FIG. 2.
Stem-loop IV-deleted DI RNA does not accumulate in helper virus-infected cells. The mfold-predicted structures of wt (pDrep1) and mutant (pSLIV-mutDel) transcripts in the positive and negative strands and their calculated free energies in kcal/mole are shown. Deleted bases in pSLIV-mutDel are represented by Δ. Accumulation was measured by Northern analysis with a reporter-specific radiolabeled oligonucleotide (see text). For Northern analyses, T7 RNA polymerase-generated transcripts of linearized plasmids were transfected into BCoV-infected cells and RNA was extracted at the indicated times posttransfection and at 48 h postinfection with VP1. Accumulation of pDrep1 progeny was considered to be 100%. The sequence of the RNA in VP1 progeny replicons was determined by bulk reverse transcription-PCR sequencing using pDrep1-specific primers as described in the text. Lanes: Uninf., mock-infected, nontransfected cells; Inf., infected, nontransfected cells; RNA, sample of the transcript.