Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Dec 4;9(1):18359.
doi: 10.1038/s41598-019-54853-0.

A functional investigation of the suppression of CpG and UpA dinucleotide frequencies in plant RNA virus genomes

Ahmad Ibrahim  1 , Jelke Fros  1   2 , Andre Bertran  2 , Ferdyansyah Sechan  1 , Valerie Odon  1 , Leslie Torrance  3 , Richard Kormelink  2 , Peter Simmonds  4
Affiliations

A functional investigation of the suppression of CpG and UpA dinucleotide frequencies in plant RNA virus genomes

Ahmad Ibrahim et al. Sci Rep. .

Abstract

Frequencies of CpG and UpA dinucleotides in most plant RNA virus genomes show degrees of suppression comparable to those of vertebrate RNA viruses. While pathways that target CpG and UpAs in HIV-1 and echovirus 7 genomes and restrict their replication have been partly characterised, whether an analogous process drives dinucleotide underrepresentation in plant viruses remains undetermined. We examined replication phenotypes of compositionally modified mutants of potato virus Y (PVY) in which CpG or UpA frequencies were maximised in non-structural genes (including helicase and polymerase encoding domains) while retaining protein coding. PYV mutants with increased CpG dinucleotide frequencies showed a dose-dependent reduction in systemic spread and pathogenicity and up to 1000-fold attenuated replication kinetics in distal sites on agroinfiltration of tobacco plants (Nicotiana benthamiana). Even more extraordinarily, comparably modified UpA-high mutants displayed no pathology and over a million-fold reduction in replication. Tobacco plants with knockdown of RDP6 displayed similar attenuation of CpG- and UpA-high mutants suggesting that restriction occurred independently of the plant siRNA antiviral responses. Despite the evolutionary gulf between plant and vertebrate genomes and encoded antiviral strategies, these findings point towards the existence of novel virus restriction pathways in plants functionally analogous to innate defence components in vertebrate cells.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
G + C content and frequencies of CpG, TpA/UpA, CHG and CHH in plant genomes. G + C content and frequencies of TpA/UpA and CpG dinucleotides and CHG and CHH motifs in genomic DNA (fragment sizes 5000 bps) and coding regions of mRNAs (>450 bases) of three different plant species and in the human genome. Dinucleotide frequencies were expressed as ratios of observed (O) numbers divided by the expected (E) numbers based on frequencies of their constituent bases or combinations of bases. Expected frequencies of CHG and CHH motifs were calculated by references to frequencies of their component dinucleotides (see Methods). For each, an O/E ratio of 1.0 (dotted line) is the expected frequency. Bar heights show mean values; error bars show ±1 standard deviation.
Figure 2
Figure 2
Comparison of CpG and UpA compositions in different plant RNA virus families. O/E ratios of CpG and UpA dinucleotides in 2612 individual protein or polyprotein genes (>450 bases) derived from 1193 plant virus genomes in the ICTV Virus Metadata Resource (https://talk.ictvonline.org/taxonomy/vmr/) divided into separate Baltimore groups and families. These comprise dsRNA RNA viruses (Baltimore group 3), negative-stranded RNA viruses (ssRNA−; Group 5), reverse transcribing viruses (RT; Group 7) and plus-stranded RNA viruses (ssRNA+; Group 4). Bar heights show mean values; error bars show ± 1 standard deviation.
Figure 3
Figure 3
CpG and TpA/UpA dinucleotide representation in N. attenuata and potyviruses. Frequencies of (A) CpG and (B) TpA/UpA dinucleotides in N. attenuata genomic DNA (5000 bp fragments), coding regions of mRNAs (>450 bases) and polyprotein genes of each potyvirus species (>450 bases). Potyviruses have been divided into those infecting dicotyledons (light green) and monocotyledons (dark green). The composition of the PYV strain used in the current study is highlighted (white diamond symbol). The O/E ratio of 1.0 is drawn as a heavier horizontal line.
Figure 4
Figure 4
