FIG. 6.

Analysis of total and capsid-associated RNA. Cells were transfected as indicated at the top of each lane. All plasmids code for a reverse transcriptase-deficient polymerase. (A) Total cytoplasmic RNA extracted from cytoplasmic lysates without micrococcal nuclease treatment. Note that similar amounts of pgRNA were produced in all cases. (B) Capsid-associated RNA isolated from immunoprecipitated capsids (IP) in the absence or presence of nuclease treatment (MN) followed by Northern blotting. Free mRNA was removed completely with both protocols as evidenced by the absence of a GAPDH (glyceraldehyde-3-phosphate dehydrogenase)-specific hybridization signal. The left panel shows a 10% aliquot of total RNA isolated from cytoplasmic lysate of wild-type transfected cells. Threefold more RNA from nuclease-treated 164 capsids was loaded relative to the amount loaded in the other lanes to highlight the absence of full-size pgRNA. sgRNA, cytoplasmic subgenomic RNA.