József Tözsér

Comparison of the HIV-1 and HIV-2 proteinases using oligopeptide substrates representing cleavage sites in Gag and Gag-Pol polyproteins

The substrate specificity of the human immunodeficiency virus type 1 (HIV‐1) and type 2 (HIV‐2) proteinases was compared using oligopeptides corresponding to cleavage sites in the Gag and Gag‐Pol polyproteins of both viruses. All peptides mimicking cleavage sites at the junction of major functional protein domains were correctly cleaved by both enzymes. However, some other peptides thought to represent secondary cleavage sites remained intact. The kinetic parameters (K m and k m) obtained for the different substrates showed several hundred‐fold variation but were similar for the same substrate.

Substitution of proline with pipecolic acid at the scissile bond converts a peptide substrate of HIV proteinase into a selective inhibitor

The nonapeptide H-Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-NH2 containing the retroviral Tyr-Pro cleavage site is a good substrate for the proteinase of human immunodeficiency viruses but it is not readily hydrolyzed by other nonviral proteinases including the structurally related pepsin-like aspartic proteinases. Replacing the Pro by L-pipecolic acid (2-piperidinecarboxylic acid) converted the substrate into an effective inhibitor of HIV-1 and HIV-2 proteinases with IC50 of approximately 1 microM. This compound showed a high degree of selectivity in that it did not inhibit cathepsin D and renin.

HIV-I protease: Maturation, enzyme specificity, and drug resistance

Publisher Summary The active form of the 99-amino-acid-long mature protease (protease retropepsin (PR)) is a homodimer. Optimal catalytic activity of the mature PR and ordered processing of the polyproteins are critical for the liberation of infective progeny virus. The mature PR has proven to be the most effective target for antiviral therapy of AIDS. However, the long-term potency of current PR inhibitors as therapeutic agents is limited by the rapid development of drug-resistant variants of the PR. Therefore, it is critical to understand the molecular mechanisms of the proteolytic processes of the wild-type and drug-resistant mutants of PR to aid the development of new inhibitors and therapeutic strategies. This chapter discusses three critical features of the HIV-1 PR— namely, autocatalytic maturation from the precursor, substrate specificity, and emergence of drug resistance, utilizing protein and peptide design, enzyme kinetics, X-ray crystallography, and nuclear magnetic resonance (NMR) spectroscopy. The formation of the intermediate precursors under suboptimal conditions for the autoprocessing reaction is similar to that observed for the conversion of the zymogen form of the gastric protease pepsin that, unlike retroviral proteases, is a monomeric enzyme. Resistance to PR inhibitors can arise by more than one mechanism. Because no direct relationship was observed between relative catalytic activity, inhibition, and structural stability of the different PR mutants, drug resistance can arise from independent changes in any one of these parameters. Prolonged exposure to the drug can result in compensating mutations that act in combination to permit optimal polyprotein processing and replicative capacity in the presence of the PR inhibitor.

Molecular basis for substrate recognition and drug resistance from 1.1 to 1.6 Å resolution crystal structures of HIV-1 protease mutants with substrate analogs

HIV‐1 protease (PR) and two drug‐resistant variants – PR with the V82A mutation (PRV82A) and PR with the I84V mutation (PRI84V) – were studied using reduced peptide analogs of five natural cleavage sites (CA‐p2, p2‐NC, p6pol‐PR, p1‐p6 and NC‐p1) to understand the structural and kinetic changes. The common drug‐resistant mutations V82A and I84V alter residues forming the substrate‐binding site. Eight crystal structures were refined at resolutions of 1.10–1.60 Å. Differences in the PR–analog interactions depended on the peptide sequence and were consistent with the relative inhibition. Analog p6pol‐PR formed more hydrogen bonds of P2 Asn with PR and fewer van der Waals contacts at P1′ Pro compared with those formed by CA‐p2 or p2‐NC in PR complexes. The P3 Gly in p1‐p6 provided fewer van der Waals contacts and hydrogen bonds at P2–P3 and more water‐mediated interactions. PRI84V showed reduced van der Waals interactions with inhibitor compared with PR, which was consistent with kinetic data. The structures suggest that the binding affinity for mutants is modulated by the conformational flexibility of the substrate analogs. The complexes of PRV82A showed smaller shifts of the main chain atoms of Ala82 relative to PR, but more movement of the peptide analog, compared to complexes with clinical inhibitors. PRV82A was able to compensate for the loss of interaction with inhibitor caused by mutation, in agreement with kinetic data, but substrate analogs have more flexibility than the drugs to accommodate the structural changes caused by mutation. Hence, these structures help to explain how HIV can develop drug resistance while retaining the ability of PR to hydrolyze natural substrates.