Synonymous variability and RNA structure predictions in the PYV genome. Scan of synonymous variability (red line; left hand axis scale) between aligned PYV sequences (O- and NTN-groups – see Methods for sequence listing) to identifiy regions of suppressed variability indicative of overlapping reading frames and structured RNA elements. A plot of MFED (blue line; right hand y-xaxis scale) has been superimposed to identify areas of thermodynamiclly favoured base pairing. The position of the mutated regions are shown underneath the genome diagram (see Fig. S5, Suppl. Data). Gene abbreviations: P1-Pro: protein required for genome replication. HC-Pro: helper component-protease. PIPO: protein required for viral replication and a recently recognized protein implicated in cell to cell movement. 6k1: exact function unknown. 6K2: membrane anchoring protein. VPg: genome-linked protein. Pro: nuclear inclusion proteinase. Coat: capsid protein.
Figure 5
Figure 5
Morphology and virus detection in N. benthamiana leaves after infection with PVX, WT PVY and PVY mutants. Phenotype comparison and green fluorescence protein (GFP) detection in the topmost third leaf of N. benthamiana agroinoculated (OD600 0.4) of PVY and compositionally altered mutants of PVY, along with PVX. Leaves were harvested at 16 dpi. Replicating PYV was visualized by fluorescence microscopy. The scale bar is 100 µm.
Figure 6
Figure 6
Quantification of pathology changes in tobacco plants infected with PYV. (A) Time course for the decline in mean leaf area of the four top most leaves of PVY mutants agroinoculated (OD600 0.4) N. benthamiana at 7, 10, and 16 dpi. (B) Comparison of the uppermost four leaves surface area harvested from three plants at 16 dpi. Bar heights show mean values; error bars show standard deviations, square data-points indicate values for separate biological replicates. Areas were compared using One-way ANOVA Tukey multiple comparisons test; significance values of differences from the WT virus are indicated above bars; *p = 0.0215; **p = 0.0099; ***p = 0.0004, ****p < 0.0001.
Figure 7
Figure 7
Quantification of PVY replication by protein ELISA and qPCR for viral RNA. (A) Quantification of viral capsid by DAS-ELISA in leaf lysates by ELISA Column at 10 dpi. Values were normalized to that of WT PVY. Bar heights show mean values of three replicates, error bars show standard deviations. (B) qPCR time-course quantitation of viral RNA at 07, 10, and 16 dpi, expressed as the ratio of viral RNA copies to those of the housekeeping gene, protein phosphatase 2A. (C) Quantitation of viral RNA by qPCR, expressed as the ratio of PYV RNA copies to those of the housekeeping gene, protein phosphatase 2A, at 10 dpi. Bar height show geometric mean ratios, error bars show standards deviation of log10 transformed data; square data-points indicate values for separate biological replicates.
Figure 8
Figure 8
Impact of RDR6 knock down on PVY replication. (A) Quantitation of PYV viral capsid by DAS-ELISA in topmost third leaf at 10 dpi of N. benthamiana with knockdown of RDR6 (right-hand column) and of a GUS control (left-hand column). Plants were infected with WT PVY and compositionally altered mutants 9 days after knockdown. Values were normalized to that of WT PVY from GUS (control)- silenced N. benthamiana. (B) Comparative effect of RDR6 k/d of PVX replication as determined by DAS-ELISA detection of PVX viral particles. Values were normalized to that of PVX from GUS (control)- silenced N. benthamiana. Bar heights show mean values of three biological replicates, error bar show standard deviations. (C) Parallel evaluation of replication of PVY WT and mutant viruses by qPCR for PYV RNA. Column heights show geometric mean tire of three biological replicates; error shows show standard deviations of log-transformed values; square data-points indicate values for separate biological replicates.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Goodfellow I, et al. Identification of a cis-acting replication element within the poliovirus coding region. J Virol. 2000;74:4590–4600. doi: 10.1128/JVI.74.10.4590-4600.2000. - DOI - PMC - PubMed
    1. Belsham GJ, Sonenberg N. RNA-protein interactions in regulation of picornavirus RNA translation. Microbiol Rev. 1996;60:499–511. - PMC - PubMed
    1. Atkinson NJ, Witteveldt J, Evans DJ, Simmonds P. The influence of CpG and UpA dinucleotide frequencies on RNA virus replication and characterization of the innate cellular pathways underlying virus attenuation and enhanced replication. Nucleic acids research. 2014;42:4527–4545. doi: 10.1093/nar/gku075. - DOI - PMC - PubMed
    1. Takata MA, et al. CG dinucleotide suppression enables antiviral defence targeting non-self RNA. Nature. 2017;550:124–127. doi: 10.1038/nature24039. - DOI - PMC - PubMed
    1. Fros, J. J. et al. CpG and UpA dinucleotides in both coding and non-coding regions of echovirus 7 inhibit replication initiation post-entry. eLife6 (2017). - PMC - PubMed