Turnip crinkle virus infection from RNA synthesized in Vitro

Genome-length cDNA clones of turnip crinkle virus (TCV) were constructed with Smal and Xbal restriction sites engineered at the 5′ and 3′ termini, respectively. The genome-length cDNAs were positioned downstream of modified λ and T7 phage promoters such that in vitro transcription resulted in RNAs with 5 extra nucleotides at the 3′ end, and 1, 2, or 14 extra nucleotides at the 5′ end depending on the construction. Transcripts with 14 extraviral 5′ nucleotides were not infectious, while transcripts with 1 or 2 additional 5' nucleotides, with or without 5′-cap analog included in transcription reactions, were biologically active. These were approximately an order of magnitude less infectious than RNA extracted from TCV virions. The additional 5′ nucleotides were not maintained in progeny viral RNAs isolated from plants.