FIG. 6.
BVDV protein folding and infectivity in NB-DNJ-treated cells. (A) MDBK cells were grown to subconfluence in six-well plates and infected with BVDV at an MOI of 1. After 1 h the inoculum was removed and the cells were grown for 24 h in medium containing either NB-DNJ or NB-DGJ at the indicated concentrations (plaque assay). The supernatants containing secreted virus were then used to infect fresh MDBK monolayers, and the plaques were counted after 24 h (yield assay). Results are the percentages of the number of plaques resulting from infection with the inhibitor-free plaque assay supernatant (considered as 100%). (B) MDBK cells were infected with BVDV at an MOI of 1 and grown in the absence or presence of increasing concentrations of NB-DNJ. Eighteen hours p.i., the cells were labeled for 15 min with [35S]methionine and [35S]cysteine and lysed. The amount of radiolabeled protein in the cell lysates was adjusted to 3 × 106 cpm/ml, before immunoprecipitation with MAb 158 and antiactin antibodies (loading control). The amount of immunoprecipitated protein was quantified by liquid scintillation counting and expressed as the percentage of counts per minute determined for the drug-free samples. (C) MDBK cells were infected with BVDV at an MOI of 1 and grown in the absence or presence of NB-DNJ at the concentrations indicated. Eighteen hours p.i., the cells were lysed and the assembly of the viral proteins was analyzed by SDS–10% PAGE under nonreducing conditions followed by Western blotting using MAb 214. Calnexin and actin were detected on the same membrane (loading control) (gels). The band intensities of E2 monomers as well as of E1-E2 and E2-E2 dimers were quantified by densitometric analysis (graph).