Sequences and Structures Involved in Replication and Recombination of TCV RNAs

点击翻译按钮获取中文摘要

9419303 Simon Turnip crinkle virus (TCV) genomic and subviral RNAs will be used to study sequences and structures involved replication and recombination. The advantages of TCV include: transcripts produced in vitro from cloned cDNA of genomic and numerous subviral RNAs are biologically active in plants and isolated plant cells (protoplasts); plant extracts containing template-dependent RNA-dependent RNA polymerase (RdRp) activity can transcribe RNA from both (+)- and (-)-strand TCV subviral RNA templates; numerous chimeric RNAs have been characterized that are the products of recombination between the genomic and subviral RNAs. RNA recombinants associated with TCV have one of three motifs at the 3' crossover point (unlike other virus recombinants), one of which is absolutely required for normal accumulation of the virus in plants and protoplasts, suggesting a link between recombination and virus replication. Preliminary results have led to numerous intriguing questions: What is the connection between replication and recombination at a particular region in the TCV genomic RNA? Is the sequence or structure of a stable TCV RNA hairpin important for recombination/replication? What are the sequences/structures required for transcription by the RdRp and is there a commonality with the crossover sites? Is there a connection between in vitro synthesis of a larger-than-full-length product using (-)-strand satellite template and recombination? What is the identity of the major smaller-than-full-length product in the same reactions, whose size varies in an unusual manner after deletion of various portions of the template? To begin answering these and other related questions, the following experiments will be performed: (1) The region involved in replication and recombination in the TCV genomic RNA will be studied by genetic manipulation and structural determination. (2) Cis-acting sequences/structures of TCV satellite RNA C that are required for transcription of (+)-and (-)-strands in vitro wil l be determined by further deletions of terminal and internal sequences, structural determinations, and modification of putative structural elements. (3) The connection between recombination and synthesis of larger-than-full-length product in vitro will be tested by using templates where the structure in the region is maintained but specific sequences, known to affect recombination, have been altered. (4) The 150 base smaller-than-full-length product will be cloned and the mechanism of its synthesis determined. Studying the sequences/structures that are recognized by the TCV RdRp during initiation of transcription or re-initiation of transcription following a crossover event, should lead to a better understanding of the process viral RNA replication. %%% Turnip crinkle virus is an icosahedral virus with and monopartite RNA genome that infects a broad range of dicot plants. The 4054 base genomic RNA encodes the viral replication and encapsidation functions. Turnip crinkle virus genomic and subviral RNAs will be used to study the RNA sequences and structures involved in viral RNA replication and recombination. As a model system, turnip crinkle virus has technical advantages including the ability to produce biologically active viral RNAs by in vitro transcription of cloned DNAs, the ability to prepare plant cell extracts that have functioning viral RNA-dependent RNA polymerase, the enzyme responsible for virus RNA replication. These experiments will greatly increase our understanding of the molecular mechanism of RNA-dependent RNA polymerase mediated replication and recombination characteristic of several plant RNA viruses. ***