Nucleotide Sequence and Infectivity of a Full-Length cDNA Clone of Panicum Mosaic Virus

Abstract

The sequence of an infectious cDNA clone of panicum mosaic virus (PMV) showed that the single-stranded RNA genome is 4326 nucleotides (nt) and a single highly abundant subgenomic (sg) RNA of 1475 nt was synthesized during PMV infection of pearl millet plants and protoplasts. Computer comparisons revealed strong similarities between the predicted amino acid sequences of the p48 and p112 open reading frames (ORFs) and replicase proteins of members of the Tombusviridae. The sgRNA has the potential to encode five proteins. Three small ORFs, p8, p8-FS, and/or p6.6 have similarity to ORFs of carmo-, necro-, and machlomoviruses thought to be involved in virus spread in plants. The sgRNA also has the potential to encode a 26-kDa capsid protein and a 15-kDa nested gene (p15) of unknown function. PMV transcripts also supported replication and movement of SPMV, the satellite virus. Genome organization, physicochemical properties, and biological features indicate that PMV is a member of the Tombusviridae family. However, PMV differs sufficiently from previously described members to warrant its placement in a new genus provisionally designated Panicovirus.

In Vitro- and In Vivo-Generated Defective RNAs of Satellite Panicum Mosaic Virus Define cis -Acting RNA Elements Required for Replication and Movement

“…To supply DNA templates for the in vitro transcription reaction, cesium chloridepurified plasmids containing full-length PMV cDNA (28) were digested with EcoICR1, and the plasmids carrying SPMV or mutagenized SPMV cDNAs were digested with BglII. Plasmids containing the SPMV D-RNA cDNA were digested with PstI and treated with DNA polymerase I large fragment (Klenow).…”

“…Approximately 3 g of total plant RNAs or the entire protoplast RNA was separated in a 1.2% agarose gel and subsequently transferred to nylon membranes (Micron Separations Inc., Westborough, Mass.). PMV and SPMV RNAs were detected by hybridizing the RNA blots with [ 32 P]dCTP-labeled PMV-or SPMV-specific probes generated by a randompriming method as previously described (28). Approximately 15 g of total proteins were electrophoresed through a sodium dodecyl sulfate-12% polyacrylamide gel and then transferred to nitrocellulose membranes (Micron Separations Inc.).…”

“…A 26-kDa CP and three other smaller proteins (p15, p8, and p6.6) have been implicated in local and systemic movement of PMV (27). RNA transcripts produced in vitro from the full-length cDNA clones of PMV and SPMV are infectious on millet host plants (9,21,28). In the present study, a comprehensive set of SPMV mutants was generated and analyzed for the ability to infect millet protoplasts and plants.…”

“…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 (3). Sequence analysis revealed that the 4,326-nt singlestranded plus-sense RNA of PMV encodes six ORFs (28). The 5Ј-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 (28).…”

“…Sequence analysis revealed that the 4,326-nt singlestranded plus-sense RNA of PMV encodes six ORFs (28). The 5Ј-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 (28). A 26-kDa CP and three other smaller proteins (p15, p8, and p6.6) have been implicated in local and systemic movement of PMV (27).…”