A schematic representation of the alphavirus genome is shown. PPMO were designed to target the terminal 5′ and 3′ untranslated regulatory regions (UTR) of alphavirus genomic (+) and antigenomic (−) RNA (labeled A-D), as well as the AUG translation start site region of the genomic (labeled E), and subgenomic promoter region of the antigenomic (labeled F), RNA. Sindbis virus (SINV)- and Venezuelan equine encephalitis virus (VEEV)-specific PPMO name designations and sequences are shown in Table 1.

BHK cells were infected with SinLuc at a multiplicity of infection (moi) of ~0.03, followed by treatment with the indicated concentrations of SINV AUG, SINV sgP or Scr PPMO (left) or SINV genomic termini targeting PPMO (right) for 24 h. Luciferase (Luc) levels were measured in triplicate, as described in Materials and Methods. The average and standard deviation are shown. Statistical analysis of luciferase levels of SINV PPMO vs Scr treatment (two-way ANOVA, α=0.05; pairwise comparison via Bonferroni post-test) was performed and results are described in the text. B) Dose-response inhibition of wild type SINV. Panel i–ii) Effect of SINV PPMO on SINV production. BHK cells were infected with SINV at an moi of ~0.03 and treated at 1 hpi with the indicated PPMO (1 or 5 μM, panel i and ii, respectively). At 24 hpi, the virus levels in supernatant samples was determined by plaque assay. Supernatants were analyzed in quadruplicate (1 μM) or triplicate (5 μM). The results of one representative experiment out of two performed is shown. Statistical analysis was performed using one-way ANOVA (α=0.05) with Dunnett’s Multiple Comparison Test in pairwise comparison of each PPMO treated group relative to the mock control. Statistically significant differences in Dunnett’s test are indicated by an asterisk (*, p-value<0.01) and the percent reduction is shown to the left. Panel iii) Effect of SINV PPMO on two control viruses, VSV and TC83. Vero cells were infected with VSV or VEEV (TC83) at an moi of 0.1 and treated at 1 hpi with the indicated PPMO at 5 μM. At 8 and 24 h, the virus production levels were determined by plaque assay. Viral titration of untreated, infected Vero cells (“No PPMO”) was also performed. The result of a single experiment is shown. C) Inhibition of viral protein expression by SINV-specific PPMO. BHK cells were treated with the indicated PPMO starting at 1 h after infection with SINV (moi of 0.03). At 24 hpi the cells were incubated with ³⁵S-methionine for one hour, and cell lysates then analyzed by SDS-PAGE. The molecular weight marker is shown. Viral capsid (C) and envelope protein (E1) and apparent molecular mass are indicated by the arrows. The percentage of C expression, relative to the mock-treated control, as determined by densitometry, is shown numerically under the lanes. D) Inhibition of viral translation by SINV-specific PPMO. In vitro transcribed SINV RNA was translated in rabbit reticulocyte lysates in the presence of a molar excess of the indicated PPMO as described in Materials and Methods. Samples were separated by SDS-PAGE and visualized by phosphor imager.

Nine-week-old NIH Swiss mice were treated with VEEV-specific P7-PPMO (combined VEEV AUG and VEEV 5′+) or the control Scr P7-PPMO at a dose of 40 μg via intranasal (i.n.) route and 160 μg via subcutaneous (s.c.) route at time points pre- (2 doses: -24 hr and -4 hr, indicated as “+pre”) and/or post-infection (5 doses: daily on day +1 through +5, indicated as “+post”). Treatment groups are indicated in the figure legend, as follows: 1) AUG & 5′+ (+pre, +post)/no VEEV: VEEV-specific PPMO at time points prior to (+pre) and following (+post) infection; graph symbol ○, 2) No PPMO/+VEEV: no treatment of VEEV-infected mice; graph symbol +, 3) Scr (+pre, +post)/+VEEV: Scr treatment at pre- and post-infection time points; graph symbol ◆, 4) AUG & 5′+ (+pre, +post)/+VEEV: VEEV-specific PPMO at pre- and post-infection time points; graph symbol ▲, 5) AUG & 5′+ (−pre, +post)/+VEEV: VEEV-specific PPMO at post-infection time points only; graph symbol △. On day 0, mice were infected via i.n. route with virulent VEEV (ZPC738) at a dose of 10³ PFU per animal (group 2–6). As a control for P7-PPMO toxicity, group 1 was treated with VEEV-specific P7-PPMO in an identical manner as group 4, but was not infected with VEEV. Mice were observed daily and deaths recorded over a 28-day period following infection.
B) Infectious virus levels in the tissues of no PPMO or VEEV-specific PPMO-treated mice. Randomly pre-selected mice (N=5 per group) from groups receiving no PPMO treatment (group 2) or VEEV-specific PPMO-treatment (group 4 and 5), described for panel A, were euthanized at 2, 3 and 4 days post-infection (dpi) for harvest of blood, brain and peripheral organs (liver, spleen and lung). Infectious virus levels were determined via plaque assay. The horizontal line depicts the limit of detection. Asterisk (*) indicates that no virus was detected for any organ sample tested in the respective group.

