FIG. 7.

MNV-1 replication is independent of COPI and COPII and insensitive to the action of BFA. (A) RAW264.7 cells were infected with MNV-1 at an MOI of 5, fixed at 12 hpi, and labeled with antibodies specific for dsRNA and for subunits of the COPI (p23; a to c) and COPII (Sec23; d to f) complexes. The antibodies were visualized with species-specific IgG conjugated to Alexa Fluor 488 or 594. No apparent colocalization was observed between dsRNA and either p23 or Sec23. (B) Western blot of lysates prepared from MNV-1-infected RAW264.7 cells that had been treated with BFA (1 μg/ml) prior to infection and at 0 and 6 hpi and cell lysates harvested at 12 hpi. The relative expression levels of MNV-1 NS7 and VP1 were assessed to determine the effect of BFA treatment on replication. None of the BFA-treated samples appear to show any significant difference in the level of either NS7 or VP1 expression. The expression level of the cellular protein actin served as a loading control. (C) Plaque assay of infected tissue culture fluid collected from MNV-1-infected RAW264.7 cells that were treated with BFA as described above at 24 hpi. No significant difference in the level of virus release was detected in any of the treated samples compared to the control.