In Vivo Restoration of Biologically Active 3' Ends of Virus-Associated RNAs by Nonhomologous RNA Recombination and Replacement of a Terminal Motif

Clifford D. Carpenter and Anne E. Simon

Department of Biochemistry and Molecular Biology and Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003

Sequences at the 3' ends of plus-strand RNA viruses and their associated subviral RNAs are important cis elements for the synthesis of minus strands in vivo and in vitro. All RNAs associated with turnip crinkle virus (TCV), including the genomic RNA (4,054 bases) and satellite RNAs (sat-RNAs) such as sat-RNA D (194 bases), terminate with the motif CCUGCCC. While investigating the ability of in vivo-generated recombinants between sat-RNA D and TCV to be amplified in planta, we discovered that sat-RNA D, although truncated by as many as 15 bases in the chimeric molecules, was released from the chimeric transcripts and amplified to high levels. The "new" sat-RNA D molecules nearly all terminated with the motif (C 1-2)UG(C 1-3) (which may begin with 1 or 2 cytosines and end with 1, 2, or 3 cytosines), which was similar or identical to the natural sat-RNA D 3' end. The new sat-RNA D also contained between 1 and 22 bases of heterogeneous sequence upstream from the terminal motif, which, in some cases, was apparently derived from internal regions of either the plus or minus strand of the TCV genomic RNA. Since most of these internal genomic RNA sequences within TCV were not adjacent to (C 1-2)UG(C 1-3), at least two steps were required to produce new sat-RNA D 3' ends: nonhomologous recombination with the TCV genomic RNA followed by the addition or modification of the terminus to generate the (C 1-2) UG(C 1-3) motif.