Resultsmentioning

confidence: 83%

Functional Correlation between a Novel Amino Acid Insertion at Codon 19 in the Protease of Human Immunodeficiency Virus Type 1 and Polymorphism in the p1/p6 Gag Cleavage Site in Drug Resistance and Replication Fitness

Brann¹,

Dewar²,

Jiang³

et al. 2006

J Virol

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Population-based sequence analysis revealed the presence of a variant of human immunodeficiency virus type 1 (HIV-1) containing an insertion of amino acid Ile in the protease gene at codon 19 (19I) and amino acid substitutions in the protease at codons 21 (E21D) and 22 (A22V) along with multiple mutations associated with drug resistance, M46I/P63L/A71V/I84V/I93L, in a patient who had failed protease inhibitor (PI) therapy. Longitudinal analysis revealed that the P63L/A71V/I93L changes were present prior to PI therapy. Polymorphisms in the Gag sequence were only seen in the p1/p6 cleavage site at the P1 position (Leu to Pro) and the P5 position (Pro to Leu). To characterize the role of these mutations in drug susceptibility and replication capacity, a chimeric HIV-1 strain containing the 19I/E21D/A22V mutations with the M46I/P63L/A71V/I84V/ I93L and p1/p6 mutations was constructed. The chimera displayed high-level resistance to multiple PIs, but not to lopinavir, and grew to 30% of that of the wild type. To determine the relative contribution of each mutation to the phenotypic characteristic of the virus, a series of mutants was constructed using site-directed mutagenesis. A high level of resistance was only seen in mutants containing the 19I/A22V and p1/p6 mutations. The E21D mutation enhanced viral replication. These results suggest that the combination of the 19I/E21D/A22V mutations may emerge and lead to high-level resistance to multiple PIs. The combination of the 19I/A22V mutations may be associated with PI resistance; however, the drug resistance may be caused by the presence of a unique set of mutations in the p1/p6 mutations. The E21D mutation contributes to replication fitness rather than drug resistance.Treatment of human immunodeficiency virus type 1 (HIV-1)-infected individuals with a combination of anti-human immunodeficiency virus (HIV) dugs is highly effective in controlling HIV-1 replication and decreases AIDS-related morbidity and mortality (29,43). Treatment reduces viral load in plasma below 50 copies per ml and increases total CD4 ϩ T-cell counts. Treatment failure, however, does occur and is typically defined as an increase in viral load, a decrease in CD4 ϩ T-cell counts, and the emergence of drug-resistant variants.Drug-resistant variants in the genetic pool of HIV-1 in patients emerge as the predominant variants under the selective pressure of anti-HIV drug. The character of the highly errorprone HIV-1 reverse transcriptase (RT) results in the emergence of a high level of mutations and increasing genetic diversity (32). This characteristic of RT makes it possible for a number of amino acid substitutions to occur in the protease (PR), RT, and envelope of HIV-1. A number of amino acid substitutions in the PR and RT have been identified as mutations conferring drug resistance (34). Recently, the emergence of variants containing amino acid insertions in RT or PR has also been reported. The insertions in RT emerge between codons 69 and 70 or near codon 103 (7,21,22,27,37,38,39). The insertion ...

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“…[1][2][3] Although studies have investigated the substrate binding and cleavage properties of proteases, [4][5][6][7][8] the factors that determine the specificity of substrate binding remain largely uncharacterized. Studies of HIV-1 protease in complex with substrates and inhibitors indicate that the drug-resistant mutants of this enzyme have altered molecular recognition abilities; they still recognize the enzyme's substrates but binding to inhibitors is disrupted.…”

Section: Introductionmentioning

confidence: 99%

Structural, kinetic, and thermodynamic studies of specificity designed HIV‐1 protease

et al. 2012

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HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT-RH over p2-NC and CA-p2. In kinetic assays, RT-RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT-RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease.

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Variability at Human Immunodeficiency Virus Type 1 Subtype C Protease Cleavage Sites: an Indication of Viral Fitness?

Cited by 58 publications

References 47 publications

Comprehensive Bioinformatic Analysis of the Specificity of Human Immunodeficiency Virus Type 1 Protease

Comprehensive Bioinformatic Analysis of the Specificity of Human Immunodeficiency Virus Type 1 Protease

Functional Correlation between a Novel Amino Acid Insertion at Codon 19 in the Protease of Human Immunodeficiency Virus Type 1 and Polymorphism in the p1/p6 Gag Cleavage Site in Drug Resistance and Replication Fitness

Structural, kinetic, and thermodynamic studies of specificity designed HIV‐1 protease

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