Human Immunodeficiency Virus Protease Cleavage Research Articles

Cognitive Framework for HIV-1 Protease Cleavage Site Classification Using Evolutionary Algorithm

Acquired immunodeficiency syndrome is the progression from human immunodeficiency virus (HIV) to symptomatic disease. The disease process starts by replicating the viral particles, and the HIV-1 protease is the causative agent behind the phenomenon. To control the replication, protease inhibitors are designed that bind to the active sites. Thus, knowledge about the HIV protease cleavage specificity is critical in the design of effective HIV inhibitors. Several classifiers and encoding techniques have been proposed for the identification of cleavage sites. A recent study shows a linear support vector machine with orthogonal encoding promulgated its sovereignty in the linearly separable peptide data. However, the performance of linear SVM degrades on nonlinear separable data. To cope with the considerable changes between linear and nonlinear separable data, we propose a cognitive framework that utilizes the dexterity of evolutionary algorithm on exploring out the best adaptive settings in the classifier and encoding techniques to improve the predictive capability of the model. Specifically, the proposed cognitive model selects the three decisive factors: physicochemical property for peptide encoding, SVM kernel selection for linear or nonlinear data and parameter tuning of the selected kernel. To validate the performance of the proposed model, the benchmark peptide data are exercised in the experimentation, and the obtained results are compared with the state-of-the-art techniques. Besides, we also analyzed the performance of the proposed model in out-of-sample test, and the extensive experimental results prove the proficiency of the proposed approach for the classification of the HIV-1 protease cleavage sites.

A genetic programming method for feature mapping to improve prediction of HIV-1 protease cleavage site

The human immunodeficiency virus (HIV) is the cause of acquired immunodeficiency syndrome (AIDS), which has profound implications in terms of both economic burden and loss of life. Modeling and examination of the HIV protease cleavage of amino acid sequences can contribute to control of this disease and production of more effective drugs. The present paper introduces a new method for encoding and characterization of amino acid sequences and a new model for the prediction of amino acid sequence cleavage by HIV protease. The proposed encoding scheme utilizes a combination of amino acids’ spatial and structural features in conjunction with 20 amino acid sequences to make sure that their physicochemical and sequencing features are all taken into account. The proposed HIV-1 amino acid cleavage prediction model is developed with the combination of genetic programming and support vector machine. The results of evaluations performed on various datasets demonstrate the superior performance of the proposed encoding and better accuracy of the proposed HIV-1 cleavage prediction model as compared to the state-of-the-art methods.

Development of a Novel Screening Strategy Designed to Discover a New Class of HIV Drugs

Current antiretroviral treatments target multiple pathways important for human immunodeficiency virus (HIV) multiplication, including viral entry, synthesis and integration of the DNA provirus, and the processing of viral polyprotein precursors. However, HIV is becoming increasingly resistant to these "combination therapies." Recent findings show that inhibition of HIV Gag protein cleavage into its two structural proteins, matrix (MA) and capsid (CA), has a devastating effect on viral production, revealing a potential new target class for HIV treatment. Unlike the widely used HIV protease inhibitors, this new class of inhibitor would target the substrate, not the protease enzyme itself. This approach offers a distinct advantage in that inhibitors of MA/CA would only need to affect a subset of the Gag molecules to disable viral replication. To discover MA/CA-specific inhibitors, we constructed a modified MA/CA fusion peptide (MA/CAΔ) that contains the HIV protease (PR) cleavage site as well as a tetracysteine motif for fluorescent labeling. The HIV PR cleavage of MA/CAΔ can then be monitored via fluorescence polarization (FP). We have adapted this FP assay for high-throughput screening and validated it according to industry standards using a 384-well plate format. We have currently tested 24,000 compounds in this assay and here detail the screening methodology and the results of this screening campaign.

