Turnip Mosaic Virus Transcriptional Slippage Dynamics and Distribution in RNA Subpopulations.

Potyviruses comprise the largest and most important group of plant positive-strand RNA viruses. The potyviral cell-to-cell movement protein P3N-PIPO is expressed via transcriptional slippage at a conserved GAAAAAA sequence, leading to insertion of an extra 'A' in a proportion of viral transcripts. Transcriptional slippage is determined by the potyviral replicase, the conserved slippery site, and its flanking nucleotides. Here, we investigate the dynamics of transcriptional slippage at different slip-site sequences, infection stages, and environmental conditions. We detect a modest increase in the level of transcripts with insertion towards later timepoints. In addition, we investigate the fate of transcripts with insertion by separately looking at different RNA subpopulations: (+)RNA, (-)RNA, translated RNA, and virion RNA. We find differences in insertional slippage between (+)RNA and (-)RNA but not other subpopulations. Our results suggest that there can be selection against the use of (-)RNAs with insertions as templates for transcription or replication and demonstrate that insertional slippage can occur at high frequency also during (-)RNA synthesis. Since transcripts with insertions are potential targets for degradation, we investigate the connection to nonsense-mediated decay (NMD). We find that these transcripts are targeted to NMD, but we only observe an impact on the level of transcripts with insertion when the insertional slippage rate is high. Together, these results further our understanding of the mechanism and elucidate the dynamics of potyviral transcriptional slippage. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Propensity of a picornavirus polymerase to slip on potyvirus-derived transcriptional slippage sites.

The substitution rates of viral polymerases have been studied extensively. However less is known about the tendency of these enzymes to 'slip' during RNA synthesis to produce progeny RNAs with nucleotide insertions or deletions. We recently described the functional utilization of programmed polymerase slippage in the family Potyviridae. This slippage results in either an insertion or a substitution, depending on whether the RNA duplex realigns following the insertion. In this study we investigated whether this phenomenon is a conserved feature of superfamily I viral RdRps, by inserting a range of potyvirus-derived slip-prone sequences into a picornavirus, Theiler's murine encephalomyelitis virus (TMEV). Deep-sequencing analysis of viral transcripts indicates that the TMEV polymerase 'slips' at the sequences U6-7 and A6-7 to insert additional nucleotides. Such sequences are under-represented within picornaviral genomes, suggesting that slip-prone sequences create a fitness cost. Nonetheless, the TMEV insertional and substitutional spectrum differed from that previously determined for the potyvirus polymerase.

Mutational analysis of the Potyviridae transcriptional slippage site utilized for expression of the P3N-PIPO and P1N-PISPO proteins.

The Potyviridae comprise the largest and most important family of RNA plant viruses. An essential overlapping ORF, termed pipo, resides in an internal region of the main polyprotein ORF. Recently, expression of pipo was shown to depend on programmed transcriptional slippage at a conserved GAAAAAA sequence, resulting in the insertion of an extra A into a proportion of viral transcripts, fusing the pipo ORF in frame with the 5' third of the polyprotein ORF. However, the sequence features that mediate slippage have not been characterized. Using a duplicate copy of the pipo slip site region fused into a different genomic location where it can be freely mutated, we investigated the sequence requirements for transcriptional slippage. We find that the leading G is not strictly required, but increased flanking sequence GC content correlates with higher insertion rates. A homopolymeric hexamer is optimal for producing mainly single-nucleotide insertions. We also identify an overabundance of G to A substitutions immediately 3'-adjacent to GAAAAAA in insertion-free transcripts, which we infer to result from a 'to-fro' form of slippage during positive-strand synthesis. Analysis of wild-type and reverse complement sequences suggests that slippage occurs preferentially during synthesis of poly(A) and therefore occurs mainly during positive-strand synthesis.

Transcriptional slippage in the positive-sense RNA virus family Potyviridae.

The family Potyviridae encompasses ~30% of plant viruses and is responsible for significant economic losses worldwide. Recently, a small overlapping coding sequence, termed pipo, was found to be conserved in the genomes of all potyvirids. PIPO is expressed as part of a frameshift protein, P3N-PIPO, which is essential for virus cell-to-cell movement. However, the frameshift expression mechanism has hitherto remained unknown. Here, we demonstrate that transcriptional slippage, specific to the viral RNA polymerase, results in a population of transcripts with an additional "A" inserted within a highly conserved GAAAAAA sequence, thus enabling expression of P3N-PIPO. The slippage efficiency is ~2% in Turnip mosaic virus and slippage is inhibited by mutations in the GAAAAAA sequence. While utilization of transcriptional slippage is well known in negative-sense RNA viruses such as Ebola, mumps and measles, to our knowledge this is the first report of its widespread utilization for gene expression in positive-sense RNA viruses.

