1. Isolation of a cRNP replicative intermediate and vRNP from influenza virus-infected cells.
Isolation of a cRNP replicative intermediate and vRNP from influenza virus-infected cells. (A) Primer extension analysis of total and purified NA-specific RNA from HEK 293T-PP7CP cells infected with wild-type (–), cRNA-PP7, or vRNA-PP7 viruses. Viral RNAs were analyzed by primer extension using primers specific for positive sense (mRNA and cRNA) and negative sense (vRNA) NA-specific viral RNAs that generate cDNAs with an expected size of 169–177 nt from mRNA (dependent on the length of the capped mRNA primer), 160 nt from cRNA, and 129 nt from vRNA. Primer extension analysis of 5S rRNA was used as a control producing a cDNA product of 100 nt. Ten times more sample of purified RNA was analyzed relative to total RNA. (B) Quantitation of primer extension analysis of purified RNA. RNA levels represent the mean, and the error bars represent SDs from three biological repeats. (C) Analysis of purified cRNP and vRNP fractions from RNA-affinity purifications and density glycerol gradients by SDS/PAGE and staining with silver. Note that lower amounts of cRNPs were purified compared with vRNPs owing to a lower accumulation of cRNA in infected cells (7, 8). In the cRNP lane the intensity of the PB1 band appears stronger than that of the PB2/PA bands owing to the presence of a contaminating cellular protein that migrates at the same position (Fig. S2). (D) Analysis of PB2 and NP in purified RNPs by Western blot.
Ashley York et al. PNAS 2013;110:45:E4238-E4245
©2013 by National Academy of Sciences