Dimerization of an umbravirus RNA genome activates subgenomic mRNA transcription.

Many eukaryotic RNA viruses transcribe subgenomic (sg) mRNAs during infections to control expression of a subset of viral genes. Such transcriptional events are commonly regulated by local or long-range intragenomic interactions that form higher-order RNA structures within these viral genomes. In contrast, here we report that an umbravirus activates sg mRNA transcription via base pair-mediated dimerization of its plus-strand RNA genome. Compelling in vivo and in vitro evidence demonstrate that this viral genome dimerizes via a kissing-loop interaction involving an RNA stem-loop structure located just upstream from its transcriptional initiation site. Both specific and non-specific features of the palindromic kissing-loop complex were found to contribute to transcriptional activation. Structural and mechanistic aspects of the process in umbraviruses are discussed and compared with genome dimerization events in other RNA viruses. Notably, probable dimer-promoting RNA stem-loop structures were also identified in a diverse group of umbra-like viruses, suggesting broader utilization of this unconventional transcriptional strategy.

-1 Programmed ribosomal frameshifting in Class 2 umbravirus-like RNAs uses multiple long-distance interactions to shift between active and inactive structures and destabilize the frameshift stimulating element.

Plus-strand RNA viruses frequently employ -1 programmed ribosomal frameshifting (-1 PRF) to maximize their coding capacity. Ribosomes can frameshift at a slippery sequence if progression is impeded by a frameshift stimulating element (FSE), which is generally a stable, complex, dynamic structure with multiple conformations that contribute to the efficiency of -1 PRF. As FSE are usually analyzed separate from the viral genome, little is known about cis-acting long-distance interactions. Using full-length genomic RNA of umbravirus-like (ula)RNA citrus yellow vein associated virus (CY1) and translation in wheat germ extracts, six tertiary interactions were found associated with the CY1 FSE that span nearly three-quarters of the 2.7 kb genomic RNA. All six tertiary interactions are conserved in other Class 2 ulaRNAs and two are conserved in all ulaRNAs. Two sets of interactions comprise local and distal pseudoknots that involve overlapping FSE nucleotides and thus are structurally incompatible, suggesting that Class 2 FSEs assume multiple conformations. Importantly, two long-distance interactions connect with sequences on opposite sides of the critical FSE central stem, which would unzip the stem and destabilize the FSE. These latter interactions could allow a frameshifting ribosome to translate through a structurally disrupted upstream FSE that no longer blocks ribosome progression.

Crystal structure of a cap-independent translation enhancer RNA.

In eukaryotic messenger RNAs, the 5' cap structure binds to the translation initiation factor 4E to facilitate early stages of translation. Although many plant viruses lack the 5' cap structure, some contain cap-independent translation elements (CITEs) in their 3' untranslated region. The PTE (Panicum mosaic virus translation element) class of CITEs contains a G-rich asymmetric bulge and a C-rich helical junction that were proposed to interact via formation of a pseudoknot. SHAPE analysis of PTE homologs reveals a highly reactive guanosine residue within the G-rich region proposed to mediate eukaryotic initiation factor 4E (eIF4E) recognition. Here we have obtained the crystal structure of the PTE from Pea enation mosaic virus 2 (PEMV2) RNA in complex with our structural chaperone, Fab BL3-6. The structure reveals that the G-rich and C-rich regions interact through a complex network of interactions distinct from those expected for a pseudoknot. The motif, which contains a short parallel duplex, provides a structural mechanism for how the guanosine is extruded from the core stack to enable eIF4E recognition. Homologous PTE elements harbor a G-rich bulge and a three-way junction and exhibit covariation at crucial positions, suggesting that the PEMV2 tertiary architecture is conserved among these homologs.

Complete genome sequence of a new putative member of the genus Pelarspovirus that infects Quercus aliena Blume in South Korea.

