Hepatitis C virus NS5A anchor peptide disrupts human immunodeficiency virus

Abstract

In the absence of an effective vaccine, there is an urgent need for safe and effective antiviral agents to prevent transmission of HIV. Here, we report that an amphipathic alpha-helical peptide derived from the hepatitis C virus NS5A anchor domain (designated C5A in this article) that has been shown to be virocidal for the hepatitis C virus (HCV) also has potent antiviral activity against HIV. C5A exhibits a broad range of antiviral activity against HIV isolates, and it prevents infection of the three in vivo targets of HIV: CD4(+) T lymphocytes, macrophages, and dendritic cells by disrupting the integrity of the viral membrane and capsid core while preserving the integrity of host membranes. C5A can interrupt an ongoing T cell infection, and it can prevent transmigration of HIV through primary genital epithelial cells, infection of mucosal target cells and transfer from dendritic cells to T cells ex vivo, justifying future experiments to determine whether C5A can prevent HIV transmission in vivo.

Fig. 1.

C5A prevents HIV infection of primary T lymphocytes (Left), macrophages (Center), and DC (Right). Blood-derived CD4⁺ T lymphocytes, macrophages, or DC (0.5 × 10⁶ cells) were exposed to NL4.3-BaL (1 ng of p24) and C5A or control peptide (5 μM) for 1 day at 37°C and washed. The amount of virus released was monitored by quantitating particulate p24 capsid protein in cell supernatants by ELISA. Error bars represent standard errors of duplicates.

Fig. 2.

C5A destabilizes HIV particles. (a) NL4.3 virus (20 ng of p24) was incubated with or without C5A for 30 min at 37°C and loaded over a sucrose density gradient (see Materials and Methods). Each gradient fraction was analyzed for capsid by p24 ELISA (Top) and immunoblot, RT by exoRT assay (Middle), and gp41 content by immunoblot. (b) Same as a except that the virus was treated with decreasing concentrations of C5A for 30 min at 37°C (Upper Left), with C5A for 15, 30, and 60 min at 37°C (Lower Left), for 30 min at 4, 25, or 37°C (Upper Right), and for 30 min at pH 8, 7, 6, and 5 (Lower Right). Gradient fractions were analyzed for HIV capsid content by p24 ELISA. (c) Same as a except that the virus was treated either with C5A or control peptide. (d) Same as a except that the virus was first trypsinized for 15 min at 37°C, incubated in 10% FCS to neutralize trypsin, microcentrifuged for 90 min at 4°C, resuspended, and loaded over a sucrose gradient for virus integrity evaluation by p24 ELISA.

Fig. 3.

Antiviral effect of C5A before, during, or after viral exposure. C5A or control peptide (5 μM) was added to TZM cells 1, 2, or 4 h before (t = −1, −2, and −4) or after (t = +1, +2, + 4, and + 8) adding HIV-1 (X4 NL4.3) (1 ng of p24) or together (t = 0) with the virus. Infection was measured 48 h after infection as β-galactosidase activity. Error bars represent standard errors of duplicates.

Fig. 4.

C5A inhibits both HIV transmigration and transmission. (a) Either cell-free (20 ng of p24) or cell-associated NL4.3 (100,000 HIV-infected Jurkat T cells washed twice just before being added to the upper chamber) was added to the apical surface of PGEC. C5A or the control peptide (5 μM) was added to PGEC 30 min after adding the virus. Amounts of transcytosed viruses in the basal chamber were quantified by p24 ELISA. Results are expressed in percentage of p24 of the original inoculum. Error bars represent standard errors of duplicates. (b) PGEC were treated twice daily with 200 μM C5A or control peptide or 0.01% saponin for a week. CellQuanti-MTTTM reagent was added, and cell viability was quantified by A₅₇₀ reading. (c–e) Epidermal sheets were infected at 37°C with NL4.3-BaL-eGFP (100 ng of p24). After 4 h, C5A, control peptide (10 μM) or medium (0.1% DMSO) was added to sheets. After 3 days, migrated cells, which exfoliated from the epidermal sheets, were isolated, washed, and incubated with Jurkat T cells (100,000 cells). Infection of migrated cells (day 3) or T cells in LC-T coculture (day 5) were analyzed for GFP expression by FACS. (c) Percentage of HIV-infected migrated cells (mainly LC) at day 3. (d) Percentage of HIV-infected Jurkat T cells (gated by using an anti-CD3 antibody) in LC-T coculture was analyzed by GFP expression on day 5. Error bars represent standard errors of duplicates. (e) DC (100,000 cells) were incubated for 2 h at 37°C with wild-type NL4.3-eGFP (X4 virus) and NL4.3-BaL-eGFP (R5 virus) viruses or with the pseudotyped NL4.3ΔEnv-eGFP/gp160 X4 Env virus (25 ng of p24). C5A or control peptide (10 μM) was added 2 h later. DC were washed 2 h after adding the peptide, Jurkat T cells (100,000 cells) were added for 3 days, and the percentage of infected Jurkat T cells was analyzed as described in d. Error bars represent standard errors of duplicates.

Fig. 5.

C5A blocks an ongoing HIV infection. (a) Primary CD4⁺ T lymphocytes (0.5 × 10⁶ cells) were exposed to NL4.3-BaL (1 ng of p24) for 1 day at 37°C and washed. Three days after infection, C5A or control peptide (5 μM) was added to infected cells for 2 h and washed. Infected cell culture supernatants were collected every 3 days, normalized for particulate p24 content, and tested for infectivity (1 ng of p24) on TZM reporter cells. Error bars represent standard errors of duplicates. (b) 293T cells (1 × 10⁶ cells) transfected with 1 μg of proviral NL4.3 or NL4.3-BaL DNA for 24 h at 37°C were treated with or without C5A or control peptide (5 μM) for 1 h at 37°C and washed. Twenty-four hours after peptide treatment, supernatants of transfected 293T cells containing newly formed viruses were tested for infectivity as described in a. Error bars represent standard errors of duplicates.