|
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. |