FIG. 2.

Distribution of nuclear fraction-associated flaviviral RdRp activity and in vitro labeled RNA following DD treatment. (A) The nuclear fractions from cells infected with JEV (lanes 1 to 3), WNV (lanes 4 to 6), and DENV (lanes 7 to 9) were subjected to an in vitro RdRp assay using [α-³²P]GTP and processed as depicted in the flowchart to obtain RNA from total (T), pellet (P), and supernatant (S) fractions. (B) Purified nuclear fractions from WNV-infected cells were processed as shown in the flowchart after treatment with DD for 1 h on ice to obtain total (T), pellet (P), and supernatant (S) fractions and then subjected to in vitro RdRp assay using [α-³²P]GTP. The activity in control nuclear fractions not treated with detergent (N) is shown in lane 1. The labeled RNA obtained from each of these fractions was resolved by urea-PAGE. Values above the lane numbers in panels A and B indicate total radioactivity incorporated by all three viral RNA species as a proportion of that detected in appropriate control assays shown in lanes T. The value of 0.45 in brackets in panel B, below lane 2, represents the proportion of radioactivity incorporated as a proportion of that detected in lane 1. The arrowheads in panels A and B indicate the positions of the three viral RNA species RI, vRNA, and RF. (C) Proteins from total (T) and DD-treated pellet (P) and supernatant (S) fractions of purified nuclei from metabolically labeled WNV-infected cells were analyzed by SDS-10% PAGE. The positions of the standard molecular weight size markers are indicated at left.