In Vitro Evolution of the Human Immunodeficiency Virus Type 1 Gag-Protease Region and Maintenance of Reverse Transcriptase Resistance following Prolonged Drug Exposure

King

et al. 2004

Maturation of human immunodeficiency virus (HIV) depends on the processing of Gag and Pol polyproteinsby 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.Human immunodeficiency virus type 1 (HIV-1) matures after the viral protease processes (35) the Gag and Pol polyproteins at 10 substrate locations (3, 15). Therefore, inhibition of HIV-1 protease represents an important avenue for antiviral therapy (13, 48). The substrate sequences cleaved by the protease are nonhomologous (3), with the sequence of the nucleocapsid-p1 (NC-p1) substrate being the most different. In spite of the poor sequence homology among the substrate sites, a series of substrate-protease crystal structures led us to hypothesize that substrate specificity in HIV-1 protease results from the enzyme's recognizing an asymmetric shape (or envelope) rather than a particular amino acid sequence (40). This shape results from the conformation that a particular substrate sequence can adopt, implying that an interdependency necessarily exists among the different substrate residue sites.All of the protease inhibitors whose designs were structure based bind competitively (8,18,28,52,53) at the active site. Since these inhibitors bind at the same site as the substrates, many protease residues contact both substrates and inhibitors. Drug resistance, which often develops in the presence of therapeutic protease inhibitors, results from high viral turnover, the infidelity of the viral reverse transcriptase (16,42,43), and selective pressure on the virus. With drug resistance, the protease no longer binds as tightly to inhibitors but r...

Section: Discussionmentioning

confidence: 99%

Section: Maturation Of Human Immunodeficiency Virus (Hiv) Depends On mentioning

confidence: 99%

mentioning

confidence: 99%

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Structural Basis for Coevolution of a Human Immunodeficiency Virus Type 1 Nucleocapsid-p1 Cleavage Site with a V82A Drug-Resistant Mutation in Viral Protease

King

et al. 2004

“…Such coevolution has occasionally been seen, although it has been primarily limited to the NC-p1 cleavage site. In this case, the P2 residue mutates from an alanine to a valine in the presence of the V82A protease drug-resistant mutation (14,21,46). These results are encouraging in that the virus may have more difficulty in developing resistance to TMC114 than to other protease inhibitors; however, with the heterogeneity of HIV in vivo this will be confirmed only when extensive clinical trials are concluded.…”

Section: Discussionmentioning

confidence: 99%

Structural and Thermodynamic Basis for the Binding of TMC114, a Next-Generation Human Immunodeficiency Virus Type 1 Protease Inhibitor

King

et al. 2004

225

284

TMC114, a newly designed human immunodeficiency virus type 1 (HIV-1) protease inhibitor, is extremely potent against both wild-type (wt) and multidrug-resistant (MDR) viruses in vitro as well as in vivo. Although chemically similar to amprenavir (APV), the potency of TMC114 is substantially greater. To examine the basis for this potency, we solved crystal structures of TMC114 complexed with wt HIV-1 protease and TMC114 and APV complexed with an MDR (L63P, V82T, and I84V) protease variant. In addition, we determined the corresponding binding thermodynamics by isothermal titration calorimetry. TMC114 binds approximately 2 orders of magnitude more tightly to the wt enzyme (K d ‫؍‬ 4.5 ؋ 10 ؊12 M) than APV (K d ‫؍‬ 3.9 ؋ 10 ؊10 M). Our X-ray data (resolution ranging from 2.2 to 1.2 Å) reveal strong interactions between the bis-tetrahydrofuranyl urethane moiety of TMC114 and main-chain atoms of D29 and D30. These interactions appear largely responsible for TMC114's very favorable binding enthalpy to the wt protease (؊12.1 kcal/mol). However, TMC114 binding to the MDR HIV-1 protease is reduced by a factor of 13.3, whereas the APV binding constant is reduced only by a factor of 5.1. However, even with the reduction in binding affinity to the MDR HIV protease, TMC114 still binds with an affinity that is more than 1.5 orders of magnitude tighter than the first-generation inhibitors. Both APV and TMC114 fit predominantly within the substrate envelope, a property that may be associated with decreased susceptibility to drug-resistant mutations relative to that of first-generation inhibitors. Overall, TMC114's potency against MDR viruses is likely a combination of its extremely high affinity and close fit within the substrate envelope.