The complete nucleotide sequence of a newly discovered virus infecting Quercus aliena Blume, tentatively named "quercus leafroll virus" (QLRV), was determined through high-throughput and Sanger sequencing. The sequence comprises 3,940 nucleotides, has five open reading frames, and has a typical pelarspovirus genome organization, with neither 3' polyadenylation nor a 5' cap. The proteins encoded by QLRV share 17.9 to 44.2% amino acid sequence identity with known pelarspovirus proteins. The highest amino acid sequence identity values for the RNA-dependent RNA polymerase (RdRp) and coat protein were 67.5% and 55.2%, respectively, which are below the current thresholds for pelarspovirus species demarcation. On the basis of these results, we propose classifying QLRV as a new member of the genus Pelarspovirus, family Tombusviridae.

Effects of Maize Chlorotic Mottle Virus and Potyvirus Resistance on Maize Lethal Necrosis Disease.

Maize lethal necrosis (MLN) is a viral disease caused by host co-infection by maize chlorotic mottle virus (MCMV) and a potyvirus, such as sugarcane mosaic virus (SCMV). The disease is most effectively managed by growing MLN-resistant varieties. However, the relative importance of MCMV and potyvirus resistance in managing this synergistic disease is poorly characterized. In this study, we evaluated the effects of SCMV and/or MCMV resistance on disease, virus titers, and synergism and explored expression patterns of known potyvirus resistance genes TrxH and ABP1. MLN disease was significantly lower in both the MCMV-resistant and SCMV-resistant inbred lines compared with the susceptible control Oh28. Prior to 14 days postinoculation (dpi), MCMV titers in resistant lines N211 and KS23-6 were more than 100,000-fold lower than found in the susceptible Oh28. However, despite no visible symptoms, titer differences between MCMV-resistant and -susceptible lines were negligible by 14 dpi. In contrast, systemic SCMV titers in the potyvirus-resistant line, Pa405, ranged from 130,000-fold to 2 million-fold lower than susceptible Oh28 as disease progressed. Initial TrxH expression was up to 49,000-fold lower in Oh28 compared with other genotypes, whereas expression of ABP1 was up to 4.5-fold lower. Measures of virus synergy indicate that whereas MCMV resistance is effective in early infection, strong potyvirus resistance is critical for reducing synergist effects of co-infection on MCMV titer. These results emphasize the importance of both potyvirus resistance and MCMV resistance in an effective breeding program for MLN management.

Structural characterization of a new subclass of panicum mosaic virus-like 3' cap-independent translation enhancer.

Canonical eukaryotic mRNA translation requires 5'cap recognition by initiation factor 4E (eIF4E). In contrast, many positive-strand RNA virus genomes lack a 5'cap and promote translation by non-canonical mechanisms. Among plant viruses, PTEs are a major class of cap-independent translation enhancers located in/near the 3'UTR that recruit eIF4E to greatly enhance viral translation. Previous work proposed a single form of PTE characterized by a Y-shaped secondary structure with two terminal stem-loops (SL1 and SL2) atop a supporting stem containing a large, G-rich asymmetric loop that forms an essential pseudoknot (PK) involving C/U residues located between SL1 and SL2. We found that PTEs with less than three consecutive cytidylates available for PK formation have an upstream stem-loop that forms a kissing loop interaction with the apical loop of SL2, important for formation/stabilization of PK. PKs found in both subclasses of PTE assume a specific conformation with a hyperreactive guanylate (G*) in SHAPE structure probing, previously found critical for binding eIF4E. While PTE PKs were proposed to be formed by Watson-Crick base-pairing, alternative chemical probing and 3D modeling indicate that the Watson-Crick faces of G* and an adjacent guanylate have high solvent accessibilities. Thus, PTE PKs are likely composed primarily of non-canonical interactions.

Identification of Novel 5' and 3' Translation Enhancers in Umbravirus-Like Coat Protein-Deficient RNA Replicons.

