Comparison of newly synthesized inhibitors of viral entry with zanamivir. Monolayers of CV-1 cells were infected with HPIV3 in the presence of increasing concentrations of the test compounds. Viral entry was assessed by plaque reduction assay. Results are presented as percent reduction in plaque number compared with no treatment, as a function of compound concentration. Each point represents the mean (±S.D.) of four experiments. Dark squares, CSC11; dark circles, CSC7; cross hatching, CSC5; and open circles, zanamivir.

Inhibitors of viral shedding from infected HAE cultures. HAE were infected at the apical surface with HPIV3 in the presence and absence of indicated compounds. The progeny virus was collected from the apical surface 72 h post-infection and titered. Results are presented as viral titer. The results are means (±S.D.) of three experiments.

Compound effect on HN-receptor interaction. Sensitivity of HN-receptor binding to increasing concentrations of viral entry inhibitors was quantified by hemadsorption on wild-type HN-expressing cells at 4 °C. The results are shown as absorbance at 405 nm reflecting RBC binding (y axis), as a function of test compound concentration (x axis). Each point represents the mean of three experiments (±S.D.) each performed in triplicate. Dark squares, CSC11; dark circles, CSC7; cross hatching, CSC5; and open circles, zanamivir.

Comparison of the mode of action of fusion inhibitors. A, monolayers of HN/F-coexpressing cells were either pre-bound to RBCs at 4 °C and then incubated at 37 °C with or without 3 mm of the indicated compounds (dark columns), or incubated at 37 °C with RBCs for 60 min in the absence or presence of the indicated compounds (white columns). B, monolayers of HN/F-coexpressing cells were incubated with the indicated compounds for 60 min at 4 °C (dark columns) or 37 °C (white columns). The compounds were removed, and the cells were incubated with RBCs at 4 °C, and then transferred to 37 °C to permit fusion. Fusion was quantitated by spectrophotometry. The results are shown as percent inhibition of fusion compared with cells unexposed to fusion inhibitory compounds. The results are means (±S.D.) of triplicate samples from a representative experiment.

The globular head of HPIV3 HN is specifically required to mediate the effect of CSC11. Monolayers of cells coexpressing HPIV3 F (untagged) and either untagged HPIV3 HN or the untagged chimeric receptor-binding protein (HPIV3 HN 1–144/NDV HN 124–571) were incubated with cycloheximide for 1 h and then exposed for 1 h to either CSC11 or control medium at 4 °C or 37 °C. The cells were then washed and allowed to bind RBCs at 4 °C for 30 min, and transferred to 37 °C for 120 min to allow activation of HPIV3 F and fusion. The medium was collected, and the cells were lysed for quantitation of the fused RBC population. The values on the y-axis reflect quantitation of the RBCs that had undergone fusion under each condition, to arrive at the percent inhibition of fusion under each condition. The values are means (± S.D.) of results from triplicate samples and are representative of the experiment repeated three times. Exposure of the HPIV3 HN/F pair to CSC11 results in a failure of subsequent fusion promotion, however exposure of the HPIV3/NDV chimera (NDV globular head)/F pair to CSC11 does not significantly impact subsequent fusion. Thus if the globular head HN is not present but substituted by another functional head, CSC11 does not result in loss of fusion promotion by HPIV3 F.

Inhibition of cell fusion. Fusion of HN/F-coexpressing cells with receptor-bearing cells in the presence of different compound concentrations was quantitated using a β-galactosidase complementation assay. Results are expressed as relative luminescence units (RLU) reflecting fusion. Each point is the mean (±S.D.) of results from triplicate wells from a representative experiment. Dark squares, CSC11; dark circles, CSC7; cross hatching, CSC5; and open circles, zanamivir.

Assessment of virucidal action. A, aliquots of HPIV3 viral preparations were incubated with 3 mm solutions of the indicated compounds or DMSO/vehicle control for 60 min at 37 °C. The compounds were removed using filtration columns, and the resulting viral infectivity was determined by plaque reduction assay. The effect of pretreatment with test compounds on infectivity is shown as plaque number compared with that obtained with virus that was not preincubated with test compounds. B, the effect of pretreatment with the test compounds on viral RNA copy number was determined using quantitative PCR. The results in A and B are means (±S.D.) of triplicate experiments.

Detection of conformational alteration in F protein promoted by CSC11. Monolayers of cells coexpressing F and HN (A) or expressing F alone (B) were incubated for 60 min in media supplemented with 3 mm CSC11 and/or 2 mm zanamivir. The cells were lysed, and the envelope glycoproteins were immunoprecipitated then incubated in the presence of increasing concentrations (0, 1×, or 2×) of proteinase K. Representative Western blots show F proteolysis, detected by polyclonal anti-F HRC antibodies. Both F0 (precursor) and F1 (processed) proteins are indicated with arrows. The relative amounts of F1 protein were quantified, and the values were normalized to the corresponding undigested sample, as shown beneath each lane. A, after receptor engagement by HN (first lane in each triplet) or CSC11 treatment (third lane in each triplet) F becomes susceptible to protease degradation. B, if HN is not present, neither the presence of receptor molecules nor CSC11 induce F to gain protease susceptibility.