Fig. 7.

MRV mRNAs escape translational shutoff when SGs are disrupted. (A) Cos-7 cells were infected with T3D, and at 24 h p.i., the cells were left untreated (No drug; top row) or treated with cycloheximide (Cyc; second row), SA (third row), or 15D-PGJ2 (15D; bottom row) for 45 min and then labeled with l-AHA for 30 min in the presence of drugs. The cells were fixed, permeabilized, labeled with biotin, and then stained with Alexa 488-conjugated streptavidin (AHA; middle column), rabbit α-μNS polyclonal antiserum (μNS; left column), and goat anti-TIAR polyclonal antibodies (TIAR; right column), followed by Alexa 350-conjugated donkey α-rabbit IgG or Alexa 594-conjugated donkey α-goat IgG. Bars = 10 μm. (B) Following treatment as in panel A, cells were counted based on their translation and SG phenotype. The percentage of cells containing each phenotype out of the total number of cells counted was calculated, and the means and standard deviations of three experimental replicates are shown. Infected groups that were statistically different from mock-infected cells are indicated by an asterisk (P < 0.005). (C) Cos-7 cells were mock infected or infected with T2J or T3D and were treated with drugs as in panel A for 60 min, at which point l-AHA was added in the presence of drugs for an additional 60 min. Proteins were labeled with biotin and then separated on SDS-PAGE and transferred to nitrocellulose by electroblotting. l-AHA-labeled proteins were detected by incubation of blots with AP-conjugated streptavidin. As protein-loading and infection controls, identical sample volumes were examined in parallel using rabbit anti-β-actin polyclonal antibodies or rabbit α-μNS polyclonal antibodies followed by AP-conjugated goat α-rabbit IgG. The positions of MRV proteins on the AHA blot are indicated.