Characterization of Ribosomal Frameshifting in Theiler's Murine Encephalomyelitis Virus.

Theiler's murine encephalomyelitis virus (TMEV) is a member of the genus Cardiovirus in the Picornaviridae, a family of positive-sense single-stranded RNA viruses. Previously, we demonstrated that in the related cardiovirus, Encephalomyocarditis virus, a programmed-1 ribosomal frameshift (1 PRF) occurs at a conserved G_GUU_UUU sequence within the 2B-encoding region of the polyprotein open reading frame (ORF). Here we show that-1 PRF occurs at a similar site during translation of the TMEV genome. In addition, we demonstrate that a predicted 3= RNA stem-loop structure at a noncanonical spacing downstream of the shift site is required for efficient frameshifting in TMEV and that frameshifting also requires virus infection. Mutating the G_GUU_UUU shift site to inhibit frameshifting results in an attenuated virus with reduced growth kinetics and a small-plaque phenotype. Frameshifting in the virus context was found to be extremely efficient at 74 to 82%, which, to our knowledge, is the highest frameshifting efficiency recorded to date for any virus. We propose that highly efficient-1 PRF in TMEV provides a mechanism to escape the confines of equimolar expression normally inherent in the single-polyprotein expression strategy of picornaviruses.

Cocksfoot mottle virus coat protein is dispensable for the systemic infection.

BACKGROUND: The Sobemovirus genome consists of polycistronic single-stranded positive-sense RNA. The first ORF encodes P1, a suppressor of RNA silencing required for virus movement. The coat protein (CP) is expressed from the 3' proximal ORF3 via subgenomic RNA. In addition to its structural role, the CP of some sobemoviruses has been reported to be required for systemic movement and to interact with P1. The aim of this study was to analyse the role of Cocksfoot mottle virus (CfMV) CP in the suppression of RNA silencing and virus movement. METHODS: Agrobacterium-mediated transient expression method was used for testing CfMV CP capacity to suppress RNA silencing. CP substitution and deletion mutants were generated to examine the role of this protein in CfMV infection, using three host plants (oat, barley and wheat). The viral movement was characterised with CfMV expressing EGFP fused to the C-terminus of CP. RESULTS: In the current study we show that CfMV CP is an additional RNA silencing suppressor. Interestingly, we observed that all CP mutant viruses were able to infect the three tested host plants systemically, although usually with reduced accumulation. CfMV expressing EGFP was detected in epidermal and mesophyll cells of inoculated leaves. Although EGFP fluorescence was not detected in upper leaves, some plants displayed CfMV symptoms. Analysis of the upper leaves revealed that the viruses had lost the EGFP sequence and sometimes also most of the CP gene. CONCLUSIONS: The present study demonstrates that CfMV CP suppresses RNA silencing but, surprisingly, is dispensable for systemic movement. Thus, CfMV does not move as virion in the tested host plants. The composition of the movement RNP complex remains to be elucidated.

Protein-RNA linkage and post-translational modifications of two sobemovirus VPgs.

Sobemoviruses possess a viral genome-linked protein (VPg) attached to the 5' end of viral RNA. VPg is processed from the viral polyprotein. In the current study, Cocksfoot mottle virus (CfMV) and Rice yellow mottle virus (RYMV) VPgs were purified from virions and analysed by mass spectrometry. The cleavage sites in the polyprotein and thereof the termini of VPg were experimentally proven. The lengths of the mature VPgs were determined to be 78 and 79 aa residues, respectively. The amino acid residues covalently linked to RNA in the two VPgs were, surprisingly, not conserved; it is a tyrosine at position 5 of CfMV VPg and serine at position 1 of RYMV VPg. Phosphorylations were identified in CfMV and RYMV VPgs with two positionally similar locations T20/S14 and S71/S72, respectively. RYMV VPg contains an additional phosphorylation site at S41.

Cocksfoot mottle sobemovirus coat protein contains two nuclear localization signals.

