Analysis of the Replication Proteins and Satellites of the Turnip Crinkle Virus

Abstract

The Turnip Crinkle Virus (TCV) is a single‐stranded, positivesense RNA plant virus that serves as a model organism for related human and animal RNA viruses, such as the West Nile, SARS‐CoV and Hepatitis C viruses. All positive‐sense RNA viruses replicate through asymmetrical replication; understanding the detailed biochemistry of this process should lead to potential drug targets for intervening in RNA viral infections.TCV provides an attractive model because satellite RNAs can accompany TCV infection. These satellite RNAs are minimal replicons that contain all cis‐acting RNA sequences required for replication. The goal of this research is to develop a model of replication of TCV satellites in the non‐host Saccharomyces cerevisiae, baker's yeast. Yeast provides numerous biochemical and genetic techniques not available in turnips.The key RNA replication proteins are p28 and p88. Their genes have been cloned into yeast shuttle vectors for expression in yeast and E. coli. The membrane binding protein, p28, has been visualized at its target membrane in yeast using fluorescence microscopy. Two natural satellites and a chimera of these two have also been cloned. The satellite constructs are bounded by self‐cleaving ribozymes that should generate active ends in vivo and in vitro for the replication system. An in vitro system for RNA recognition and eventually full replication will be established.