BHK cells were incubated with the 0.1, 0.5, 1, 2.5, 5, 7.5, 10, or 20 μM of the indicated with the P3-conjugated SINV-specific PPMO (SINV 5′+, SINV AUG, or SINV sgP, see Table 1), or as a negative control, the P3-conjugated Scr PPMO (a random nucleotide sequence), for 24 hours. Reduction of cell viability, measured by MTT assay (described in Materials and Methods), is expressed relative to mock-treated cells. B) VEEV PPMO. Vero cells were incubated with the P7- or P7-AMCA-conjugated VEEV 5′+ PPMO (see Table 1), or as a negative control, the P7- or P7-AMCA-conjugated Scr PPMO (a random nucleotide sequence) with a range of PPMO concentrations (0.1, 0.5, 1, 2.5, 5, 7.5, 10, or 20 μM) for different time points (0, 4, 10, 20, 24, 36, 48 h). The average of quadruplicate treatments, measured by CellTiter-Blue® assay (described in Materials and Methods), is shown. Two-way ANOVA (α=0.05) with Bonferroni post-test for pairwise comparison of PPMO treated vs. untreated (“No PPMO”) controls by respective time point and concentration was performed.

Vero cells were infected at a moi of 0.1 with the indicated strain of VEEV (TC-83, SH3, ZPC738, or 68U201) or, as a negative control, with vesicular stomatitis virus (VSV) for 2 h prior to the addition of the indicated PPMO at 5 μM. At 8 (left) or 24 hpi (right), supernatants were harvested for analysis of viral titer via plaque assay. The VEEV serotype is indicated in parentheses. SH3 and TC-83 are epizootic (or epizootic derivative) strains; 68U201 and ZPC738 are enzootic strains. The horizontal line depicts the limit of detection. Asterisk (*) indicates that no virus was detected for any sample in that group. The result of a single experiment is shown. B) PPMO dose-response inhibition of VEEV. Vero cells were infected at a moi of 0.1 with VEEV (TC-83 strain) for 1 h prior to the addition of the indicated AMCA-conjugated PPMO at doses in the range of 0.5 to 7.5 μM. At various time points post-infection (indicated in graph), supernatants were harvested for analysis of viral titer via plaque assay. The result of a single experiment is shown. C) Effect of VEEV PPMO on VEEV and on the control virus, VSV (eight replicates). Vero cells were infected at a moi of 0.1 with VEEV (TC-83 strain), or as a control, with VSV, for 1 h prior to the addition of the indicated P7-AMCA-conjugated PPMO at 5 μM. Supernatants were harvested at 8 and 24 hpi for analysis of viral titer via plaque assay, performed in eight replicates at each time point for VEEV or VSV. Statistical analysis was performed on log₁₀-transformed viral titer values comparing all treatments using one-way analysis of variance (α=0.05, GraphPad Prism). Pairwise comparison of the log₁₀-titer values by treatment group was performed using Bonferroni post-test. Bars with asterisk indicate a statistically significant difference (Bonferroni, p-value<0.001). The average percent change was calculated from log₁₀ titers as: 100* (VEEV PPMO-average control)÷average control. A summary table showing the average percent change, relative to either the untreated (“No PPMO”) or Scr controls is shown. D) Effect of combined VEEV 5′+ and VEEV AUG PPMO on VEEV. Vero cells were infected at a moi of 0.1 with VEEV (TC-83 strain), or as a control, with VSV, for 1 h prior to the addition of the indicated P7-AMCA-conjugated PPMO at 7.5 and 10 μM (each PPMO). Supernatants were harvested at 8 and 24 hpi (and for VSV, at 8, 24, and 48 hpi) for analysis of viral titer via plaque assay. The result of a single experiment is shown.