FIG 10.

VLS formation by NSP2 CTR mutants. (A to F) Cos-7 cells on glass coverslips were transfected with plasmids expressing untagged NSP5 and either GFP only (A) or the following NSP2-GFP fusion proteins: NSP2_WT (B), NSP2_ΔCTR (C), NSP2_K294E (D), NSP2_K294A (E), or NSP2_K294R (F). At 48 h posttransfection, cells were fixed with methanol and stained using αNSP5 and an Alexa-546-conjugated secondary antibody. Confocal microscopy was used to determine the localization of GFP signal (488 nm; green) relative to αNSP5 staining (561 nm; red). Colocalization of green and red is shown as yellow in the merged images. The locations of punctate VLS formed from NSP2 and NSP5 are indicated with closed arrowheads, while aggregates of NSP2 fusion proteins are indicated with open arrowheads. Scale bar = 10 μm.