Translation of plant plus-strand RNA viral genomes that lack a 5' cap frequently requires the use of cap-independent translation enhancers (CITEs) located in or near the 3' untranslated region (UTR). 3'CITEs are grouped based on secondary structure and ability to interact with different translation initiation factors or ribosomal subunits, which assemble a complex at the 3' end that is nearly always transferred to the 5' end via a long-distance kissing-loop interaction between sequences in the 3'CITE and 5' hairpins. We report here the identification of a novel 3'CITE in coat protein-deficient RNA replicons that are related to umbraviruses. Umbra-like associated RNAs (ulaRNAs), such as citrus yellow vein-associated virus (CYVaV), are a new type of subviral RNA that do not encode movement proteins, coat proteins, or silencing suppressors but can independently replicate using their encoded RNA-dependent RNA polymerase. An extended hairpin structure containing multiple internal loops in the 3' UTR of CYVaV is strongly conserved in the most closely related ulaRNAs and structurally resembles an I-shaped structure (ISS) 3'CITE. However, unlike ISS, the CYVaV structure binds to eIF4G and no long-distance interaction is discernible between the CYVaV ISS-like structure and sequences at or near the 5' end. We also report that the ∼30-nucleotide (nt) 5' terminal hairpin of CYVaV and related ulaRNAs can enhance translation of reporter constructs when associated with either the CYVaV 3'CITE or the 3'CITEs of umbravirus pea enation mosaic virus (PEMV2) and even independent of a 3'CITE. These findings introduce a new type of 3'CITE and provide the first information on translation of ulaRNAs. IMPORTANCE Umbra-like associated RNAs (ulaRNAs) are a recently discovered type of subviral RNA that use their encoded RNA-dependent RNA polymerase for replication but do not encode any coat proteins, movement proteins, or silencing suppressors yet can be found in plants in the absence of any discernible helper virus. We report the first analysis of their translation using class 2 ulaRNA citrus yellow vein-associated virus (CYVaV). CYVaV uses a novel eIF4G-binding I-shaped structure as its 3' cap-independent translation enhancer (3'CITE), which does not connect with the 5' end by a long-distance RNA:RNA interaction that is typical of 3'CITEs. ulaRNA 5' terminal hairpins can also enhance translation in association with cognate 3'CITEs or those of related ulaRNAs and, to a lesser extent, with 3'CITEs of umbraviruses, or even independent of a 3'CITE. These findings introduce a new type of 3'CITE and provide the first information on translation of ulaRNAs.

Effects of the Noncoding Subgenomic RNA of Red Clover Necrotic Mosaic Virus in Virus Infection.

In recent years, a new class of viral noncoding subgenomic RNA (ncsgRNA) has been identified. This RNA is generated as a stable degradation product via an exoribonuclease-resistant RNA (xrRNA) structure, which blocks the progression of 5'→3' exoribonuclease on viral RNAs in infected cells. Here, we assess the effects of the ncsgRNA of red clover necrotic mosaic virus (RCNMV), called SR1f, in infected plants. We demonstrate the following: (i) the absence of SR1f reduces symptoms and decreases viral RNA accumulation in Nicotiana benthamiana and Arabidopsis thaliana plants; (ii) SR1f has an essential function other than suppression of RNA silencing; and (iii) the cytoplasmic exoribonuclease involved in mRNA turnover, XRN4, is not required for SR1f production or virus infection. A comparative transcriptomic analysis in N. benthamiana infected with wild-type RCNMV or an SR1f-deficient mutant RCNMV revealed that wild-type RCNMV infection, which produces SR1f and much higher levels of virus, has a greater and more significant impact on cellular gene expression than the SR1f-deficient mutant. Upregulated pathways include plant hormone signaling, plant-pathogen interaction, MAPK signaling, and several metabolic pathways, while photosynthesis-related genes were downregulated. We compare this to host genes known to participate in infection by other tombusvirids. Viral reads revealed a 10- to 100-fold ratio of positive to negative strand, and the abundance of reads of both strands mapping to the 3' region of RCNMV RNA1 support the premature transcription termination mechanism of synthesis for the coding sgRNA. These results provide a framework for future studies of the interactions and functions of noncoding RNAs of plant viruses. IMPORTANCE Knowledge of how RNA viruses manipulate host and viral gene expression is crucial to our understanding of infection and disease. Unlike viral protein-host interactions, little is known about the control of gene expression by viral RNA. Here, we begin to address this question by investigating the noncoding subgenomic RNA (ncsgRNA) of red clover necrotic mosaic virus (RCNMV), called SR1f. Similar exoribonuclease-resistant RNAs of flaviviruses are well studied, but the roles of plant viral ncsgRNAs, and how they arise, are poorly understood. Surprisingly, we find the likely exonuclease candidate, XRN4, is not required to generate SR1f, and we assess the effects of SR1f on virus accumulation and symptom development. Finally, we compare the effects of infection by wild-type RCNMV versus an SR1f-deficient mutant on host gene expression in Nicotiana benthamiana, which reveals that ncsgRNAs such as SR1f are key players in virus-host interactions to facilitate productive infection.

