Antiviral peptides targeting the west nile virus envelope protein

Abstract

West Nile virus (WNV) can cause fatal murine and human encephalitis. The viral envelope protein interacts with host cells. A murine brain cDNA phage display library was therefore probed with WNV envelope protein, resulting in the identification of several adherent peptides. Of these, peptide 1 prevented WNV infection in vitro with a 50% inhibition concentration of 67 muM and also inhibited infection of a related flavivirus, dengue virus. Peptide 9, a derivative of peptide 1, was a particularly potent inhibitor of WNV in vitro, with a 50% inhibition concentration of 2.6 muM. Moreover, mice challenged with WNV that had been incubated with peptide 9 had reduced viremia and fatality compared with control animals. Peptide 9 penetrated the murine blood-brain barrier and was found in the brain parenchyma, implying that it may have antiviral activity in the central nervous system. These short peptides serve as the basis for developing new therapeutics for West Nile encephalitis and, potentially, other flaviviruses.

FIG. 1.

(a and b) Peptide (P1, P2, and P3) inhibition of WNV infection in Vero cells at 24 h (a) and 48 h (b). P1 (1 mM) significantly (P < 0.03) inhibited WNV infection at 24 h, as measured by Q-PCR. (c and d) Q-PCR (c) and PRNT (d) demonstrated that DMSO does not influence the inhibition of WNV. DMSO groups contained the same concentration of DMSO as the 1 mM P1 sample. Statistical significance for Q-PCR: overall, P < 0.02; 1 mM versus PBS, P < 0.05; DMSO versus PBS, P > 0.05. For PRNT: overall, P < 0.0001; 1 mM, 100 μM, and 10 μM versus PBS, P < 0.001; DMSO versus PBS, P > 0.05. Any average value below 0 is considered no inhibition. All the experiments were repeated at least twice, and all the measurements were made in triplicate, with the mean ± standard deviation shown. Statistical analysis was done by one-way analysis of variance, in some cases followed by Tukey's test.

FIG. 2.

Evaluation of peptides derived from P1 against WNV infection of cultured Vero cells. (a) Selected peptides (P8, P9, P10, and P11) inhibited viral infectivity in Vero cell cultures. Two concentrations (100 μM and 10 μM) of each peptide were tested, and 100 μM P1 was included as a positive control. WNV RNA levels 24 h postinfection were determined by Q-PCR. All the groups significantly inhibited WNV infectivity compared with the DMSO vehicle-only control. *, P < 0.01; **, P < 0.001. (b and c) P9 inhibited WNV infection in Vero cell cultures in a dose-dependent manner as determined by both Q-PCR (b) and by a plaque formation assay (c) for viral titers in the cell supernatant 24 h postinfection. (d) P9 reduced WNV attachment or entry into Vero cells after 1 h of binding at 4°C (P < 0.001). (e and f) P9 inhibited WNV infection in HeLa cell cultures as measured both by Q-PCR (e) and a plaque formation assay (f) for viral titer in the cell supernatant. (g) P9 inhibited WNV infection in cultured Neuro 2a cells as measured by Q-PCR. (h) P9 did not inhibit WNV in C6/36 cells in the plaque formation assay (P = 0.96). All the experiments were repeated at least twice, and all the measurements were made in triplicate, with the mean ± standard deviation shown. Data were analyzed by one-way analysis of variance followed by Tukey's test.

FIG. 3.

P1 and P9 inhibition of WNV or dengue virus infection of Vero cells. (a) Dose-response curves of P1 (IC₅₀ of 67 μM) and P9 (IC₅₀ of 2.6 μM) against WNV infection in vitro. Serially diluted P1 and P9 were preincubated with 100 PFU of WNV for 1 h at room temperature and then examined in a PRNT. Plots were made using GraphPad Prism. (b) P1 inhibited dengue virus infection in Vero cells, as measured by Q-PCR 24 h postinfection. All the measurements were made in triplicate, with the mean ± standard deviation shown. (c) P1 and P9 are not cytotoxic. Vero cells were exposed to peptide (1 μM to 500 μM) for 72 h, and LDH levels in cell supernatants were monitored. Each sample was assayed in duplicate, and data represent the mean ± standard deviation. No statistical difference was observed by one-way analysis of variance (P > 0.05). (d) Scrambled P9 showed no inhibition compared with P9, at either 100 μM or 10 μM, as measured by PRNT. (e) Cyclized P9 (at ≤100 μM) did not demonstrate inhibitory activity. Serially diluted peptides were tested and compared between P9 (linear) and P9 cyclized with an intramolecular disulfide bond. Any average value below zero in panels b and e is considered no inhibition. P9 binds to WNV E protein. (f and g) P9 binds to WNV E protein with a K_d of approximately 6 μM. P2 did not show affinity for the WNV E protein. The black line indicates the theoretical fit. All the experiments were repeated at least twice.

FIG. 4.

P9 inhibited WNV infectivity in vivo. (a) Mixing P9 and WNV prior to challenge significantly reduced murine viremia at day 3 (P < 0.0001; Student's t test). (b) Indirect immunofluorescence revealed a reduced viral burden in brains of P9-treated mice. The red signal represents the WNV E protein, and the green signal indicates microtubule-associated protein 2 (MAP2; a neuron marker). Bar, 100 μm. (c) Preincubation of P9 with WNV significantly increased murine survival (n = 35; P < 0.0001, log rank test). P2 was used as a noninhibitory control peptide. Data were combined from seven independent experiments. Control mice were treated with noninhibitory P2. (d) P9 entered the brain after i.p. injection. Two hundred μl of 1 mM biotin-labeled P9 was injected via the i.p. route of administration. Mice were sacrificed at various time points postinjection, and brain and spleen tissues were collected after perfusion. Biotin-labeled P9 was detected using an enzymatic tyramide signal amplification method. Biotin-labeled P9 was detected in the cerebellum (cblm) and in the cerebral cortex (ctx). At 6 h, most of the signal was associated with cerebral blood vessels, but thereafter signal relocalized into the brain parenchyma. (e) Biotin-labeled P9 was detected in the spleen as early as 6 h postinjection and declined in intensity thereafter.