FIG. 6.

Analysis of RNA products in the membrane-based BVDV polymerase assay. (A) [³³P]CTP-labeled RNA products from mock-infected (lanes 1, 2, and 5) or virus-infected (lanes 3, 4, and 6) cell membranes were phenol-chloroform extracted and ethanol precipitated and then either directly loaded (lanes 1 to 4) or treated with S1 nuclease (lanes 5 and 6) before loading on a 1% agarose gel. Positions of the RNA size marker are indicated on the left. A position (12K) indicated by an arrow on the right was extrapolated on the basis of the positions of the RNA size markers. (B) RNA fragments used in a reverse RNase protection assay. + and − refer to positive- and negative-sense polarities of RNA relative to the BVDV genome. Numbers refer to nucleotide positions in the BVDV genome. (C) RNA products after RNase digestion. Lane 1, the RNA product from the membrane assay (no RNase digestion); lane 2, the RNA product from the membrane digested with RNase; lanes 3, 4, 5, and 6, RNA products after hybridization with the protective RNAs indicated in panel B and digestion with RNase. The positions of protective RNA (according to the results of toluidine blue staining) are indicated by arrows.