“…Further studies provided structural insights into why a prime drug-resistant mutation, V82A (5,8,10,31,46), has less effect on substrate binding than on inhibitor binding as Val82 interacts more closely with the drugs than with natural substrates (41). The nucleocapsid-p1 (NC-p1) substrate, however, coevolves (AlaP2Val) in a correlated manner with the V82A mutation (3,6,9,23,56). Processing of the NC-p1 substrate is the slowest and rate-determining cleavage step in the maturation of Gag (38,50,55).…”

mentioning

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Mechanism of Substrate Recognition by Drug-Resistant Human Immunodeficiency Virus Type 1 Protease Variants Revealed by a Novel Structural Intermediate

Romano

et al. 2006

Maturation of human immunodeficiency virus type 1 (HIV-1) is achieved by the proteolytic processing of Gag and Gag-Pol sites at at least 10 nonhomologous substrate sites by a viral protease (4,14,36). This processing leads to the release of viral enzymes and structural proteins. In the absence of this proteolysis, immature noninfectious virions are produced (7). For this reason, HIV-1 protease is a prime target for antiviral therapy (48, 54).The three-dimensional structure of this 22-kDa dimeric protease comprises a terminal region, an active site, and a core domain (29,53). The active site residues are located at the dimer interface, with one catalytic aspartate (Asp25/25Ј) donated by each monomer (53, 54). The substrates bind the active site in an extended conformation forming mainly backbone hydrogen bonds (39, 51). On the opposite side of Asp25, two ␤-hairpins, known as the flaps, one from each monomer, wrap around the substrates. In addition to its interactions with the substrates, the flap tips (Ile50-Gly51) also participate in intermolecular interactions. As a common feature of aspartyl proteases for ligand binding to occur, several structural rearrangements must take place (13,15,24,28,45). The flaps open to allow substrate binding, and upon substrate recognition, they must close to attain the canonical "closed" conformation of HIV-1 protease. Whether the flap movements are synchronized between the monomers, as HIV-1 protease is homodimeric, or whether they move in an asynchronous fashion, as found in molecular dynamics simulations (22,37,45), is still to be verified structurally. The opening and the closing of the flaps are not likely random events but involve a few or several discrete structural intermediates. Nuclear magnetic resonance (NMR) experiments proposed that an ensemble of flap conformations are possible between open and closed stages of the flaps (12, 16). Whether other regions of the protease also move in concert with the flaps is not known. However, trapping any of these intermediates in a crystal is a challenge.Our crystallographic investigations of wild-type (WT) and drug-resistant variants of HIV-1 protease complexed with several of its substrates have revealed a conserved shape we defined as the "substrate envelope," which we hypothesize is crucial for substrate specificity (39)(40)(41)(42). Further studies provided structural insights into why a prime drug-resistant mutation, V82A (5,8,10,31,46), has less effect on substrate binding than on inhibitor binding as Val82 interacts more closely with the drugs than with natural substrates (41). The nucleocapsid-p1 (NC-p1) substrate, however, coevolves (AlaP2Val) in a correlated manner with the V82A mutation (3,6,9,23,56). Processing of the NC-p1 substrate is the slowest and rate-determining cleavage step in the maturation of Gag (38,50,55). Unlike in other substrate-V82A protease complexes, PheP1Ј forms hydrophobic interactions with Val82, which are lost in the V82A complex (40). The AlaP2 in WT NC-p1 does not fill the S2 pocket, which is t...