Trans-complementation of ∆-NS1-WNV with non-translated NS1 or ectopically expressed WNV NS1′. A. Scheme for construction of a VEEV replicon that encodes a non-translated form of NS1 containing an internal stop codon at the 5′ end. B. Agarose gel electrophoresis showing PCR of the cDNA of full-length NS1 and NS1 with an internal stop codon. The bands are slightly different in size due to the use of distinct primer sets. C. Western blot of NS1 in BHK21 cells propagating replicons encoding NS1-IS (internal stop), NS1, or NS1′. NS1 migrates as a doublet reflecting intracellular NS1 (non-glycosylated, lower band) and cell-surface NS1 (glycosylated, upper band). NS1′ in cell lysates reflects only an intracellular non-glycosylated form, as it is not expressed appreciably on the cell surface (data not shown). D. Growth kinetics of ∆-NS1-WNV in BHK21-VEEV-WNV NS1, BHK21-VEEV-WNV-NS1′, or BHK21-VEEV WNV NS1 IS cells. Cells were infected at an MOI of 1, and supernatants were titrated on BHK21-VEEV-WNV NS1 cells by focus-forming assay. The dashed line indicates the limit of detection of the assay. Results are the average of two independent experiments performed in triplicate. Error bards indicate standard deviations. E. Expression of NS1 and NS1′ in the VEEV replicon in BHK21 cells, as judged by (left) flow cytometry. Experiments in panels B, C, and E reflect one of several independent experiments.