Cocksfoot mottle virus (CfMV) coat protein (CP) localization was studied in plant and mammalian cells. Fusion of the full-length CP with enhanced green fluorescent protein (EGFP) localized to the cell nucleus whereas similar constructs lacking the first 33 N-terminal amino acids of CP localized to the cytoplasm. CP and EGFP fusions containing mutations in the arginine-rich motif of CP localized to the cytoplasm and to the nucleus in plant cells indicating the involvement of the motif in nuclear localization. In mammalian cells, mutations in the arginine-rich region were sufficient to completely abolish nuclear transport. The analysis of deletions of amino acid residues 1-11, 1-22, and 22-33 of CP demonstrated that there were two separate nuclear localization signals (NLS) within the N-terminus--a strong NLS1 in the arginine-rich region (residues 22-33) and a weaker NLS2 within residues 1-22. Analysis of point mutants revealed that the basic amino acid residues in the region of the two NLSs were individually not sufficient to direct CP to the nucleus. Additional microinjection studies with fluorescently labeled RNA and CP purified from CfMV particles demonstrated that the wild-type CP was capable of transporting the RNA to the nucleus. This feature was not sequence-specific in transient assays since both CfMV and GFP mRNA were transported to the cell nucleus by CfMV CP. Together the results suggest that the nucleus may be involved in CfMV infection.

Identification of a novel member of 2H phosphoesterases, 2',5'-oligoadenylate degrading ribonuclease from the oyster Crassostrea gigas.

Several genes of IFN-mediated pathways in vertebrates, among them the genes that participate in the 2',5'-oligoadenylate synthetase (OAS)/RNase L pathway, have been identified in C. gigas. In the present study, we identified genes, which encode proteins having 2',5'-oligoadenylate degrading activity in C. gigas. These proteins belong to the 2H phosphoesterase superfamily and have sequence similarity to the mammalian A kinase anchoring protein 7 (AKAP7) central domain, which is responsible for the 2',5'-phosphodiesterase (2',5'-PDE) activity. Comparison of the genomic structures of C. gigas proteins with that of AKAP7 suggests that these enzymes originate from a direct common ancestor. However, the identified nucleases are not typical 2',5'-PDEs. The found enzymes catalyse the degradation of 2',5'-linked oligoadenylates in a metal-ion-independent way, yielding products with 2',3' -cyclic phosphate and 5'-OH termini similarly to the 3'-5' bond cleavage in RNA, catalyzed by metal-independent ribonucleases. 3',5'-linked oligoadenylates are not substrates for them. The preferred substrates for the C. gigas enzymes are 5'-triphosphorylated 2',5'-oligoadenylates, whose major cleavage reaction results in the removal of the 5'-triphosphorylated 2',3'-cyclic phosphate derivative, leaving behind the respective unphosphorylated 2',5'-oligoadenylate. Such a cleavage reaction results in the direct inactivation of the biologically active 2-5A molecule. The 2',5'-ribonucleases (2',5'-RNases) from C. gigas could be members of the ancient group of ribonucleases, specific to 2'-5' phosphodiester bond, together with the enzyme that was characterized previously from the marine sponge Tethya aurantium. The novel 2',5'-RNases may play a role in the control of cellular 2-5A levels, thereby limiting damage to host cells after viral infection.

Protein-RNA linkage and posttranslational modifications of feline calicivirus and murine norovirus VPg proteins.

Members of the Caliciviridae family of positive sense RNA viruses cause a wide range of diseases in both humans and animals. The detailed characterization of the calicivirus life cycle had been hampered due to the lack of robust cell culture systems and experimental tools for many of the members of the family. However, a number of caliciviruses replicate efficiently in cell culture and have robust reverse genetics systems available, most notably feline calicivirus (FCV) and murine norovirus (MNV). These are therefore widely used as representative members with which to examine the mechanistic details of calicivirus genome translation and replication. The replication of the calicivirus RNA genome occurs via a double-stranded RNA intermediate that is then used as a template for the production of new positive sense viral RNA, which is covalently linked to the virus-encoded protein VPg. The covalent linkage to VPg occurs during genome replication via the nucleotidylylation activity of the viral RNA-dependent RNA polymerase. Using FCV and MNV, we used mass spectrometry-based approach to identify the specific amino acid linked to the 5' end of the viral nucleic acid. We observed that both VPg proteins are covalently linked to guanosine diphosphate (GDP) moieties via tyrosine positions 24 and 26 for FCV and MNV respectively. These data fit with previous observations indicating that mutations introduced into these specific amino acids are deleterious for viral replication and fail to produce infectious virus. In addition, we also detected serine phosphorylation sites within the FCV VPg protein with positions 80 and 107 found consistently phosphorylated on VPg-linked viral RNA isolated from infected cells. This work provides the first direct experimental characterization of the linkage of infectious calicivirus viral RNA to the VPg protein and highlights that post-translational modifications of VPg may also occur during the viral life cycle.