Maize miR167-ARF3/30-polyamine oxidase 1 module-regulated H2O2 production confers resistance to maize chlorotic mottle virus.

Maize chlorotic mottle virus (MCMV) is the key pathogen causing maize lethal necrosis (MLN). Due to the sharply increased incidence of MLN in many countries, there is an urgent need to identify resistant lines and uncover the underlying resistance mechanism. Here, we showed that the abundance of maize (Zea mays) microR167 (Zma-miR167) positively modulates the degree of resistance to MCMV. Zma-miR167 directly targets Auxin Response Factor3 (ZmARF3) and ZmARF30, both of which negatively regulate resistance to MCMV. RNA-sequencing coupled with gene expression assays revealed that both ZmARF3 and ZmARF30 directly bind the promoter of Polyamine Oxidase 1 (ZmPAO1) and activate its expression. Knockdown or inhibition of enzymatic activity of ZmPAO1 suppressed MCMV infection. Nevertheless, MCMV-encoded p31 protein directly targets ZmPAO1 and enhances the enzyme activity to counteract Zma-miR167-mediated defense to some degree. We uncovered a role of the Zma-miR167-ZmARF3/30 module for restricting MCMV infection by regulating ZmPAO1 expression, while MCMV employs p31 to counteract this defense.

Molecular characterization of a novel gammacarmovirus infecting cucurbits in India.

In this study, we describe the identification of a new gammacarmovirus infecting Cucurbita pepo plants showing a range of mosaic, stunting, yellowing, and wilting symptoms. The virus had a narrow host range and mostly produced chlorotic and necrotic local lesions in the majority of the tested plants. However, Nicotiana benthamiana showed systemic symptoms under laboratory conditions. Using a combination of Sanger sequencing and rapid amplification of cDNA ends (RACE), the complete genome sequence of the virus was determined to be 4274 nucleotides (nt) in length. Its genome organization is similar to that of members of the genus Gammacarmovirus in the family Tombusviridae, consisting of five overlapping open reading frames (ORFs) encoding p28, replicase, p7A, p7B, and coat protein (CP), respectively. The genome is flanked by short 5' and 3' non-coding regions (NCR) at either end. In pairwise comparisons of replicase and CP sequences, the virus showed the highest amino acid sequence identity of 71.55% and 54.86%, respectively, to melon necrotic spot virus (MNSV), the type member of the genus Gammacarmovirus. Since the sequence identity values are below the species demarcation threshold suggested by the International Committee on Taxonomy of Viruses (ICTV), the virus from Cucurbita pepo plants, for which the name "cucurbit carmovirus" (CuCV) is proposed, represents a new species. In phylogenetic analysis based on the replicase and CP amino acid sequences, CuCV clustered with MNSV but formed a distinct branch, further confirming that the virus is a distinct member of the genus Gammacarmovirus.

Exploring the diversity and evolution of tombus-like viruses: phylogenetic analysis, recombination events, and suppressor protein homologs.

