FIG. 3.

Characteristics of RNA synthesized from endogenous templates in FCV RCs. The RC pellet was isolated from mock- and FCV-infected cells and analyzed in a replication assay that included [α-³²P]CTP. Total RNA was extracted from the mixture and analyzed on a 1% agarose gel that was dried and exposed to film. (A) Lanes: 1, RNA from FCV-infected (FCV-inf) RC pellet assay; 2, RNA from mock-infected RC pellet assay; 3, total ³²P-labeled RNA isolated from FCV-infected CRFK cells by oligo(dT) selection. (B) Proteins associated with TLs and fractions obtained during purification of RCs (as described in Materials and Methods) were subjected to SDS-PAGE in a 10 to 20% Tris-glycine gel, followed by staining with Coomassie blue. Lanes: 1, Mark 12 protein standards (Invitrogen); 2, mock-infected CRFK TL; 3, mock-infected P1 nuclear pellet; 4, mock-infected supernatant (S) from P2 pellet; 5, mock-infected P2 RC pellet; 6, FCV-infected TL; 7, FCV-infected P1; 8, FCV-infected S; 9, FCV P2 RC pellet. (C) The mock- and FCV-infected TLs and fractions shown in panel B were each analyzed in a replication assay that contained [α-³²P]UTP. The radiolabeled RNA was purified and subjected to electrophoresis in a 1% agarose gel, and the signal was measured on a PhosphorImager. The RNA was derived from replication assays with the following samples: lane 2, mock-infected CRFK TL; lane 3, mock-infected P1 nuclear pellet; lane 4, mock-infected supernatant (S) from P2 pellet; lane 5, mock-infected P2 RC pellet; lane 6, FCV-infected TL; lane 7, FCV-infected P1; lane 8, FCV-infected S; lane 9, FCV P2 RCs. The amounts of total protein in each replication assay (which used 18 μl of sample) in lanes 2 to 9 were 6.8, 10.1, 3.7, 11.1, 9.9, 7.0, 5.2, and 17.7 μg, respectively, and reflected the nonadjusted yield of protein in each sample from the isolation procedures described in Materials and Methods. Lane 1 depicts the RNA Ladder (Invitrogen) from the same gel stained with ethidium bromide (Eth.Br.).