Involvement of tryptase-related cellular protease(s) in human immunodeficiency virus type 1 infection

Trypstatin, a new cellular Kunitz-type protease inhibitor purified from rat mast cells, inhibited syncytium formation in human immunodeficiency virus type 1 (HIV-1)-infected CCRF-CEM and uninfected Molt-4 clone 8 at a concentration of 1 microM. Anti-rat tongue mast cell tryptase antibodies reacted with Molt-4 clone 8 cells, as determined by Western blot and by immunofluorescence. In addition, the antibody inhibited syncytium formation. These findings along with homologous sequences with trypstatin and a neutralizing epitope of gp120 of HIV-1 suggest that a tryptase-like cellular enzyme(s) is involved in HIV-1 infection.     

Fitness landscape of human immunodeficiency virus type 1 protease quasispecies

Here we show, at a high resolution (1%), the human immunodeficiency virus type 1 (HIV-1) protease gene quasispecies landscape from three infected na?ve individuals. A huge range of genetic configurations was found (67%, 71%, and 80% of the nucleotide clones from the three individuals, respectively, were different), and these configurations created a dense net that linked different parts of the viral population. Similarly, a vast diversity of different protease activities was also found. Importantly, 65% of the analyzed enzymes had detectable protease activity, and 11% of the minority individual variants showed similar or better fitness than the master (most abundant) enzyme, suggesting that the viral complexity in this genomic region does not exclusively depend on the enzyme's catalytic efficiency. Several high-fitness minority variants had only one substitution compared to the master sequence, supporting the possibility that the rugged HIV-1 protease quasispecies fitness landscape may be formed by a continuous network that can be traversed by single mutational steps without passing through defective or less-adapted proteins.     

Replicative fitness of protease inhibitor-resistant mutants of human immunodeficiency virus type 1

The relative replicative fitness of human immunodeficiency virus type 1 (HIV-1) mutants selected by different protease inhibitors (PIs) in vivo was determined. Each mutant was compared to wild type (WT), NL4-3, in the absence of drugs by several methods, including clonal genotyping of cultures infected with two competing viral variants, kinetics of viral antigen production, and viral infectivity/virion particle ratios. A nelfinavir-selected protease D30N substitution substantially decreased replicative capacity relative to WT, while a saquinavir-selected L90M substitution moderately decreased fitness. The D30N mutant virus was also outcompeted by the L90M mutant in the absence of drugs. A major natural polymorphism of the HIV-1 protease, L63P, compensated well for the impairment of fitness caused by L90M but only slightly improved the fitness of D30N. Multiply substituted indinavir-selected mutants M46I/L63P/V82T/I84V and L10R/M46I/L63P/V82T/I84V were just as fit as WT. These results indicate that the mutations which are usually initially selected by nelfinavir and saquinavir, D30N and L90M, respectively, impair fitness. However, additional mutations may improve the replicative capacity of these and other drug-resistant mutants. Hypotheses based on the greater fitness impairment of the nelfinavir-selected D30N mutant are suggested to explain observations that prolonged responses to delayed salvage regimens, including alternate PIs, may be relatively common after nelfinavir failure.     

Conditional human immunodeficiency virus type 1 protease mutants show no role for the viral protease early in virus replication.

The human immunodeficiency virus type 1 protease plays a critical role in the proteolytic processing of precursor polyproteins during virion maturation. Contradictory evidence has been obtained for a possible role for the protease early after infection, i.e., during DNA synthesis and/or integration. We have reexamined this question by using conditional mutants of the protease. In one set of experiments, protease mutants that confer a temperature-sensitive phenotype for processing were used to assess the need for protease activity early after infection. No significant difference from results with wild-type virus was seen when infections were carried out at either 35 or 40 degrees C. In a separate set of experiments, infections were carried out in the presence of a protease inhibitor. In this case, both wild-type virus and a drug-resistant variant were used, the latter as a control to ensure a specific effect of the inhibitor. Infection with either virus was not inhibited at drug concentrations that were up to 10-fold higher than those needed to inhibit intracellular processing by the viral protease. The results obtained by both of these experimental protocols provide evidence that the human immunodeficiency virus type 1 protease does not play a role early after infection.     

Degradation of cytoskeletal proteins by the human immunodeficiency virus type 1 protease.

