Extraction of nuclei from sonchus yellow net rhabdovirus-infected plants yields a polymerase that synthesizes viral mRNAs and polyadenylated plus-strand leader RNA.
Although the primary sequence of the genome of the plant rhabdovirus sonchus yellow net virus (SYNV) has been determined, little is known about the composition of the viral polymerase or the mechanics of viral transcription and replication. In this paper, we report the partial isolation and characterization of an active SYNV polymerase from nuclei of SYNV-infected leaf tissue. A salt extraction procedure is shown to be an effective purification step for recovery of the polymerase from the nuclei. Full-length, polyadenylated SYNV N and M2 mRNAs and plus-strand leader RNA are among the products of the in vitro polymerase reactions. Polyadenylation of the plus-strand leader RNA in vitro is shown with RNase H and specific oligonucleotides. This is the first report of a polyadenylated plus-strand leader RNA for a minus-strand RNA virus, a feature that may reflect adaptation of SYNV to replication in the nucleus. Analysis of the SYNV proteins present in the polymerase extract suggests that the N, M2, and L proteins are components of the transcription complex. Overall, the system we developed promises to be useful for an in-depth characterization of the mechanics of SYNV RNA synthesis.
Department of Plant Biology, University of California, Berkeley 94720, USA.
J D
JD
Wagner
T J
TJ
Choi
A O
AO
Jackson
D001483:Base Sequence; D002213:Capsid / Q000235:genetics; D002467:Cell Nucleus / Q000201:enzymology / Q000821:virology*; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000235:genetics; D018515:Plant Leaves / Q000821:virology; D010942:Plant Viruses / Q000201:enzymology*; D010947:Plants, Toxic; D012333:RNA, Messenger / Q000096:biosynthesis*; D012367:RNA, Viral / Q000096:biosynthesis*; D012209:Rhabdoviridae / Q000201:enzymology*; D014026:Nicotiana / Q000821:virology; D014158:Transcription, Genetic; D014758:Viral Core Proteins / Q000235:genetics
D010750:Phosphoproteins; D012333:RNA, Messenger; D012367:RNA, Viral; D014758:Viral Core Proteins; D012321:DNA-Directed RNA Polymerases