Predicting human immunodeficiency virus protease cleavage sites in nonlinear projection space

HIV-1 protease has a broad and complex substrate specificity. The discovery of an accurate, robust, and rapid method for predicting the cleavage sites in proteins by HIV protease would greatly expedite the search for inhibitors of HIV protease. During the last two decades, various methods have been developed to explore the specificity of HIV protease cleavage activity. However, because little advancement has been made in the understanding of HIV-1 protease cleavage site specificity, not much progress has been reported in either extracting effective methods or maintaining high prediction accuracy. In this article, a theoretical framework is developed, based on the kernel method for dimensionality reduction and prediction for HIV-1 protease cleavage site specificity. A nonlinear dimensionality reduction kernel method, based on manifold learning, is proposed to reduce the high dimensions of protease specificity. A support vector machine is applied to predict the protease cleavage. Superior performance in comparison to that previously published in literature is obtained using numerical simulations showing that the basic specificities of the HIV-1 protease are maintained in reduction feature space, and by combining the nonlinear dimensionality reduction algorithm with a support vector machine classifier.

HIVcleave: a web-server for predicting human immunodeficiency virus protease cleavage sites in proteins

According to the ‘‘distorted key theory’’ [K.C. Chou, Analytical Biochemistry, 233 (1996) 1–14], the information of cleavage sites of proteins by HIV (human immunodeficiency virus) protease is very useful for finding effective inhibitors against HIV, the culprit of AIDS (acquired immunodeficiency syndrome). To meet the increasing need in this regard, a web-server called HIVcleave was established at http://chou.med.harvard.edu/bioinf/HIV/. In this note we provide a step-to-step guide for how to use HIVcleave to identify the cleavage sites of a query protein sequence by HIV-1 and HIV-2 proteases, respectively.

Rational Design of p53, an Intrinsically Unstructured Protein, for the Fabrication of Novel Molecular Sensors

The dominant paradigm of protein engineering is structure-based site-directed mutagenesis. This rational approach is generally more effective for the engineering of local properties, such as substrate specificity, than global ones such as allostery. Previous workers have modified normally unregulated reporter enzymes, including beta-galactosidase, alkaline phosphatase, and beta-lactamase, so that the engineered versions are activated (up to 4-fold) by monoclonal antibodies. A reporter that could easily be "reprogrammed" for the facile detection of novel effectors (binding or modifying activities) would be useful in high throughput screens for directed evolution or drug discovery. Here we describe a straightforward and general solution to this potentially difficult design problem. The transcription factor p53 is normally regulated by a variety of post-translational modifications. The insertion of peptides into intrinsically unstructured domains of p53 generated variants that were activated up to 100-fold by novel effectors (proteases or antibodies). An engineered p53 was incorporated into an existing high throughput screen for the detection of human immunodeficiency virus protease, an arbitrarily chosen novel effector. These results suggest that the molecular recognition properties of intrinsically unstructured proteins are relatively easy to engineer and that the absence of crystal structures should not deter the rational engineering of this class of proteins.

Human immunodeficiency virus type 1 protease cleavage site mutations associated with protease inhibitor cross-resistance selected by indinavir, ritonavir, and/or saquinavir.

We examined the prevalence of cleavage site mutations, both within and outside the gag region, in 28 protease inhibitor (PI) cross-resistant patients treated with indinavir, ritonavir, and/or saquinavir compared to control patients treated with reverse transcriptase inhibitors. Three human immunodeficiency virus protease cleavage sites within gag (p2/NC, NC/p1, and NC/TFP) showed considerable in vivo evolution before and after therapy with indinavir, ritonavir, and/or saquinavir. Another gag cleavage site (p1/p6(gag)) showed a trend compared to matched controls. The other eight recognized cleavage sites showed relatively little difference between PI-resistant cases and controls. An A-->V substitution at the P2 position of the NC/p1 and NC/TFP cleavage sites was the most common (29%) change selected by the PIs used in this study.

Kinetics Analysis of Consecutive HIV Proteolytic Cleavages of the Gag-Pol Polyprotein

The ordered, sequential cleavages of the Gag-Pol polyprotein by human immunodeficiency virus (HIV) protease present the virus with severe limitations on viable mutations of the enzyme. An extension of the method of Kuchel et al. (Kuchel, P. W., Nichol, L. W., and Jeffrey, P. D. (1974) J. Theor. Biol. 48, 39-49) for the analysis of consecutive enzyme reactions leads to a simple description of the catalytic efficiency of mutant and wild type HIV protease in the presence or absence of inhibitors. The overall catalytic efficiency of a mutant HIV protease relative to the wild type enzyme is given by the product of the ratios of their respective efficiencies for the 8 obligatory cleavages. Under no conditions is HIV viable when the geometric mean efficiency of a mutant HIV protease is less than 61% of the wild type activity for each cleavage. The lower catalytic efficiencies of the mutant enzymes coupled with the exponential dependence on 1/(1 + [I]/Ki) more than offset the inhibitor resistance acquired by HIV protease. The conclusion of this analysis is that inhibitor-resistant mutant HIV proteases are very unlikely to contribute to viral viability in vivo. The results strongly suggest that future protease inhibitor clinical trials should measure the infectivity of the virions in blood plasma instead of relying on viral RNA levels.