A novel sweet potato potyvirus open reading frame (ORF) is expressed via polymerase slippage and suppresses RNA silencing.

The single-stranded, positive-sense RNA genome of viruses in the genus Potyvirus encodes a large polyprotein that is cleaved to yield 10 mature proteins. The first three cleavage products are P1, HCpro and P3. An additional short open reading frame (ORF), called pipo, overlaps the P3 region of the polyprotein ORF. Four related potyviruses infecting sweet potato (Ipomoea batatas) are predicted to contain a third ORF, called pispo, which overlaps the 3' third of the P1 region. Recently, pipo has been shown to be expressed via polymerase slippage at a conserved GA6 sequence. Here, we show that pispo is also expressed via polymerase slippage at a GA6 sequence, with higher slippage efficiency (∼5%) than at the pipo site (∼1%). Transient expression of recombinant P1 or the 'transframe' product, P1N-PISPO, in Nicotiana benthamiana suppressed local RNA silencing (RNAi), but only P1N-PISPO inhibited short-distance movement of the silencing signal. These results reveal that polymerase slippage in potyviruses is not limited to pipo expression, but can be co-opted for the evolution and expression of further novel gene products.

Cocksfoot mottle sobemovirus establishes infection through the phloem.

Cocksfoot mottle virus (CfMV) localization in oat plants was analyzed during three weeks post infection by immunohistochemical staining to follow its spread through different tissues. In early stages of infection, the virus was first detectable in phloem parenchyma and bundle sheath cells of inoculated leaves. Bundle sheath and phloem parenchyma were also the cell types where the virus was first detected in stems and systemic leaves of infected plants. In later stages of infection, CfMV spread also into the mesophyll surrounding vascular bundles and was seldom detected in xylem parenchyma of inoculated leaves. In systemic leaves, CfMV was not detected from xylem. Moreover, sometimes it was found from phloem only. In straw and roots, CfMV was detected both from phloem and xylem. According to our observations, CfMV predominantly moves through phloem, which makes the systemic movement of CfMV different from that of another monocot-infecting sobemovirus, Rice yellow mottle virus (RYMV).

Sobemovirus RNA linked to VPg over a threonine residue.

Positive sense ssRNA virus genomes from several genera have a viral protein genome-linked (VPg) attached over a phosphodiester bond to the 5' end of the genome. The VPgs of Southern bean mosaic virus (SBMV) and Ryegrass mottle virus (RGMoV) were purified from virions and analyzed by mass spectrometry. SBMV VPg was determined to be linked to RNA through a threonine residue at position one, whereas RGMoV VPg was linked to RNA through a serine also at the first position. In addition, we identified the termini of the corresponding VPgs and discovered three and seven phosphorylation sites in SBMV and RGMoV VPgs, respectively. This is the first report on the use of threonine for linking RNA to VPg.

Stem-loop structure of Cocksfoot mottle virus RNA is indispensable for programmed -1 ribosomal frameshifting.

The -1 programmed ribosomal frameshifting (-1 PRF) mechanism utilized by many viruses is dependent on a heptanucleotide slippery sequence and a downstream secondary structure element. In the current study, the RNA structure downstream from the slippery site of cocksfoot mottle sobemovirus (CfMV) was proven to be a 12bp stem-loop with a single bulge and a tetranucleotide loop. Several deletion and insertion mutants with altered stem-loop structures were tested in wheat germ extract (WGE) for frameshifting efficiency. The impact of the same mutations on virus infectivity was tested in oat plants. Mutations shortening or destabilizing the stem region reduced significantly but did not abolish -1 PRF in WGE. The same mutations proved to be deleterious for virus infection. However, extending the loop region to seven nucleotides had no significant effect on frameshifting efficiency in WGE and did not hamper virus replication in infected leaves. This is the first report about the experimentally proven RNA secondary structure directing -1 PRF of sobemoviruses.

P1 protein of Cocksfoot mottle virus is indispensable for the systemic spread of the virus.

Cocksfoot mottle sobemovirus (CfMV) encodes a non-conserved protein P1 from the 5' ORF1 of genomic RNA. The functions of CfMV P1 are unknown. In the current study we show that P1-deficient CfMV can replicate both in oat leaves and barley suspension culture cells but can not infect oat plants systemically. However, the absence of P1 reduces the efficiency of virus accumulation considerably. The infectivity of the mutant virus restores as a result of the spontaneous transversion. CfMV P1:EGFP shows a very limited cell-to-cell movement in leaf epidermal cells. In Sf9 insect cells CfMV P1 localizes in the fraction of membranes and inclusions but not in soluble cytoplasmic protein fraction.