Cereal plant disease virus (CYDV) RNA features a 5′-terminal genome-linked protein (VPg). we’ve expressed the VPg region of the CYDV genome in bacteria and used the purified protein (bVPg) to boost an antiserum which was ready to detect free VPg in extracts of CYDV-infected oat plants. A template-dependent RNA-dependent RNA polymerase (RdRp) has been produced from a CYDV membrane-bound RNA polymerase by treatment with BAL 31 nuclease. The RdRp was template specific, having the ability to utilize templates from CYDV plus- and minus-strand RNAs but not those of three unrelated viruses, purple clover necrotic mosaic virus, Cucumber mosaic virus, and mosaic virus.
RNA synthesis catalyzed by the RdRp required a 3′-terminal GU sequence and therefore the presence of bVPg. Additionally, synthesis of minus-strand RNA on a plus-strand RNA template required the presence of a putative stem-loop structure near the 3′ terminus of CYDV RNA. The base-paired stem, a single-nucleotide (A) bulge within the stem, and therefore the sequence of a tetraloop were all required for the template activity. Evidence was produced showing that minus-strand synthesis in vitro was initiated by priming by bVPg at the 3′ end of the template. the info are according to a model during which the RdRp binds to the stem-loop structure which positions the site to acknowledge the 3′-terminal GU sequence for initiation of RNA synthesis by the addition of an A residue to VPg.
In vitro RNA-dependent RNA polymerase (RdRp) systems are established for several plus-strand RNA plant viruses and, along side in vivo methods, are useful in elucidating the mechanisms of the virus RNA replication. Generally the isolated RdRp complexes are comprised of the virus RdRp intrinsically , other virus replication proteins, and host proteins. In many cases, these RdRps are template specific and are ready to initiate RNA synthesis de novo at or near the 3′ end of the template