Abstract
BackgroundInfectious cDNA clones are a powerful tool for studies on RNA viruses using reverse genetics. Potato virus S (PVS) is a carlavirus with a worldwide distribution. Although the complete genome sequences of many PVS isolates have been reported, the construction of an infectious cDNA clone of PVS is yet to be reported. The aim of this study is the development and molecular characterization of an infectious cDNA clone of PVS.MethodsA full-length cDNA clone pPVS-H-FL-AB was constructed by connecting eight cDNA clones of PVS isolate H95. Capped RNA transcripts from pPVS-H-FL-AB and a modified clone pPVS-H-FL-H, containing the consensus genome sequence of PVS-H95, proved to be non-infectious. Therefore, a full-length cDNA clone pPVS-H-FL-β was reconstructed from PVS-H00, isolated from PVS-H95 populations by repeating a single local lesion isolation in Chenopodium quinoa three times; PVS-H00 appeared to be a selected variant that survived genetic bottlenecks. The sequence of cDNA clone pPVS-H-FL-β was determined as the genome sequence of PVS-H00 and compared with the consensus sequence of PVS-H95 genome.ResultsAll Nicotiana occidentalis plants inoculated with ≥0.2 μg capped RNA transcripts from pPVS-H-FL-β developed symptoms on upper leaves, as observed with PVS-H00 inoculation. Similar levels of viral genomic and subgenomic RNAs and coat protein were detected in systemically infected leaves. Sequence comparison of PVS-H95 and PVS-H00 revealed 370 nucleotide polymorphisms (4.4% of the entire genome sequence), causing 91 amino acid substitutions in six open reading frames (ORFs). The infectivity of chimeric RNAs derived from recombinants between the two cDNA clones revealed that the lack of infectivity of pPVS-H-FL-H transcripts was due to ORF1, which encodes replicase and harbors 80 amino acid substitutions compared with pPVS-H-FL-β. Approximately 71.3% amino acid substitutions in replicase were located within the variable region of unknown function between the putative methyltransferase and ovarian tumor-like protease domains.ConclusionsThis is the first report of the development of an infectious cDNA clone of PVS. Our analyses suggest that PVS population within a plant exists as quasispecies and the replicase sequence diversity of PVS obstruct the construction of a full-length infectious cDNA clone.
Highlights
Infectious cDNA clones are a powerful tool for studies on RNA viruses using reverse genetics
Three amino acid substitutions, including Ser44Cys, Ile660Val, and Ser908Pro, due to nucleotide substitutions A192T, A2040G, and T2784C, respectively, were generated in ORF1 of pPVS-H-FL-AB compared with the consensus sequence of Potato virus S (PVS)-H95 genome (Fig. 1)
PPVS -H-FL-AB was modifed to obtain an infectious cDNA clone. pPVS-H-FL-V with Ser-44 and pPVS-H-FL-C with Ile-660 and Ser-908 were constructed by the modification of ORF1; capped RNA transcripts derived from the two clones failed to infect N. occidentalis
Summary
Infectious cDNA clones are a powerful tool for studies on RNA viruses using reverse genetics. Two major biologically distinct strains of PVS, PVSO (ordinary) and PVSA (Andean), have been identified, based on the ability to systemically infect Chenopodium quinoa and C. amaranticolor plants. Unlike PVSO, 13 out of 44 Tasmanian isolates cause local but asymptomatic infection only on inoculated leaves of C. quinoa. Nine isolates, unlike PVSA, cause systemic infection without the typical symptoms in C. quinoa. Of these nine isolates, five induce symptoms on inoculated leaves only, and four cause asymptomatic systemic infection on inoculated and uninoculated upper leaves [5]. There is no evidence that CP is involved in systemic infection on Chenopodium spp
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