A deletion of WNV NS1 affects viral RNA synthesis and formation of the replication complex. A-B. Measurement of negative (A) or positive (B) strand viral RNA in BHK21-VEEV-NS1 or BHK21-VEEV-NS1-IS (premature stop codon) cells infected with WNV-WT or ∆-NS1-WNV. The indicated cells were infected with WNV-WT or ∆-NS1-WNV at an MOI of 5. At the indicated time points, viral RNA was harvested and strand-specific qRT-PCR [31, 32] was performed. The results are the average of two independent performed in triplicate. The very low levels of negative strand viral RNA at input (t = 0) have been reported previously for DENV and WNV [33, 34], and possibly reflect delivery of viral RNA in exosomes or defective viruses with inappropriate packing of RNA intermediates. C-E. Vero cells were transfected with CMV-launched infectious cDNA clones of (C) wild type KUNV (pKUN1-wild type), (D) KUNV with a mutation in NS5 that abolishes RNA polymerase activity (pKUN1-NS5 GDD/GAA), or (E) ∆-NS1-WNV KUNV (pKUN1-∆-NS1). 48 hours later, cells were co-stained with antibodies against (top panels) NS3 and dsRNA, (middle panels) NS3 and NS1, or (bottom panels) NS4A and NS5. Nuclei were stained blue with DAPI. Images were processed by confocal microscopy. Representative images from three independent experiments are shown.