Cell fractionation and analysis of the distribution of RNPs and M protein during virus entry.

<p>rVSV-wt was bound to cells in the cold for 90 minutes in the presence of cycloheximide to prevent new protein synthesis, the inoculum was removed and the cells were washed and then either removed from the dish immediately (t-0) or warmed to 37°C and then harvested for fractionation at the times indicated. Fractions were subjected to immunoblot analysis using polyclonal anti-VSV sera. Relevant regions of the immunoblot are shown. NV (no virus) indicates cells that were mock infected and harvested at t-0. Graphs below the blots show quantification of N or M protein in each fraction. (A) N protein detected in the P16 (plasma membrane-associated virions) and NDG pellet (detergent-resistant nucleocapsids) fractions. Times post-entry are shown above the immunoblot. VSV N is a lane containing purified virus. (B) M protein detected in the S16 (plasma membrane and mitochondrial membrane) and NDG supernatant (endosomal and nuclear membrane) fraction. NV (no virus) mock infected cells. VSV M is the lane containing purified virus. (C) The four fractions probed with antibodies to the indicated cellular markers.