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Replication and 3'-end repair of a subviral RNA associated with turnip crinkle virus
Replication of plus (+)-strand RNA viruses proceeds through minus (−)-strand intermediates. Satellite RNA C (satC), one of the nonessential subviral RNAs of Turnip crinkle virus (TCV), is dependent on the TCV-encoded RdRp for its replication. Earlier work showed that a stem-loop structure at the 3′ end of (+)-strand satC is required for synthesis of (−)-strands (Song and Simon, 1995a). Using an in vitro RdRp assay, I defined two separate cis-acting elements on satC (−)-strands that can promote complementary strand synthesis. One element comprises 11 bases and is located near the 3′ end (3′-proximal), and the other consists of 14 bases and is located 41 bases from the 5′ end (5′ -proximal). Both elements contain multiple consecutive C residues followed by multiple consecutive purines. ^ In vivo mutagenesis and genetic selection (SELEX) studies were carried out to investigate the functional significance of the two elements as well as the satC (−)-strand 3′ terminus (3 ′ OH-CCCUAU), which contains the (−)-strand 3′ -end sequence 3′ OH-CC1–2(A/U)(A/U)(A/U) found in all carmovirus RNAs (named the carmovirus consensus sequence or CCS). My results indicate that the 3′-terminal CCS and the 5′-proximal element are highly conserved and required for satC (+)-strand synthesis. Although mutations introduced into the 3′-proximal element were tolerable, this element preferentially contains a sequence similar to the CCS and/or polypurines, suggesting that this element may also contribute to satC accumulation in vivo. ^ All RNAs associated with TCV terminate with the motif CCUGCCC-3 ′ at the 3′ end. Transcripts of satC containing a deletion of the motif, or the 3′-terminal 6 bases, are nearly always repaired to wild-type in vivo by RdRp-mediated primer extension of oligoribonucleotides synthesized by abortive initiation and complementary to the 3′ end of TCV genomic RNA (Nagy et al., 1997). In this thesis, I provide evidence that two additional mechanisms are used by the TCV RdRp to repair shorter deletions of the 3 ′-end motif of satC. Deletion of the 3′-terminal CCC residues along with addition of 8 non-template bases is repaired in vivo mainly by homologous recombination between the similar 3 ′ ends of satC and TCV. Deletion of the 3′-terminal 4 or 5 bases, in the presence or absence of non-template bases, led to recovery of progeny containing a mixture of wild-type 3′ ends and non-wild-type 3′ ends that included base alterations, deletions and insertions. Assays using an in vitro RdRp transcription system indicate that the TCV RdRp is likely able to polymerize nucleotides in a template-independent, non-random fashion before initiating transcription of deletion-containing satC. The existence of 3 different repair mechanisms associated with a single virus suggests an intrinsic need for 3′ -end reconstruction in the cellular environment. ^
"Replication and 3'-end repair of a subviral RNA associated with turnip crinkle virus"
(January 1, 2000).
Electronic Doctoral Dissertations for UMass Amherst.