PA-457: a potent HIV inhibitor that disrupts core condensation by targeting a late step in Gag processing

Abstract

New HIV therapies are urgently needed to address the growing problem of drug resistance. In this article, we characterize the anti-HIV drug candidate 3-O-(3',3'-dimethylsuccinyl) betulinic acid (PA-457). We show that PA-457 potently inhibits replication of both WT and drug-resistant HIV-1 isolates and demonstrate that the compound acts by disrupting a late step in Gag processing involving conversion of the capsid precursor (p25) to mature capsid protein (p24). We find that virions from PA-457-treated cultures are noninfectious and exhibit an aberrant particle morphology characterized by a spherical, acentric core and a crescent-shaped, electron-dense shell lying just inside the viral membrane. To identify the determinants of compound activity we selected for PA-457-resistant virus in vitro. Consistent with the effect on Gag processing, we found that mutations conferring resistance to PA-457 map to the p25 to p24 cleavage site. PA-457 represents a unique class of anti-HIV compounds termed maturation inhibitors that exploit a previously unidentified viral target, providing additional opportunities for HIV drug discovery.

Fig. 1.

(A) The chemical structure of PA-457 (FW 584). (B) In vitro assays show that PA-457 does not affect HIV-1 PR function. After a 30-min incubation, PR-mediated processing of baculovirus-expressed Pr55^Gag in the presence of high concentrations of PA-457 (dissolved in DMSO) is identical to that observed with no compound and compound (DMSO only) controls. Contrast these results with the complete block to PR function observed in the presence of the PR inhibitor indinavir at 0.5 μg/ml.

Fig. 2.

Effect of PA-457 on virus particle production and Gag processing. HeLa cells were transfected with pNL4-3 and cultured in the absence or presence of indicated concentrations of PA-457. Two days posttransfection, cells were metabolically labeled for 2 h with ³⁵S-Met/Cys. Cell lysates (Upper) and virus lysates (Lower) were immunoprecipitated with HIV Ig as described (11). The positions of virally encoded proteins gp160, gp120, Pr55^Gag, p41^Gag, p25, and p24 are indicated. Note the accumulation of p25 in the presence of PA-457.

Fig. 3.

PA-457 does not inhibit the Gag-TSG101 interaction. HeLa cells were transfected with pNL4-3 alone or cotransfected with a 1:1 DNA ratio of pNL4-3 and the TSG-5′ expression vector, pcGNM2/TSG-5′, which expresses an hemagglutinin (HA)-tagged N-terminal TSG101 fragment (11, 32). Cells, either not treated or treated with the indicated concentration of PA-457, were metabolically labeled overnight with ³⁵S-Met/Cys. Virus lysates were immunoprecipitated with HIV Ig (Upper). Virus lysates were also immunoprecipitated with anti-HA antiserum (11) (Lower). The positions of virally encoded proteins p25, and p24 and TSG-5′ are indicated.

Fig. 4.

Thin-section EM analysis of virions produced from PA-457-treated cells. HeLa cells were transfected with pNL4-3 (A and B) or pNL4-3/CA-5 (C) and were not treated (A and C) or treated (B) with PA-457. Two days posttransfection, cells were fixed and analyzed by thin-section EM. Arrowheads in A indicate mature, conical cores; arrows in B and C indicate the crescent-shaped, electron-dense layer inside the viral membrane that results from inhibition of p25 processing. (Bar: ≈100 nm.)

Fig. 5.

Virus selected for resistance to PA-457 contains a single amino acid change in the CA/SP1 cleavage site. (A) Single amino acid mutation was identified in the resistant virus that was not present in the virus passaged in the absence of PA-457, an alanine to valine substitution at the N terminus of SP1 (SP1/A1V). Arrows indicate the site of CA-SP1 cleavage by HIV-1 PR. (B) Effect of PA-457 on Gag processing of the PA-457-resistant mutant SP1/A1V. HeLa cells were transfected with pNL4-3 or the pNL4-3 SP1/A1V mutant and cultured in the absence or the presence of the indicated concentrations of PA-457. Two days posttransfection, cells were metabolically labeled for 2 h with ³⁵S-Met/Cys. Cell lysates (Upper) and virus lysates (Lower) were immunoprecipitated with HIV Ig. The positions of virally encoded proteins gp160, gp120, Pr55^Gag, p41^Gag, p25, and p24 are indicated.