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

Abstract

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...

Gag Determinants of Fitness and Drug Susceptibility in Protease Inhibitor-Resistant Human Immunodeficiency Virus Type 1

“…In contrast, the coevolved Gag protein and PI-resistant protease act synergistically, suggesting that they function together, as has been shown for the A431V CSM. The A431V CSM has been shown to function due to increased contact of the cleavage site with the enzyme, but only in the presence of resistance mutations in protease (32). However, our results and those of others suggest that CSM can function as PI resistance mutations in the absence of protease mutations, both after in vitro selection (28) and in clinical isolates (our results and those of Dam et al [8]).…”

Structural and Thermodynamic Basis of Amprenavir/Darunavir and Atazanavir Resistance in HIV-1 Protease with Mutations at Residue 50

“…The WT protease gene was generated as previously described (20), with the Q7K substitution introduced to prevent autoproteolysis (21). I50V/A71V and I50L/A71V variants were generated by introducing the appropriate mutations into the wild-type gene by site-directed mutagenesis using a Stratagene QuikChange site-directed mutagenesis kit (Agilent Technologies, La Jolla, CA).…”

The Effect of Clade-Specific Sequence Polymorphisms on HIV-1 Protease Activity and Inhibitor Resistance Pathways

“…The clade B wild-type (B-WT) protease gene was generated as previously described (34). The AE wild-type (AE-WT) protease gene was synthesized in fragments (Integrated DNA Technologies, Coralville, IA), with codons optimized for expression in Escherichia coli.…”