FIG 2.

Antibodies exhibiting cell type-dependent neutralization patterns are sensitive to changes in the maturation state of the virion. The efficiency of maturation of infectious WNV was increased by propagation in an HEK-293T cell line that overexpresses human furin (293T-FIRB). Viruses produced in these cells are referred to as WNV-Furin. (A) The levels of furin expression in 293T-FIRB and HEK-293T cells were evaluated along with recombinant furin (rFurin) (R&D Systems, Minneapolis, MN) by Western blotting using the furin-reactive antibody MON-152 (Enzo Life Sciences, Farmingdale, NY). (B) The efficiency of prM cleavage in preparations of WNV-Furin was compared to that in standard WNV produced using HEK-293T cells (WNV-Std) by Western blotting of pelleted virions using an anti-prM antibody (Abcam, Cambridge, MA) and the E protein-specific MAb 4G2. (C to F) Neutralization studies with the indicated antibodies were performed using WNV-Std and WNV-Furin on Raji-DC-SIGNR cells as described for Fig. 1. Dose-response curves of MAbs E16 (C), E53 (D), E113 (E), and E121 (F) are shown. In each case, experiments are representative of four or five independent experiments. Error bars represent the range of data from duplicate wells within a single experiment. (G) Summary of the fractions of neutralization-resistant WNV-Std or WNV-Furin at saturating concentrations of the indicated antibodies. Error bars represent standard errors from four or five independent experiments. A paired t test was used to compare the sizes of the neutralization-resistant fractions of WNV-Std and WNV-Furin.