CHARACTERIZATION OF THE COMPONENTS AND ACTIVITY OF SONCHUS YELLOW NETRHABDOVIRUS POLYMERASE

Sonchus yellow net virus (SYNV) is the best-characterized member of a group of plant rhabdoviruses that replicate in the host cell nucleus. Using a recently developed method for partial purification of active S YNV polymerase by salt extraction of nuclei from infected plant tissue (J. D. O. Wagner et al, J. Virol. 70:468-477, 1996), we have identifi ed the nucleocapsid (N), M2, and L proteins as polymerase complex comp onents (based on copurification with the polymerase activity and by co immunoprecipitation assays). Furthermore, the L protein was shown by a ntibody inhibition analysis to be a functional component of the polyme rase. A second complex of M2 and L proteins, thought to be a precursor to the polymerase complex, was also identified. In addition, we condu cted a detailed characterization of SYNV RNA synthesis in vitro. The r esults demonstrate that the RNAs are transcribed sequentially, beginni ng with the N mRNA and followed successively by the remaining five mRN As in the order of their genome organization. Gene expression conforms to a cascade pattern, with synthesis of the 3'-proximal N mRNA occurr ing at the highest level, followed by consecutively lower levels of tr anscription from each subsequent gene. The reaction conditions favor t ranscription over minus-sense RNA replication, which, we posit, is inh ibited near specific signal sequences located on the antigenomic templ ate. The results support the concept that the mechanism of transcripti on is highly conserved among diverse rhabdoviruses and are compatible with a unified model for the regulation of genomic and antigenomic RNA synthesis.