FIG 4.

Analysis of nucleic acids in vac2- and vac2-C^KO-infected cells and in purified vac2 and vac2-C^KO particles. (A) Two-step gradient purification of MV particles. Virus bands are indicated by brackets. (B) Titers (numbers of TCID₅₀s per milliliter) of virus stocks prior to and after purification. (C) RNA protection assay with purified virions. Samples were treated with the indicated combinations of trypsin, Triton X-100, and Benzonase; immunoblots were against MV H and N proteins; RT-PCR/PCR analysis was performed for DI-RNAs (A1/A2 primers) and full-length RNA (A1/B1 primers). (D) Northern blot analysis of total RNA from about 3 × 10⁵ uninfected (UI; lane 1) or infected (lanes 2 and 3) Vero cells harvested at 32 h postinfection, as well as RNA extracted from 1 × 10⁶ TCID₅₀s of purified (pur.) and Benzonase-treated vac2 (lane 4) and vac2-C^KO (lane 5) particles. The positions of the antigenome and L, H-L, and F-H-L mRNAs (+probe) are indicated on the right side of the left panel. The identities of the genomic RNA and the DI-RNAs (−probe) mapped by PCR amplification are indicated on the right side of the right panel. Lane M, molecular size marker. The numbers on the left side of the left panel indicate the sizes of the marker bands (in bases).