FIG. 6.

RNA-binding activities of mutant NPs. (A) COS-1 cells were infected with vTF7-3 and transfected with either plasmid pGEM-NP (NP) or mock-transfected (mock). Cell extracts were prepared and resolved by centrifugation in a CsCl-glycerol gradient, and fractions were harvested from the top (sample 1) to the bottom (sample 15). Aliquots of fractions 9 to 13 (as indicated in the figure) were incubated with a ³²P-labeled RNA, irradiated with UV light, treated with RNase A, resolved by SDS-PAGE, and the proteins containing residual cross-linked radioactive nucleotides were visualized by autoradiography. Lane NPv corresponds to a sample containing NP purified from virions which was also included in the cross-linking analysis. (B) The NPs indicated in each panel were expressed and resolved by CsCl-glycerol centrifugation as described in panel A. Fractions 9 to 11 of each gradient were pooled and cross-linked to a labeled RNA. The mixtures were then resolved by SDS-PAGE and electroblotted onto Immobilon-P paper. In each panel, the autoradiography of the membrane (³²P), as well as the result of developing the same membrane by using the ECL kit with a rabbit serum, which recognizes the C-terminal region of NP (WB), are shown. Lane NP corresponds to the wild-type NP protein. In the central panel, in addition to the standard wild-type NP sample, three serial twofold dilutions prepared from this sample were also included in the gel.