Fullerene Derivatives Prevent Packaging of Viral Genomic RNA into HIV-1 Particles by Binding Nucleocapsid Protein

Abstract

Fullerene derivatives with hydrophilic substituents have been shown to exhibit a range of biological activities, including antiviral ones. For a long time, the anti-HIV activity of fullerene derivatives was believed to be due to their binding into the hydrophobic pocket of HIV-1 protease, thereby blocking its activity. Recent work, however, brought new evidence of a novel, protease-independent mechanism of fullerene derivatives' action. We studied in more detail the mechanism of the anti-HIV-1 activity of N,N-dimethyl[70]fulleropyrrolidinium iodide fullerene derivatives. By using a combination of in vitro and cell-based approaches, we showed that these C₇₀ derivatives inhibited neither HIV-1 protease nor HIV-1 maturation. Instead, our data indicate effects of fullerene C₇₀ derivatives on viral genomic RNA packaging and HIV-1 cDNA synthesis during reverse transcription-without impairing reverse transcriptase activity though. Molecularly, this could be explained by a strong binding affinity of these fullerene derivatives to HIV-1 nucleocapsid domain, preventing its proper interaction with viral genomic RNA, thereby blocking reverse transcription and HIV-1 infectivity. Moreover, the fullerene derivatives' oxidative activity and fluorescence quenching, which could be one of the reasons for the inconsistency among reported anti-HIV-1 mechanisms, are discussed herein.

Keywords: HIV-1; RNA packaging; fullerene; inhibition; nucleocapsid.

Figure 1

Fullerene derivatives and their effects on the HIV-1 life cycle. (a) Structures of fullerene derivatives 1–5. (b) HEK 293 cells produced VSV-G-pseudotyped GFP-HIV-1 in the presence of DMSO or fullerene derivatives at indicated concentrations. At 48 h post-transfection, a normalized amount of the VSV-G pseudotyped GFP-HIV-1 released into the culture media was used for infection of fresh HEK 293 cells, and 48 h later, the GFP-positive cells were quantified using a flow cytometer. (c) Effects of fullerenes on the early stage of HIV-1 life cycle. ELISA-normalized amounts of VSV-G pseudotyped GFP-HIV-1 viruses were used for infection of HEK 293 cells in the presence of DMSO or indicated concentrations of fullerene derivatives. Forty-eight hours later, the GFP-positive cells were quantified using a flow cytometer. (d) Immunoanalysis of intracellular VSV-G pseudotyped GFP-HIV-1 virus produced in HEK 293 cells in the presence of DMSO, fullerene derivatives, or bevirimat at the indicated concentrations. (e) Immunoanalysis of the VSV-G pseudotyped GFP-HIV-1 virus released from the HEK 293 cells.

Figure 2

Effect of fullerene 1 on in vitro and virus-based maturation. (a) Western blot analysis of products of Δ16-99MA-CA-SP1-NC-SP2 and CA-SP1-NC cleavage by HIV-1 PR in the absence or presence of ritonavir. Western blot analysis of maturation of (b) MA-CA-SP1-NC-SP2 and (c) CA-SP1-NC mediated by HIV-1 PR in the absence or presence of 5 µM fullerene 1. (d) Expression (pulse), maturation, and release (pulse chase and chase) of HIV-1 Gag and Gag–Pol polyproteins. HEK 293 cells were pulse-labeled for 30 min with Tran35S-label and chased overnight in complete DMEM. Viral proteins from the cell lysates (pulse and pulse-chase) and media (chase) were immunoprecipitated by anti-HIV CA antibody. Proteins were separated by SDS-PAGE and detected by using a Typhoon PhosphorImager.

Figure 3

Effect of fullerene 1 on the assembly of immature and mature HIV-1 particles. (a) Representative TEM pictures of negatively stained particles assembled from (panels (A–D)) Δ16-99MACANCSP2 and (panels (E–H)) CANC in the absence of tqON (panels (A,E)), in the presence of tqON (panels (B,F)), in the presence of fullerene 1 (panels (C,G)), and in the presence of assembly inhibitor CAI (panels (D,H)). (b) TEM analysis of HEK 293 cell producing VSV-G-HIV-1 in the absence (A,C) or presence of fullerene 1 (B,D). Bars represent 100 nm.