Predicting human immunodeficiency virus protease cleavage sites in proteins by a discriminant function method.

Based on the sequence-coupled (Markov chain) model and vector-projection principle, a discriminant function method is proposed to predict sites in protein substrates that should be susceptible to cleavage by the HIV-1 protease. The discriminant function is defined by delta = phi+ - phi-, where phi+ and phi- are the cleavable and noncleavable attributes for a given peptide, and they can be derived from two complementary sets of peptides, S+ and S-, known to be cleavable and noncleavable, respectively, by the enzyme. The rate of correct prediction by the method for the 62 cleavable peptides and 239 noncleavable peptides in the training set are 100 and 96.7%, respectively. Application of the method to the 55 sequences which are outside the training set and known to be cleaved by the HIV-1 protease accurately predicted 100% of the peptides as substrates of the enzyme. The method also predicted all but one of the sites hydrolyzed by the protease in native HIV-1 and HIV-2 reverse transcriptases, where the HIV-1 protease discriminates between nearly identical sequences in a very subtle fashion. Finally, the algorithm predicts correctly all of the HIV-1 protease processing sites in the native gag and gag/pol HIV-1 polyproteins, and all of the cleavage sites identified in denatured protease and reverse transcriptase. The new predictive algorithm provides a novel route toward understanding the specificity of this important therapeutic target.

Human immunodeficiency virus reverse transcriptase. Expression in Escherichia coli, purification, and characterization of a functionally and structurally asymmetric dimeric polymerase

Human immunodeficiency virus (HIV) reverse transcriptase isolated from viral particles contains two subunits, p51 and p66. We have produced both subunits in separate Escherichia coli strains using expression vectors. Stop codons were placed immediately after the codon for the carboxyl-terminal residue of the mature processed p51 and p66 subunits found in viral particles. Insertion of a methionine in front of the HIV protease cleavage site in the recombinant protein enabled synthesis of both subunits with the natural amino-terminal proline, since E. coli methionine aminopeptidase cleaves a Met-Pro amino-terminal linkage. That this occurred to an extent greater than 95% was confirmed by sequencing the purified subunits. Examination of the activities of the individual p51 and p66 subunits on a variety of templates and under solution conditions optimized for each subunit revealed a significant catalytic activity for the natural p51 subunit. This result contrasts to results reported earlier for many recombinant forms without the natural amino and/or carboxyl termini. As expected from earlier work, the optimal homopolymeric template for the p66 subunit was poly(rA). For the p51 subunit, poly(dC) was found to be the optimal template; its activity is 2- to 4-fold greater than p66 on poly(dC). The p51 subunit is 13- to 50-fold less active on poly(rC). These findings are discussed in the context of our earlier hypothesis (McHenry, C. S. (1989) in Molecular Biology of Chromosome Function (Adolph, K., ed) Chap. 5, Springer-Verlag, New York) that the HIV reverse transcriptase might be functionally asymmetric with distinct plus- and minus-strand polymerases.

Beta-Galactosidase containing a human immunodeficiency virus protease cleavage site is cleaved and inactivated by human immunodeficiency virus protease.

A "cleavage cassette" specifying a decapeptide human immunodeficiency virus (HIV) protease cleavage site was introduced into six different locations of beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) in Escherichia coli. Four of these constructs retained beta-galactosidase activity despite the insertion of the cleavage cassette. Of these four constructs, one was cleaved by HIV protease, resulting in the inactivation of beta-galactosidase both in vivo and in vitro. This cleavage was inhibited by pepstatin A, a known inhibitor of HIV protease. Thus, beta-galactosidase has been converted into an easily assayed substrate for HIV protease. An analogous construct of beta-galactosidase containing a polio protease cleavage site was cleaved likewise by polio protease, suggesting that this system may be generic for monitoring cleavage by a variety of proteases.