FIG 8.

Hsc70 promotes the formation of viral replication complexes and viral DNA replication. (A) 3T3 fibroblasts were treated with vehicle (DMSO) or VER-155008 (50 μM) for 1 h prior to and during infection with 73.GFP MHV68 at an MOI of 5 PFU/cell. Cells were fixed at 18 h postinfection and stained with antibodies directed against GFP to detect mLANA-GFP or ORF59 as a marker for viral replication complexes. DNA was detected with DAPI. Images were analyzed by fluorescence microscopy at a magnification of ×60. White arrowheads indicate representative viral replication complexes. (B) Viral replication complexes from the experiment for panel A were quantified by counting the number of cells containing ORF59 foci and dividing by the total number of cells exhibiting GFP⁺ nuclei for a field of view at a magnification of ×20. Results represent mean percentages of ORF59⁺ GFP⁺ cells for 15 randomly selected fields of view for each condition. Error bars represent standard errors of the means. *, P = 0.0001. (C) 3T3 fibroblasts were infected with MHV68 at an MOI of 5 PFU/cell in the presence of vehicle, VER-155008 (50 μM), or PAA (200 μg/ml). Total DNA was harvested at 4 h and 18 h postinfection, and viral genomes were measured by quantitative PCR. Data represent the change in viral DNA copy number normalized to the cellular GAPDH gene between 4 h and 18 h postinfection using the ΔΔC_T method. Results are means for biological duplicate samples analyzed in technical triplicate. Error bars represent standard deviations.