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Replication and 3'-end repair of a subviral RNA associated with turnip crinkle virus
Abstract
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.
Subject Area
Molecular biology
Recommended Citation
Guan, Hancheng, "Replication and 3'-end repair of a subviral RNA associated with turnip crinkle virus" (2000). Doctoral Dissertations Available from Proquest. AAI9978502.
https://scholarworks.umass.edu/dissertations/AAI9978502