In vitro- and in vivo-generated defective RNAs of satellite panicum mosaicvirus define cis-acting RNA elements required for replication and movement

Satellite panicum mosaic virus (SPMV) depends on its helper virus, panicum mosaic virus (PMV), to provide trans-acting proteins for replication and mo vement, The 824-nucleotide (nt) genome of SPMV possesses an open reading fr ame encoding a 17.5-kDa capsid protein (CP), which is shown to be dispensab le for SPMV replication. To localize cis-acting RNA elements required for r eplication and movement, a comprehensive set of SPMV cDNA deletion mutants was generated. The results showed that the 263-nt 3' untranslated region (U TR) plus 73 nt upstream of the CP stop codon and the first 16 nt in the 5' UTR are required for SPMV RNA amplification and/or systemic spread. A regio n from nt 17 to 67 within the 5' UTR may have an accessory role in RNA accu mulation, and a fragment bracketing nt 68 to 104 appears to be involved in the systemic movement of SPMV RNA in a host-dependent manner. Unexpectedly, defective RNAs (D-RNAs) accumulated de novo in millet plants coinfected wi th PMV and either of two SPMV mutants: SPMV-91, which is incapable of expre ssing the 17.5-kDa CP, and SPMV-GUG, which expresses low levels of the 17.5 -kDa CP. The D-RNA derived from SPMV-91 was isolated from infected plants a nd used as a template to generate a cDNA clone, RNA transcripts derived fro m this 399-nt cDNA replicated and moved in millet plants coinoculated with PMV, The characterization of this D-RNA provided a biological confirmation that the critical RNA domains identified by the reverse genetic strategy ar e essential for SPMV replication and movement, The results additionally sug gest that a potential "trigger" for spontaneous D-RNA accumulation may be a ssociated with the absence or reduced accumulation of the 17.5-kDa SPMV CP. This represents the first report of a D-RNA associated with a satellite vi rus.