Studies on the role of the S4 substrate binding site of HIV proteinases

Kinetic analysis of the hydrolysis of the peptide H‐Vat‐Ser‐Gin‐Asn‐Tyr*Pro‐He‐Val‐Gin‐NH2 and its analogs obtained by varying the length and introducing substitutions at the P4 site was carried out with both HIV‐1 and HIV‐2 proteinases. Deletion of the terminal Val and Gin had only moderate effect on the substrate hydrolysis, while the deletion of the P4, Ser as well as P3 Val greatly reduced the substrate hydrolysis. This is predicted to be due to the loss of interactions between main chains of the enzyme and the substrate. Substitution of the P4 Ser by amino acids having a high frequency of occurrence in β turns resulted in good substrates, while large amino acids were unfavourable in this position. The two proteinases acted similarly, except for substrates having Thr, Val and Leu substitutions, which were better accommodated in the HIV‐2 substrate binding pocket.

Atomic resolution crystal structures of HIV-1 protease and mutants V82A and I84V with saquinavir

Saquinavir (SQV), the first antiviral HIV‐1 protease (PR) inhibitor approved for AIDS therapy, has been studied in complexes with PR and the variants PRI84V and PRV82A containing the single mutations I84V and V82A that provide resistance to all the clinical inhibitors. Atomic resolution crystal structures (0.97–1.25 Å) of the SQV complexes were analyzed in comparison to the protease complexes with darunavir, a new drug that targets resistant HIV, in order to understand the molecular basis of drug resistance. PRI84V and PRV82A complexes were obtained in both the space groups P21212 and P212121, which provided experimental limits for the conformational flexibility. The SQV interactions with PR were very similar in the mutant complexes, consistent with the similar inhibition constants. The mutation from bigger to smaller amino acids allows more space to accommodate the large group at P1′ of SQV, unlike the reduced interactions observed in darunavir complexes. The residues 79–82 have adjusted to accommodate the large hydrophobic groups of SQV, suggesting that these residues are intrinsically flexible and their conformation depends more on the nature of the inhibitor than on the mutations in this region. This analysis will assist with development of more effective antiviral inhibitors. Proteins 2007.

Stabilization from Autoproteolysis and Kinetic Characterization of the Human T-cell Leukemia Virus Type 1 Proteinase

We have developed a system for expression and purification of wild-type human T-cell leukemia virus type 1 (HTLV-1) proteinase to attain sufficient quantities for structural, kinetic, and biophysical investigations. However, similar to the human immunodeficiency virus type 1 (HIV-1) proteinase, HTLV-1 proteinase also undergoes autoproteolysis rapidly upon renaturation to produce two products. The site of this autoproteolytic cleavage was mapped, and a resistant HTLV-1 proteinase construct (L40I) as well as another construct, wherein the two cysteine residues were exchanged to alanines, were expressed and purified. Oligopeptide substrates representing the naturally occurring cleavage sites in HTLV-1 were good substrates of the HTLV-1 proteinase. The kinetic parametersk cat and K m were nearly identical for all the three enzymes. Although three of four peptides representing HTLV-1 proteinase cleavage sites were fairly good substrates of HIV-1 proteinase, only two of nine peptides representing HIV-1 proteinase cleavage sites were hydrolyzed by the HTLV-1 proteinase, suggesting substantial differences in the specificity of the two enzymes. The large difference in the specificity of the two enzymes was also demonstrated by inhibition studies. Of the several inhibitors of HIV-1 or other retroviral proteinases that were tested on HTLV-1 proteinase, only two inhibit the enzyme with aK i lower than 100 nm.