This study focuses on the phylogenetic analysis of previously unclassified tombus-like viruses, which are characterized by the presence of homologs of the suppressor protein p19. The primary objectives of this research were to investigate the evolutionary relationships among these viruses and to explore the impact of suppressor proteins and recombination events on their evolution. A dataset comprising 94 viral sequences was analyzed to achieve these goals. The phylogenetic analysis revealed the presence of two distinct clusters within the tombus-like virus group. One cluster consisted of viruses that encoded p19-like RNA suppressors, while the other cluster comprised viruses encoding p14-like suppressors. Based on these findings, we propose the classification of PGT-pt108 as an isolate of carnation Italian ringspot virus (CIRV), and both Tombusviridae sp. s48-k141_139792 and Tombusviridae sp. s51-k141_185213 as isolates of tomato bushy stunt virus (TBSV). Furthermore, this study suggests the establishment of two new genera within the family Tombusviridae, based on the observed divergence and distinct characteristics of these tombus-like viruses. Through the analysis of recombination events, we provide insights into the interspecies movement of CIRV, which is reflected in its phylogenetic positioning. This research contributes to our understanding of the evolutionary dynamics and classification of tombus-like viruses, shedding light on the role of suppressor proteins and recombination events in their evolution and interspecies transmission.

The complete genome sequence of Neckar River virus confirms it to be a distinct member of the genus Tombusvirus in the family Tombusviridae.

Neckar River virus (NRV), first isolated from a water sample of the Neckar River (Germany) in the 1980s, was serologically characterized as a novel tombusvirus. In this study, the complete genome sequence was determined, and an infectious full-length cDNA clone was constructed. The genome organization of NRV (DSMZ PV-0270) resembles that of tombusviruses. The genome consists of 4739 nucleotides and contains five open reading frames (ORFs) and one additional putative ORF (pX) in the 3'-terminal region. Phylogenetic analysis and sequence comparisons confirmed NRV to be a member of the species Tombusvirus neckarfluminis in the genus Tombusvirus. The infectious full-length cDNA clone was constructed using Gibson assembly and subsequent infection of Nicotiana benthamiana plants by Rhizobium radiobacter inoculation. The virus derived from the full-length cDNA clone caused symptoms resembling those caused by the wild-type virus, but slightly milder.

Genomic characterization of two new viruses infecting Ageratum conyzoides in China.

Two new RNA viruses were identified in Ageratum conyzoides in China using high-throughput sequencing, and their genome sequences were determined using PCR and rapid amplification of cDNA ends. The new viruses, which have positive-sense, single-stranded RNA genomes, were provisionally named "ageratum virus 1" (AgV1) and "ageratum virus 2" (AgV2). AgV1 has a genome of 3,526 nucleotides with three open reading frames (ORFs) and shares 49.9% nucleotide sequence identity with the complete genome of Ethiopian tobacco bushy top virus (genus Umbravirus, family Tombusviridae). The genome of AgV2 consists of 5,523 nucleotides and contains five ORFs that are commonly observed in members of the genus Enamovirus of the family Solemoviridae. Proteins encoded by AgV2 exhibited the highest amino acid sequence similarity (31.7-75.0% identity) to the corresponding proteins of pepper enamovirus R1 (an unclassified enamovirus) and citrus vein enation virus (genus Enamovirus). Based on their genome organization, sequence, and phylogenetic relationships, AgV1 is proposed to be a new umbra-like virus of the family Tombusviridae, and AgV2 is proposed to be a new member of the genus Enamovirus of the family Solemoviridae.

The complete genome sequence of Stellaria aquatica virus A, a new member of the genus Alphacarmovirus, family Tombusviridae.

A new member of the genus Alphacarmovirus was detected in Stellaria aquatica using high-throughput RNA sequencing analysis. The complete genome sequence of this new virus isolate, tentatively named "Stellaria aquatica virus A" (StAV-A), comprises 4,017 nucleotides with five predicted open reading frames (ORFs) and has a typical alphacarmovirus genome organization. Pairwise comparison of StAV-A with selected members of family Tombusviridae showed 44-58%, 32-64%, and 19-49% sequence identity for the overall nucleotide sequence, polymerase, and coat protein, respectively. Phylogenetic analysis of polymerase sequences places StAV-A alongside other members of the genus Alphacarmovirus in the family Tombusviridae.

