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. 2019 Aug 27;10(4):e01900-19.
doi: 10.1128/mBio.01900-19.

Panicum Mosaic Virus and Its Satellites Acquire RNA Modifications Associated with Host-Mediated Antiviral Degradation

Jesse D Pyle  1 , Kranthi K Mandadi  1   2 , Karen-Beth G Scholthof  3
Affiliations

Panicum Mosaic Virus and Its Satellites Acquire RNA Modifications Associated with Host-Mediated Antiviral Degradation

Jesse D Pyle et al. mBio. .

Abstract

Positive-sense RNA viruses in the Tombusviridae family have genomes lacking a 5' cap structure and prototypical 3' polyadenylation sequence. Instead, these viruses utilize an extensive network of intramolecular RNA-RNA interactions to direct viral replication and gene expression. Here we demonstrate that the genomic RNAs of Panicum mosaic virus (PMV) and its satellites undergo sequence modifications at their 3' ends upon infection of host cells. Changes to the viral and subviral genomes arise de novo within Brachypodium distachyon (herein called Brachypodium) and proso millet, two alternative hosts of PMV, and exist in the infections of a native host, St. Augustinegrass. These modifications are defined by polyadenylation [poly(A)] events and significant truncations of the helper virus 3' untranslated region-a region containing satellite RNA recombination motifs and conserved viral translational enhancer elements. The genomes of PMV and its satellite virus (SPMV) were reconstructed from multiple poly(A)-selected Brachypodium transcriptome data sets. Moreover, the polyadenylated forms of PMV and SPMV RNAs copurify with their respective mature icosahedral virions. The changes to viral and subviral genomes upon infection are discussed in the context of a previously understudied poly(A)-mediated antiviral RNA degradation pathway and the potential impact on virus evolution.IMPORTANCE The genomes of positive-sense RNA viruses have an intrinsic capacity to serve directly as mRNAs upon viral entry into a host cell. These RNAs often lack a 5' cap structure and 3' polyadenylation sequence, requiring unconventional strategies for cap-independent translation and subversion of the cellular RNA degradation machinery. For tombusviruses, critical translational regulatory elements are encoded within the 3' untranslated region of the viral genomes. Here we describe RNA modifications occurring within the genomes of Panicum mosaic virus (PMV), a prototypical tombusvirus, and its satellite agents (i.e., satellite virus and noncoding satellite RNAs), all of which depend on the PMV-encoded RNA polymerase for replication. The atypical RNAs are defined by terminal polyadenylation and truncation events within the 3' untranslated region of the PMV genome. These modifications are reminiscent of host-mediated RNA degradation strategies and likely represent a previously underappreciated defense mechanism against invasive nucleic acids.

Keywords: Panicum mosaic virus; RNA degradation; polyadenylation; positive-sense RNA virus; satellite virus.