Triton X-100-extracted human skin fibroblasts were exposed to human immunodeficiency virus type 1 protease and analysed by 2D-gel electrophoresis and immunofluorescence microscopy. Vimentin, two of the tropomyosin isoforms, a protein with M_r ∼ 90,000 and a protein with M_r ∼ 200,000 were found to be degraded. Structurally, this was accompanied by the disintegration of the vimentin filament network and the disappearance of the microfilament network. In contrast to our in vivo observations (Höner et al., 1991), prominent stress fibers and chromatin structure seemed to be rather resistant to the action of this protease.     

Zinc inhibition of renin and the protease from human immunodeficiency virus type 1

We report here for the first time that Zn2+ is an effective inhibitor of renin and the protease from HIV-1, two aspartyl proteinases of considerable physiological importance. Inhibition of renin is noncompetitive and is accompanied by binding of 1 mol of Zn2+/mol of enzyme. Depending on the substrate, inhibition of the HIV protease by Zn2+ can be either competitive or noncompetitive, but in neither case is loss of activity due to disruption of the protease dimer. Inhibition of both enzymes is first order with respect to Zn2+ and is rapidly reversed by addition of EDTA. Ki values are strongly pH dependent and optimal in the range of 20 microM at or above pH 7. All of the data in hand suggest that the inhibitory effect of Zn2+ is a consequence of its binding at, or near, the active-site carboxyl groups of these aspartyl proteinases. This inhibition of the viral enzyme may help to explain some of the beneficial effects seen in AIDS patients who have received Zn2+ therapy.     

Comprehensive bioinformatic analysis of the specificity of human immunodeficiency virus type 1 protease

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.     

Insights into cellular microRNAs and human immunodeficiency virus type 1 (HIV-1)

Recent findings suggest that mammalian microRNAs (miRNAs) may influence viral replication in host cells. Studies on HIV-1 infection have contributed in part to the development of this notion. Herein, we review, in brief, some of the evidence supportive of an interplay between human miRNAs and HIV-1 in cells. Several cellular miRNAs potentially act to restrict HIV-1 replication, and the virus has countermeasures to evade such restriction.     

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.     

Hydrophobic sliding: a possible mechanism for drug resistance in human immunodeficiency virus type 1 protease

Hydrophobic residues outside the active site of HIV-1 protease frequently mutate in patients undergoing protease inhibitor therapy; however, the mechanism by which these mutations confer drug resistance is not understood. From analysis of molecular dynamics simulations, 19 core hydrophobic residues appear to facilitate the conformational changes that occur in HIV-1 protease. The hydrophobic core residues slide by each other, exchanging one hydrophobic van der Waal contact for another, with little energy penalty, while maintaining many structurally important hydrogen bonds. Such hydrophobic sliding may represent a general mechanism by which proteins undergo conformational changes. Mutation of these residues in HIV-1 protease would alter the packing of the hydrophobic core, affecting the conformational flexibility of the protease. Therefore these residues impact the dynamic balance between processing substrates and binding inhibitors, and thus contribute to drug resistance.     

X-ray crystal structures of human immunodeficiency virus type 1 protease mutants complexed with atazanavir

Atazanavir, which is marketed as REYATAZ, is the first human immunodeficiency virus type 1 (HIV-1) protease inhibitor approved for once-daily administration. As previously reported, atazanavir offers improved inhibitory profiles against several common variants of HIV-1 protease over those of the other peptidomimetic inhibitors currently on the market. This work describes the X-ray crystal structures of complexes of atazanavir with two HIV-1 protease variants, namely, (i) an enzyme optimized for resistance to autolysis and oxidation, referred to as the cleavage-resistant mutant (CRM); and (ii) the M46I/V82F/I84V/L90M mutant of the CRM enzyme, which is resistant to all approved HIV-1 protease inhibitors, referred to as the inhibitor-resistant mutant. In these two complexes, atazanavir adopts distinct bound conformations in response to the V82F substitution, which may explain why this substitution, at least in isolation, has yet to be selected in vitro or in the clinic. Because of its nearly symmetrical chemical structure, atazanavir is able to make several analogous contacts with each monomer of the biological dimer.     

