FIG. 6.

MHV replication is reduced in cells depleted of ubiquitin. HeLa-CEACAM1a cells grown in 96-wells clusters were transfected with 10 nM siRNAs targeting the indicated ubiquitin genes (i.e., UBA52 and Rps27A). Mock- and scrambled siRNA-transfected cells were used as controls. (A) At 72 h after transfection, the cells were lysed and processed for Western blotting with antibodies against ubiquitin and β-actin (loading control). The levels of free ubiquitin and ubiquitin conjugates were quantified and are expressed as a percentages relative to mock-transfected cells. Note that the values were corrected for the loading control. (B) The siRNA-transfected cells were inoculated with MHV-EFLM. At 6 h postinfection, the cells were fixed and stained for viral antigen using the polyclonal anti-MHV serum. The total numbers of MHV-infected cells per well are indicated, as well as the standard deviations (n = 3). (C) In parallel, the intracellular luciferase expression levels measured at 6 h postinfection are shown as a percentage relative to the control (scrambled siRNA-transfected cells). Note that the RLU values are corrected for cell viability. *, P < 0.05 as determined by statistical analysis using the Student t test.