9630191 Simon Virus-encoded RNA-dependent RNA polymerases (RdRp) are responsible for a two-stage replication process: the RdRp synthesizes complementary minus-strands that in turn become templates for plus-strand synthesis. A number of different structural elements have been implicated in replication including pseudoknots, stem-loops and tRNA-like structures. Less understood viruses whose genomic RNA(s) end in a 3'-terminal hydroxyl group such as turnip crinkle virus (TCV) will be utilized as a model to study sequences and structures involved in replication. TCV is among the smallest and simplest of the plant RNA viruses and is associated with subviral sat-RNAs and DI RNAs, making possible the use of these simple, non-protein-coding RNAs as templates for replication studies. Protoplast and in vitro systems have been developed to study replication of genomic and subviral RNAs and virus-encoded RdRp subunits have been expressed in insect cells using baculovirus vectors. A 29 base sequence at the 3' end of plus-strand satC (356 bases), which includes a stable hairpin and the CCUGCCC motif found at the ends of all TCV-associated RNAs, is required and sufficient for minus-strand synthesis in vitro. Mutagenesis studies indicated that the stability of the stem, and not the sequence of the stem or loop, is important for promoter function. Minus-strand satC was found to have two independent promoters, one within the 3' terminal 49 nucleotides and a second near positions 310 to 334. The following experiments will be performed: (1) The sequence/structural requirements for plus-strand and minus-strand promoters of satC will be determined using further mutagenesis and an in vitro selection strategy (SELEX), where a large pool of random polynucleotide sequences are "selected" for further amplification if they comprise a functional promoter. (2) Cis-signals involved in replication in vivo and transcription in vitro for satD will be determined using deletions and site-directed mutagenesis. (3) The importa nce of the sequence and location of the CCUGCCC 3'-terminal motif in relation to the 3' hairpin for satC and TCV will be examined. In addition, we will determine if deletion of the 3' end of TCV and satC also result in the formation of "new" RNAs in vivo with replacement of the CCUGCCC terminal motif as was found for satD. (4) Binding studies using the TCV-encoded RdRp subunits p28 and p88 to promoter sequences associated with satC and satD will be performed using gel retardation analysis, filter binding, and hydroxyl radical footprinting. (5) The effect of coat protein on replication of TCV subviral RNA plus and minus strands in protoplasts will be determined using wild-type TCV and TCV with a coat protein open reading frame deletion (TCV(CP). In addition, using RdRp-active extracts from protoplasts inoculated with TCV(CP, we will assay for the effect of different amounts of highly purified TCV(CP on in vitro transcription of satC and satD plus- and minus -strands. %%% The process by which a plant RNA virus can reproduce itself by copying its gene is very complex. Turnip crinkle virus RNA replication can be carried out in a test tube. These studies will focus on the contribution of specific sequences of the RNA interacting with the enzymatic activities that produce new viral RNA copies. These studies may lead to understanding how to manipulate and control RNA virus activities in plant and animal systems. ***
