FIG. 4.

Time course of p28 localization during MHV infection. MHV-infected DBT cells grown on glass coverslips were fixed at the times p.i. shown on the right of the images and stained with antibodies against viral replicase and structural proteins. (A) p28 colocalizes with Hel over the course of MHV infection. Individual coverslips were stained with antibodies against p28 (GP3) (red), Hel (green), and M (purple), a marker for sites of virion assembly. (B) p28 is associated with Hel and N at 9 h p.i. Coverslips from the 9-h time point were stained with antibodies against p28 (GP3) (red), Hel (green), and N (purple). Colocalization of all three colors is shown as white pixels. (C) p28 is distinct from replication complexes at 9 h p.i. Coverslips from the 9-h time point were incubated with antibodies against p28 (GP3) (red), M (purple), and the replicase protein p65 (green) as a marker for replication complex staining at late times p.i. Colocalization of red and purple is shown as pink pixels. (D) Quantitation of percent colocalization of p28, Hel, and M. Percent colocalization was determined using Metamorph Imaging software. The background fluorescence was determined empirically by staining mock-infected cells with immune sera. The lower-limit threshold was set to exclude pixels below background. The upper-limit level was set to exclude saturated pixels. For colocalization measurements of each protein pair at each time point, three independent images (∼5 cells per image) were acquired and processed. The error bars indicate standard deviations.