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Figures

FIG 1
FIG 1
The tripartite panicovirus pathosystem. (A) Genome organization of Panicum mosaic virus (top), satellite panicum mosaic virus (bottom left), and the PMV satellite RNAs (bottom right). Open reading frames and protein names are indicated within the colored boxes. The position of the UAG amber stop codon in the PMV genome is indicated by an asterisk. The SPMV putative ORF2 of unknown function is indicated by a question mark. The region of shared sequence similarity between the 3′ ends of PMV and satC (nt positions 347 to 444) RNAs is shown on the satC genome in red. Surface representations of the PMV (PDB 4V99) and SPMV (PDB 5CW0) virion biological assemblies are shown in forest green (top) and pale green (bottom), respectively. (B) Diseased St. Augustinegrass (Stenotaphrum secundatum) turfgrass with typical symptoms of St. Augustine decline caused by PMV and its satellite agents.
FIG 2
FIG 2
PMV and SPMV RNAs are polyadenylated in vivo during infection of Brachypodium. (A) Amplification of PMV and SPMV cDNAs from transcript-inoculated Brachypodium. Primers corresponding to the three PMV ORFs (CP, p6.6, and p8) and the three regions of the SPMV genome (positions 87 to 297, 297 to 541, and 87 to 541) were used to amplify cDNA from mRNA-enriched transcripts purified from infected plant tissues. Lanes M, reaction mixtures containing cDNA from mock-infected plant tissues; lanes I, reaction mixtures containing cDNA from PMV-SPMV-infected plant tissues. (B) PMV RNA containing the p48 ORF is polyadenylated during infection of Brachypodium. (Top) A genome schematic for PMV, indicating the forward and reverse primer position for amplification of the p48 ORF (arrows). The subgenomic RNA promoter region is indicated by a bent arrow. (Bottom) Amplification of the PMV p48 ORF from mock- and PMV-SPMV-infected Brachypodium cDNA at 10, 21, or 42 days postinoculation (dpi). Lane +, a PCR mixture containing the PMV infectious cDNA plasmid as a control for amplification; lane -RT, a reaction mixture with PMV-SPMV cDNA at 42 dpi where no reverse transcriptase enzyme was included for the cDNA synthesis reaction. Lanes L for panels A and B contain a DNA molecular weight marker.
FIG 3
FIG 3
The PMV 3′ UTR is notably edited in Brachypodium. (A) mRNA-enriched PMV RNAs contain major truncations and A/U-rich sequences in their 3′ UTRs. The 3′ end of the PMV capsid protein (CP) ORF is shown in green, followed by a horizontal line representing the 3′ UTR. Relative positions of the 3′ termini from 12 mRNA-enriched clones are indicated with vertical lines. The position of the truncated termini within the PMV genome is indicated by subscript numbers. Terminal residues corresponding to the truncation point in the wild-type genome are underlined. Nonviral sequences are shown to the right of the underlined residue, followed by an indicator for the RT-PCR oligo(dT) reverse primer ([AAAAAx30]). (B) Insertion and substitution events within the PMV 3′ UTR identified from short reads in the SRA. The scale of the UTR is the same as described for panel A. “Conflict” indicates substitution or deletion events; “insertion” indicates base insertion events.
FIG 4
FIG 4
PMV, SPMV, and satC RNAs are polyadenylated during natural infections of St. Augustinegrass. (Top) Three asymptomatic (A1 to A3) and symptomatic (S1 to S3) St. Augustinegrass leaf samples were collected from the Texas A&M University campus. Symptomatic samples were selected based on the typical chlorotic mottling associated with PMV infection and St. Augustine decline disease. (Bottom) Semiquanititative RT-PCR detection of PMV, SPMV, and satC cDNA from mRNA-enriched RNA purified from the six St. Augustinegrass tissue samples. Sample cDNAs were synthesized using oligo(dT) primers for reverse transcription. Primers for the PMV capsid protein ORF (PMV-CP), the SPMV capsid protein ORF (SPCP), and satC were used for amplification of PMV, SPMV, and satC cDNAs, respectively (see Table S2 in the supplemental material). Lane +, PCR mixtures containing the PMV, SPMV, or satC infectious cDNAs as positive controls for amplification.
FIG 5
FIG 5
The polyadenylated RNAs of PMV and SPMV are associated with purified virions from infected proso millet (Panicum miliaceum) hosts. (A) Purification of PMV and SPMV virions from infected proso millet. (Left panel) A comparison of symptoms among mock-, PMV-, and PMV-SPMV-infected proso millet. (Right panel) Typical sucrose density gradient showing the sedimentation patterns of purified SPMV (42S) and PMV (109S) virions. (B) Semiquantitative RT-PCR analyses of PMV and SPMV RNAs from purified virions. Purified virion RNAs were subjected to cDNA synthesis primed with random hexamers or oligo(dT) primers. PMV p8 and SPCP cDNA ORFs were amplified for detection of the PMV and SPMV RNAs, respectively. Lane L, DNA molecular weight marker; lane +, products from reaction mixtures containing PMV or SPMV cDNA plasmid templates as positive controls for the PCRs. (C) Relative percentage of poly(A) RNAs packaged within PMV and SPMV virions. Quantification was performed by comparing the relative abundance ratios of amplified cDNA products from oligo(dT)- and random hexamer-primed RT reactions for each of the purified virions. Products of cDNA amplification were quantified using ImageJ.
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References

    1. Nagy PD, Barajas D, Pogany J. 2012. Host factors with regulatory roles in tombusvirus replication. Curr Opin Virol 2:691–698. doi:10.1016/j.coviro.2012.10.004. - DOI - PubMed
    1. Nagy PD. 2016. Tombusvirus-host interactions: co-opted evolutionarily conserved host factors take center court. Annu Rev Virol 3:491–515. doi:10.1146/annurev-virology-110615-042312. - DOI - PubMed
    1. Hearne PQ, Knorr DA, Hillman BI, Morris TJ. 1990. The complete genome structure and synthesis of infectious RNA from clones of tomato bushy stunt virus. Virology 177:141–151. doi:10.1016/0042-6822(90)90468-7. - DOI - PubMed
    1. Sit TL, Lommel SA. 2015. Tombusviridae, p 1–9. In Encyclopedia of Life Sciences (eLS). John Wiley & Sons, Ltd, Chichester.
    1. Gunawardene CD, Donaldson LW, White KA. 2017. Tombusvirus polymerase: structure and function. Virus Res 234:74–86. doi:10.1016/j.virusres.2017.01.012. - DOI - PubMed

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