Figure 4

The effects of fullerene 1 on the selected steps of early phase of HIV-1 replication cycle—HIV-1 uncoating. (a,b) CsA washout assay. OMK cells were infected by normalized amounts of VSV-G pseudotyped HIV-1 produced in the presence of indicated concentration of fullerene 1, or 5 µM PF74 was added. At the indicated times post-infection, the CsA-containing medium was replaced with fresh, CsA-free culture medium. The percentage of GFP-positive OMK cells was determined by flow cytometry and (a) normalized to the number of DMSO-containing non-drug control cells, which was considered 100%, or (b) normalized by setting the number of GFP-positive cells to 100% for HIV-1 reverse transcription. Progress of reverse transcription, (c) early stage, (d) intermediate stage, and (e) late stage. VSV-G pseudotyped HIV-1 particles produced in the absence or presence of 5 µM fullerene 1 were used to infect fresh HEK 293 cells, which were harvested 2, 4, 6, 10, 24, 33, and 48 h post-infection. Real time PCR analysis of isolated DNA was used to detect different reverse transcription products. The results were normalized for CA content using semi-quantitative Western blotting and two housekeeping genes: phospholipase A and C-C chemokine receptor type 5—CCR5. (f) Activity of reverse transcriptase was measured using a reverse transcriptase assay, colorimetric. Released virions produced in HEK 293 cells in the presence of indicated concentrations of fullerene 1 were harvested and centrifuged 48 h post-transfection. The subsequent steps were performed according to the manufacturer’s protocol (Merck). (g) Amount of viral gRNA incorporated into HIV-1 particles. The amount of gRNA from HIV-1 particles released from HEK 293 cells treated by DMSO or the indicated amount of 1 was quantified by real-time PCR analysis. If not stated otherwise, results represent two independent experiments.

Figure 5

Interactions of fullerene 1 with HIV-1 CANC, CA, NC, and PR analyzed by microscale thermophoresis measurement (MST) and electrophoretic mobility shift assay (EMSA). (a) SDS PAGE of HIV-1 proteins used in MST and EMSA analysis. (b) HIV-1 PR following THS-RED labeling was tested for its proteolytic activity using the indicated samples. (c) Dose–response plot of different concentrations of fullerene 1 after the interaction with fluorescently labeled HIV-1 proteins and nucleic acids calculated from MST analysis. Error bars represent standard deviations. (d) Dissociation constants (K_D) calculated from MST for fullerene 1 and indicated HIV-1 proteins. (e) EMSA: the same amounts of DNA (200 ng) and HIV-1 protein (1 μM) were used in all samples. The concentrations of 1 (1, 2, and 5 μM) at the final molar ratios of protein:fullerene 1 corresponding to 1:1, 1:2, and 1:5 were used. Orange panel: DNA was incubated with DMSO (1%) (lane 1) or with various amounts of 1, corresponding to 1, 2, and 5 μM in 1% DMSO (lanes 2–4); green panel: HIV-1 CA was incubated with DNA (lane 5) or with various amounts of 1, corresponding to 1, 2, and 5 μM (lanes 6–8). To analyze the interactions among HIV-1 NC, 1, and nucleic acid (blue panel), two experiments were performed. In one, NC was first incubated with DNA and then with 1 (1, 2, or 5 μM) at the final molar ratios 1:1, 1:2, and 1:5 (lanes 12, 15, 18 respectively). In the second, NC was first preincubated with the various amounts of 1, and then incubated with DNA (lanes 13, 16, and19). As controls, NC was incubated with DMSO (1%) in the absence of 1 (lane 10), and DNA was incubated with the various amounts of 1 (lanes 11, 14, and 17). CANC’s interactions with 1 and DNA (yellow panel) were tested identically as described for NC: CANC was preincubated with DNA, and then with 1 (lanes 23, 26, and 29), or CANC was preincubated with various amounts of 1, and then incubated with DNA (lanes 24, 27, and 30). DNA binding to various amounts of 1 was also analyzed (22, 25, and 28). All samples were incubated for 40 min at RT and then analyzed using 0.8% agarose gel electrophoresis, stained with Gel Red, and visualized with a Quantum gel documentation imaging system (Vilbert Lourmat).

Figure 6

Docking of fullerene 1 into HIV-1 NC. Docking into NC (PDB 1F6U) was carried out in two programs, PLANTS and AutoDock Vina: (a) Heat map of NC with docked fullerene 1 (blue: hydrophilic, gold: hydrophobic). (b) A detailed illustration of fullerene 1 and the NC hydrophobic plateau formed by Val13, Phe16, Ile24, and Ala25 of the NC proximal ZF (the hydrophobic residues are drawn in capped sticks and marked by labels).