Human Immunodeficiency Virus Type 1 Resistance to the Small Molecule Maturation Inhibitor 3- O -(3′,3′-Dimethylsuccinyl)-Betulinic Acid Is Conferred by a Variety of Single Amino Acid Substitutions at the CA-SP1 Cleavage Site in Gag

Abstract

The compound 3-O-(3,3-dimethylsuccinyl)-betulinic acid (DSB) potently and specifically inhibits human immunodeficiency virus type 1 (HIV-1) replication by delaying the cleavage of the CA-SP1 junction in Gag, leading to impaired maturation of the viral core. In this study, we investigated HIV-1 resistance to DSB by analyzing HIV-1 mutants encoding a variety of individual amino acid substitutions in the CA-SP1 cleavage site. Three of the substitutions were lethal to HIV-1 replication owing to a deleterious effect on particle assembly. The remaining mutants exhibited a range of replication efficiencies; however, each mutant was capable of replicating in the presence of concentrations of DSB that effectively inhibited wild-type HIV-1. Mutations conferring resistance to DSB also led to impaired binding of the compound to immature HIV-1 virions and loss of DSB-mediated inhibition of cleavage of Gag. Surprisingly, two of the DSB-resistant mutants retained an intermediate ability to bind the compound, suggesting that binding of DSB to immature HIV-1 particles may not be sufficient for antiviral activity. Overall, our results indicate that Gag amino acids L363 and A364 are critical for inhibition of HIV-1 replication by DSB and suggest that these residues form key contacts with the drug in the context of the assembling HIV-1 particle. These results have implications for the design of and screening for novel inhibitors of HIV-1 maturation.

The structural biology of HIV assembly

“…One class of maturation inhibitors is exemplified by 3-O-(3′,3′-dimethylsuccinyl)betulinic acid (also called PA-457) [113,114], which inhibits HIV-1 replication in tissue culture and animal model systems, and is currently in Phase 2 clinical trials [115]. PA-457 inhibits Gag processing at the CA-SP1 junction and appears to bind directly to this site because the inhibitor is incorporated into assembling virions and because mutations at the CA-SP1 junction induce drug resistance [115][116][117][118]. PA-457 does not bind free Gag, however, and therefore presumably recognizes SP1 in its assembled conformation.…”

Impact of Human Immunodeficiency Virus Type 1 Resistance to Protease Inhibitors on Evolution of Resistance to the Maturation Inhibitor Bevirimat (PA-457)

“…Swapping residues between HIV-1 and SIVmac at the CA-SP1 junction renders SIVmac sensitive to BVM (47). The incorporation of BVM into immature HIV-1 particles has been demonstrated, and this incorporation is reduced by some of the reported BVM resistance mutations (46,48). The observations that BVM does not inhibit the processing of monomeric Gag in solution (21), is specifically incorporated into immature particles (48), and requires immature virus assembly for activity (21,32) suggest that BVM binds an undefined pocket in Gag that is formed upon Gag oligomerization.…”

“…However, several lines of evidence suggest that BVM directly targets the CA-SP1 junction in Gag. All mutations reported thus far to confer resistance to BVM map to the CA-SP1 junction (3,21,22,46,49). HIV-2 and simian immunodeficiency virus from rhesus macaques (SIVmac) are naturally resistant to BVM (49); the amino acid sequence at the CA-SP1 junction of these lentiviruses diverges from that of HIV-1.…”

HIV-1 Maturation Inhibitor Bevirimat Stabilizes the Immature Gag Lattice

“…Additionally, it has been demonstrated that BVM is incorporated into assembling virus particles in a Gag-dependent manner (55). Furthermore, nearly all reported BVM resistance mutations have mapped to the CA-SP1 junction or within SP1 (2,37,38,53,56), and the resistance mutants that have been tested incorporate less BVM into particles (53,55). Finally, BVM is able to prevent cleavage of CA from SP1 only in Gag assembled into particles, not in free Gag in solution (37,46).…”