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Replication and 3'-end repair of a subviral RNA associated with turnip crinkle virus

Hancheng Guan, University of Massachusetts - Amherst

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

Biology, Molecular

Recommended Citation

Hancheng Guan, "Replication and 3'-end repair of a subviral RNA associated with turnip crinkle virus" (January 1, 2000). Doctoral Dissertations Available from Proquest. Paper AAI9978502.
http://scholarworks.umass.edu/dissertations/AAI9978502

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