In Vitro- and In Vivo-Generated Defective RNAs of Satellite Panicum Mosaic Virus 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 movement. The 824-nucleotide (nt) genome of SPMV possesses an open reading frame encoding a 17.5-kDa capsid protein (CP), which is shown to be dispensable for SPMV replication. To localize cis-acting RNA elements required for replication and movement, a comprehensive set of SPMV cDNA deletion mutants was generated. The results showed that the 263-nt 3' untranslated region (UTR) 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 region from nt 17 to 67 within the 5' UTR may have an accessory role in RNA accumulation, 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 with PMV and either of two SPMV mutants: SPMV-91, which is incapable of expressing 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 and used as a template to generate a cDNA clone. RNA transcripts derived from 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 are essential for SPMV replication and movement. The results additionally suggest that a potential "trigger" for spontaneous D-RNA accumulation may be associated 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 virus.

Four plant satellite viruses have been identified in association with helper viruses from natural infections, including satellite tobacco necrosis virus (STNV), satellite panicum mosaic virus (SPMV), satellite maize white line mosaic virus, and satellite tobacco mosaic virus (STMV). Satellite viruses are valuable tools for dissecting sequence or structural features of RNA molecules that are essential for biological and biochemical functions, such as replication, symptom induction, and spread, without perturbing the helper virus genome. These "molecular parasites" can also help us to understand the intricately coordinated interactions between helper virus-encoded proteins, the satellite virus RNAs, and the host cells.

The SPMV genome is composed of a single-stranded, plus-sense RNA of 824 nucleotides (nt). Four open reading frames (ORFs) were originally identified, two on the viral plus-sense strand and two on the viral complementary strand. An ORF from nt 88 to 561 on the viral sense RNA encodes a 17.5-kDa capsid protein (CP). The 5' untranslated region (UTR) is composed of 87 nt preceding the SPMV CP start codon, and the 3' UTR extends from nt 562 to 824. Although strong secondary structures were predicted in the 5' and 3' UTRs and implicated in the replication of the SPMV genome, cis-acting sequences essential for the replication and/or systemic movement of SPMV have not been experimentally identified.

SPMV depends on its helper virus, panicum mosaic virus (PMV), for replication and systemic spread. PMV has the unusual characteristic of supporting two distinct types of subviral entities, SPMV and satellite RNAs, which is reflected in the naturally occurring viral complex in infected St. Augustinegrass lawns along the Gulf Coast of the United States. Sequence analysis revealed that the 4,326-nt single-stranded plus-sense RNA of PMV encodes six ORFs. The 5'-proximal p48 and p112 read-through proteins provide the core components of the PMV replicase complex, which is also proposed to be essential for the replication of SPMV and the satellite RNAs. A 26-kDa CP and three other smaller proteins (p15, p8, and p6.6) have been implicated in local and systemic movement of PMV.

RNA transcripts produced in vitro from the full-length cDNA clones of PMV and SPMV are infectious on millet host plants. In the present study, a comprehensive set of SPMV mutants was generated and analyzed for the ability to infect millet protoplasts and plants. Infectivity assays with these mutants mapped thecis-acting elements that are required for SPMV replication and movement. Furthermore, the characterization of an infectious cDNA clone derived from a defective SPMV RNA (D-RNA), which accumulated de novo in the plants coinoculated with PMV and SPMV CP-deficient mutants, also defined the cis-acting elements required by SPMV for its viability in plants.

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Journal of virology