Mapping protease inhibitor resistance to human immunodeficiency virus type 1 sequence polymorphisms within patients

Resistance genotyping provides an important resource for the clinical management of patients infected with human immunodeficiency virus type 1 (HIV-1). However, resistance to protease (PR) inhibitors (PIs) is a complex phenotype shaped by interactions among nearly half of the residues in HIV-1 PR. Previous studies of the genetic basis of PI resistance focused on fixed substitutions among populations of HIV-1, i.e., host-specific adaptations. Consequently, they are susceptible to a high false discovery rate due to founder effects. Here, we employ sequencing “mixtures” (i.e., ambiguous base calls) as a site-specific marker of genetic variation within patients that is independent of the phylogeny. We demonstrate that the transient response to selection by PIs is manifested as an excess of nonsynonymous mixtures. Using a sample of 5,651 PR sequences isolated from both PI-naive and -treated patients, we analyze the joint distribution of mixtures and eight PIs as a Bayesian network, which distinguishes residue-residue interactions from direct associations with PIs. We find that selection for resistance is associated with the emergence of nonsynonymous mixtures in two distinct groups of codon sites clustered along the substrate cleft and distal regions of PR, respectively. Within-patient evolution at several positions is independent of PIs, including those formerly postulated to be involved in resistance. These positions are under strong positive selection in the PI-naive patient population, implying that other factors can produce spurious associations with resistance, e.g., mutational escape from the immune response.     

Regulation of human immunodeficiency virus type 1 Env-mediated membrane fusion by viral protease activity

We and others have presented evidence for a direct interaction between the matrix (MA) domain of the human immunodeficiency virus type 1 (HIV-1) Gag protein and the cytoplasmic tail of the transmembrane envelope (Env) glycoprotein gp41. In addition, it has been postulated that the MA domain of Gag undergoes a conformational change following Gag processing, and the cytoplasmic tail of gp41 has been shown to modulate Env-mediated membrane fusion activity. Together, these results raise the possibility that the interaction between the gp41 cytoplasmic tail and MA is regulated by protease (PR)-mediated Gag processing, perhaps affecting Env function. To examine whether Gag processing affects Env-mediated fusion, we compared the ability of wild-type (WT) HIV-1 Env and a mutant lacking the gp41 cytoplasmic tail to induce fusion in the context of an active (PR(+)) or inactive (PR(-)) viral PR. We observed that PR(-) virions bearing WT Env displayed defects in cell-cell fusion. Impaired fusion did not appear to be due to differences in the levels of virion-associated Env, in CD4-dependent binding to target cells, or in the formation of the CD4-induced gp41 six-helix bundle. Interestingly, truncation of the gp41 cytoplasmic tail reversed the fusion defect. These results suggest that interactions between unprocessed Gag and the gp41 cytoplasmic tail suppress fusion.     

A new sequence representation as applied in better specificity elucidation for human immunodeficiency virus type 1 protease

Factor analysis scales of generalized amino acid information (FASGAI) involving hydrophobicity, alpha and turn propensities, bulky properties, compositional characteristics, local flexibility, and electronic properties were derived from 516 property parameters of 20-coded amino acids, and was then employed to represent sequence structures of 746 peptides with 8 amino acid residues. Cleavage site prediction models for human immunodeficiency virus type 1 protease by linear discriminant analysis and support vector machine with radial basis function kernel were constructed to identify if they could be cleaved or not, and were further utilized to investigate the cleavage specificity. These diversified properties, including the bulky properties, secondary conformation characteristics, electronic properties, and hydrophobicity at the first, the second, the fourth, the fifth, and the sixth residue, are possibly important factors in determining HIV PR cleavage or not. Particularly, maximal positive and negative influences result from the bulky properties of different sites. Further results from analysis of variance also likely reflect that the HIV PR recognizes diversified key properties of various sites in the octameric sequences. Satisfactory results show that FASGAI can not only be used to represent sequence structures of various functional peptides, but alsoprovide a potential feasible measure for exploring relationship between protein motif sequences and their functions.     

Characterization of an active single polypeptide form of the human immunodeficiency virus type 1 protease

The pepsin-like aspartyl proteases consist of a single polypeptide chain with topologically similar amino- and carboxyl-terminal domains, each of which contributes 1 aspartic acid residue to the active site. This structure has been proposed to have evolved by gene duplication and fusion from a dimeric enzyme composed of two identical polypeptide chains, such as the aspartyl protease (PRT) of human immunodeficiency virus type 1 (HIV-1). To determine if a single polypeptide form of the HIV-1 protease would be enzymatically active, two protease coding regions were linked to form a dimeric gene (pFGGP). Expression of this gene in Escherichia coli yielded a protein with the expected molecular mass of 22 kDa. The in vitro kinetic parameters of PRT and FGGP (where FGGP is the single polypeptide form of the HIV-1 protease with 2 glycine residues connecting the two subunits) for three peptide substrates are similar. Construction and analysis of a CheY-GAG-FGGP fusion protein demonstrated that FGGP is capable of precursor processing in vivo. Mutation of one or both of the active site aspartates to either asparagine or glutamate rendered the enzyme inactive, demonstrating that both active site aspartate residues are required for enzymatic activity.