Combining mutations in HIV-1 protease to understand mechanisms of resistance.

HIV‐1 develops resistance to protease inhibitors predominantly by selecting mutations in the protease gene. Studies of resistant mutants of HIV‐1 protease with single amino acid substitutions have shown a range of independent effects on specificity, inhibition, and stability. Four double mutants, K45I/L90M, K45I/V82S, D30N/V82S, and N88D/L90M were selected for analysis on the basis of observations of increased or decreased stability or enzymatic activity for the respective single mutants. The double mutants were assayed for catalysis, inhibition, and stability. Crystal structures were analyzed for the double mutants at resolutions of 2.2–1.2 Å to determine the associated molecular changes. Sequence‐dependent changes in protease‐inhibitor interactions were observed in the crystal structures. Mutations D30N, K45I, and V82S showed altered interactions with inhibitor residues at P2/P2′, P3/P3′/P4/P4′, and P1/P1′, respectively. One of the conformations of Met90 in K45I/L90M has an unfavorably close contact with the carbonyl oxygen of Asp25, as observed previously in the L90M single mutant. The observed catalytic efficiency and inhibition for the double mutants depended on the specific substrate or inhibitor. In particular, large variation in cleavage of p6pol‐PR substrate was observed, which is likely to result in defects in the maturation of the protease from the Gag‐Pol precursor and hence viral replication. Three of the double mutants showed values for stability that were intermediate between the values observed for the respective single mutants. D30N/V82S mutant showed lower stability than either of the two individual mutations, which is possibly due to concerted changes in the central P2‐P2′ and S2‐S2′ sites. The complex effects of combining mutations are discussed. Proteins 2002;48:107–116.

Comparison of the effect of FK506 and cyclosporin A on virus production in H9 cells chronically and newly infected by HIV-1

Summary The presence of FK506-binding protein-12 was demonstrated in virions of HIV-1, although its concentration was lower than that of cyclophilin A. The effect of two inhibitors of the peptidyl-prolyl cis-trans isomerases FK506 and cyclosporin A (CsA) was studied in H9 cells that were chronically infe- cted by HIV-1. Both drugs inhibited virus production in the infected cells in a concentration-dependent manner, by decreasing the number of the producing cells. FK506 did not have an effect on Gag processing, based on the p24 antigen content of virions produced in the presence of this drug. Furthermore, FK506 treatment of uninfected H9 cells did not diminish their susceptibility toward HIV-1 infection, whereas CsA treatment decreased the degree of HIV-1 infection with an IC50 of 1–2 μg/ml. Also, pretreatment of the virus with CsA decreased its infectivity in HeLaCD4-LTR/β-gal cells; in contrast, at concentrations up to 10 μg/ml, FK506 did not have an effect. Our findings on the antiviral activity of FK506 and CsA suggest that FK506 is effective only in chronically infected cells, by selectively inhibiting the growth of HIV-1 infected cells, whereas CsA has a specific effect on virus replication.

Comparison of the substrate specificity of two potyvirus proteases.

The substrate specificity of the nuclear inclusion protein a (NIa) proteolytic enzymes from two potyviruses, the tobacco etch virus (TEV) and tobacco vein mottling virus (TVMV), was compared using oligopeptide substrates. Mutations were introduced into TEV protease in an effort to identify key determinants of substrate specificity. The specificity of the mutant enzymes was assessed by using peptides with complementary substitutions. The crystal structure of TEV protease and a homology model of TVMV protease were used to interpret the kinetic data. A comparison of the two structures and the experimental data suggested that the differences in the specificity of the two enzymes may be mainly due to the variation in their S4 and S3 binding subsites. Two key residues predicted to be important for these differences were replaced in TEV protease with the corresponding residues of TVMV protease. Kinetic analyses of the mutants confirmed that these residues play a role in the specificity of the two enzymes. Additional residues in the substrate‐binding subsites of TEV protease were also mutated in an effort to alter the specificity of the enzyme.