Fullerene Derivatives Strongly Inhibit HIV-1 Replication by Affecting Virus Maturation without Impairing Protease Activity

Abstract

Three compounds (1, 2, and 3) previously reported to inhibit HIV-1 replication and/or in vitro activity of reverse transcriptase were studied, but only fullerene derivatives 1 and 2 showed strong antiviral activity on the replication of HIV-1 in human CD4(+) T cells. However, these compounds did not inhibit infection by single-round infection vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped viruses, indicating no effect on the early steps of the viral life cycle. In contrast, analysis of single-round infection VSV-G-pseudotyped HIV-1 produced in the presence of compound 1 or 2 showed a complete lack of infectivity in human CD4(+) T cells, suggesting that the late stages of the HIV-1 life cycle were affected. Quantification of virion-associated viral RNA and p24 indicates that RNA packaging and viral production were unremarkable in these viruses. However, Gag and Gag-Pol processing was affected, as evidenced by immunoblot analysis with an anti-p24 antibody and the measurement of virion-associated reverse transcriptase activity, ratifying the effect of the fullerene derivatives on virion maturation of the HIV-1 life cycle. Surprisingly, fullerenes 1 and 2 did not inhibit HIV-1 protease in an in vitro assay at the doses that potently blocked viral infectivity, suggesting a protease-independent mechanism of action. Highlighting the potential therapeutic relevance of fullerene derivatives, these compounds block infection by HIV-1 resistant to protease and maturation inhibitors.

FIG 1

Structures of fullerene derivatives 1 and 2, C₆₀-bis(N,N-dimethylpyrrolidinium iodide), derivative 3, fullereno-C₆₀-pyrrole-2,5-dicarboxylic acid-1-(carboxymethyl)-1,5-dihydro, and derivative 4, cis-2-C₆₀-bis(N,N-ethylmethylpyrrolidinium iodide).

FIG 2

Effects of fullerene derivatives on HIV-1 replication. SupT1 cells were treated with DMSO (+) or compound 1 (a) and the regioisomeric mixture 2 (b) at 1 μM (■) or 3 μM (Δ) at the time of infection with HIV-1 NL4-3. Twenty-four hours later, the compounds and virus were removed, and infected cells were cultured for up to 2 weeks. The amount of HIV-1 p24 antigen was determined in cell supernatant by ELISA. Results from one experiment are shown.

FIG 3

Assessing the cytotoxicity of compounds 1 and 2. SupT1 cells were treated with DMSO, compound 1 (■), or regioisomeric mixture 2 (×) at various concentrations for 24 h, and the amounts of viable cells were determined by the tetrazolium dye reduction assay. Cell viability values were normalized to DMSO-treated cells. Results shown are representative of two independent experiments.

FIG 4

Analysis of the viral life cycle step affected by compounds 1, 2, 3, and 4. (a) Effects on the early stages of the HIV-1 viral life cycle. SupT1 cells were infected with single-round infection HIV-1 viruses (c) in the presence of DMSO or fullerene derivatives (10 μM) and analyzed for luciferase expression and cellular viability (ATP content) 3 days later. Luciferase was normalized to cellular viability. (b) Effects on the late phase of the HIV-1 infection. Single-round infection HIV-1 was produced in the presence of DMSO, indinavir (Ind; 0.1 μM), or fullerene derivatives (3 μM), and their infectivity was analyzed in single-round infection assays using HIV-1 p24-normalized viruses. Results shown are the averages and standard deviations of triplicate readings of one experiment representative of three independent experiments. (c) The HIV-1 reporter virus used was previously described (26, 27, 51) and includes a frameshift mutation in vpr (circle) and a deletion of 430 nucleotides in env (oval); the luciferase open reading frame is replacing nef.

FIG 5

Effects of compounds 1, 2, 3, and 4 on virion production. VSV-G-pseudotyped, single-round HIV-1 expressing luciferase was produced in the presence of DMSO or fullerene derivatives (3 μM) and then concentrated by ultracentrifugation and analyzed. Indinavir (Ind; 0.1 μM) was used as a control. (a) Virion production as determined by HIV-1 p24 levels quantified by ELISA. (b) Virion-associated RNA as quantified by real-time PCR analysis of reverse-transcribed cDNA using primers that hybridize to Gag. Results represent two experiments; standard deviations indicate the variability of multiple readings.

FIG 6

Evaluation of the early steps of the HIV-1 life cycle of virions produced in cells treated with compound 1 or 2. SupT1 cells were infected with HIV-1 p24-normalized, DNase-treated, single-round HIV-1 viruses produced in the presence of DMSO, indinavir (0.1 μM), or fullerene derivatives (3 μM). DNA was extracted from infected cells 24 h (a) and 4 days (b) postinfection and used to detect total HIV cDNA (I), 2LTR junctions (II), and proviruses (III). Results shown are the averages of triplicate readings of one experiment.

FIG 7

Effects of compounds 1 and 2 on Gag and Gag-Pol processing. Protease-mediated processing of capsid (p24) (a) and integrase (b) was evaluated in virions by immunoblotting. (c) Reverse transcriptase (RT) activity of virions was measured by the exogenous reverse transcription assay. Results are representative of one (a and b) or three (c) independent experiments.

FIG 8

Effect of compounds 1 and 2 on the in vitro activity of HIV-1 protease. The cleavage of an HIV-derived FRET peptide by recombinant HIV-1 protease in the presence of compounds 1 (Δ) and 2 (×) at 3 μM (a) or 10 μM (b) was determined by fluorescence measurements. DMSO (◆) and indinavir (■) were used as negative and positive controls, respectively. Experiments were performed in duplicates.

FIG 9

Activity of compound 1 on the infectivity of HIV-1 harboring multi-protease-inhibitor-resistant protease mutants. (a) HIV-1 viruses harboring wild-type or mutant protease were produced in the presence of DMSO, compound 1 (3 μM), or indinavir (0.1 μM), and their infectivity was evaluated in single-round infection assays. (b and c) SupT1 cells were infected with HIV-1 NL4-3 harboring a wild-type (b) or a multi-protease-inhibitor-resistant protease mutant (virus 11803) (c) in the presence of DMSO (●) or compound 1 (3 μM) (■). (d) Infectivity in TZM-bl cells of HIV-1-harboring CA mutants resistant to maturation inhibitors (lanes 1 and 2) or wild-type HIV-1 (lane 3) produced in the presence of DMSO or fullerene 1. Results are representative of one (a), two (b), more than four (c), or three (d) independent experiments.