FIG. 3.

Comparison of RNA and protein syntheses from the RNA replication-competent and RNA replication-defective KUN replicon DNA vectors. (A) Northern blot hybridization analysis of total RNA isolated from BHK21 cells 36 h after transfection with equal amounts of pKUNrep2 (lane 1) or pKUNrep2(dGDD) (lane 2) plasmid DNAs, or untransfected cells (lane 3). The probe was a [³²P]dCTP-labeled cDNA fragment representing the last 761 nucleotides of the KUN genome (24). (B) RIP analysis with KUN anti-NS3 antibodies of BHK21 cells radiolabeled for 1 h at 30 h after transfection with pKUNrep2 (lanes 1 and 2) or pKUNrep2(dGDD) (lanes 3 and 4) plasmid DNAs. Shown are RIP samples from cells labeled in the absence (−) (lanes 1 and 3) or presence (+) (lanes 2 and 4) of ACD. The specificity of KUN anti-NS3 antibodies in RIP and IF analyses was demonstrated previously (45). Relative phosphorimager counts of the radiolabeled NS3 bands in corresponding RIP samples are shown with the background level deducted. DNA transfection, Northern blot hybridization, protein labeling, ACD treatment, and RIP procedures were performed as described in Materials and Methods.