Proc. Natl. Acad. Sci. USA Vol. 91, pp. 8792-8796, September 1994 Biochemistry

RNA-dependent RNA polymerase from plants infected with turnip crinkle virus can transcribe (+) and (-) strands of virus-associated RNAs

Chuanzheng Song and Anne E. Simon

Department of Biochemistry and Molecular Biology and Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, MA 01003 Communicated by George Bruening, May 2, 1994

RNA-dependent RNA polymerase (RdRp) was solubilized from membranes of turnip infected with turnip crinkle virus (TCV), a single-stranded, monopartite RNA virus. The RdRp activity could be separated into three peaks by Sephacryl S500HR chromatography. RdRp from peak I, which contained substantial amounts of endogenous TCV genomic RNA, and peak II were template-specific, synthesizing full-length complementary strands of exogenous TCV subviral RNAs but not control RNA templates. Peak III RdRp was nonspecific, synthesizing full-sized products for all added RNA templates. Peak II RdRp transcribed several different TCV satellite (sat) and defective interfering RNA templates in both (+)- and ( -- )-sense orientations but did not transcribe (+)strands of satellite RNAs associated with unrelated viruses. Monomeric-length sat-RNA C was synthesized from a template containing as many as 220 nonsatellite bases at the 3' ends of either (+)- or (-)-strands, indicating that the RdRp was able to recognize 3'-end sequences in an internal location. Deletion of 95-242 bases from the 3' end of (+)-strand sat-RNA C abolished the synthesis of template-length product. However, transcription of template-length products was not affected by the deletion of at least 257 bases from the 3' end of (-) strand sat-RNA C template (leaving only the 100 5'-terminal residues), implying that different mechanisms exist for synthesis of (+) and (-) strand satellite RNA in vitro.