Molecular characterization of a novel umbra-like virus from Thuja orientalis (arborvitae) in South Korea.

A novel umbra-like virus was identified in arborvitae in South Korea using RNA sequencing (RNA-seq). The virus identified was tentatively named "arborvitae umbra-like virus" (AULV) and contained a 4,300-nucleotide genome organized into four non-structural open reading frames (ORFs). Cloning and Sanger sequencing were used to confirm the viral contig sequence and determine the size of the genome. Genome analysis indicated that ORF2 encodes an RNA-dependent RNA polymerase that is probably expressed through ribosomal frameshifting. ORF3 encodes a putative long-distance movement protein, while the functions of ORFs 1 and 4 are unknown. The virus lacks a coat protein gene. The genome of AULV shares 27.3%-48.4% nucleotide sequence identity with closely related umbraviruses. Phylogenetic analysis based on the complete genome sequences and amino acid sequences of the RNA-dependent RNA polymerase revealed that AULV forms a monophyletic lineage with Guiyang paspalum paspaloides tombus-like virus (GPpTV1). We suggest that AULV is a novel umbra-like virus belonging to the family Tombusviridae.

The mitochondrial and chloroplast dual targeting of a multifunctional plant viral protein modulates chloroplast-to-nucleus communication, RNA silencing suppressor activity, encapsidation, pathogenesis and tissue tropism.

Plant defense against melon necrotic spot virus (MNSV) is triggered by the viral auxiliary replicase p29 that is targeted to mitochondrial membranes causing morphological alterations, oxidative burst and necrosis. Here we show that MNSV coat protein (CP) was also targeted to mitochondria and mitochondrial-derived replication complexes [viral replication factories or complex (VRC)], in close association with p29, in addition to chloroplasts. CP import resulted in the cleavage of the R/arm domain previously implicated in genome binding during encapsidation and RNA silencing suppression (RSS). We also show that CP organelle import inhibition enhanced RSS activity, CP accumulation and VRC biogenesis but resulted in inhibition of systemic spreading, indicating that MNSV whole-plant infection requires CP organelle import. We hypothesize that to alleviate the p29 impact on host physiology, MNSV could moderate its replication and p29 accumulation by regulating CP RSS activity through organelle targeting and, consequently, eluding early-triggered antiviral response. Cellular and molecular events also suggested that S/P domains, which correspond to processed CP in chloroplast stroma or mitochondrion matrix, could mitigate host response inhibiting p29-induced necrosis. S/P deletion mainly resulted in a precarious balance between defense and counter-defense responses, generating either cytopathic alterations and MNSV cell-to-cell movement restriction or some degree of local movement. In addition, local necrosis and defense responses were dampened when RSS activity but not S/P organelle targeting was affected. Based on a robust biochemical and cellular analysis, we established that the mitochondrial and chloroplast dual targeting of MNSV CP profoundly impacts the viral infection cycle.

Metagenomic identification of novel viruses of maize and teosinte in North America.

BACKGROUND: Maize-infecting viruses are known to inflict significant agronomic yield loss throughout the world annually. Identification of known or novel causal agents of disease prior to outbreak is imperative to preserve food security via future crop protection efforts. Toward this goal, a large-scale metagenomic approach utilizing high throughput sequencing (HTS) was employed to identify novel viruses with the potential to contribute to yield loss of graminaceous species, particularly maize, in North America. RESULTS: Here we present four novel viruses discovered by HTS and individually validated by Sanger sequencing. Three of these viruses are RNA viruses belonging to either the Betaflexiviridae or Tombusviridae families. Additionally, a novel DNA virus belonging to the Geminiviridae family was discovered, the first Mastrevirus identified in North American maize. CONCLUSIONS: Metagenomic studies of crop and crop-related species such as this may be useful for the identification and surveillance of known and novel viral pathogens of crops. Monitoring related species may prove useful in identifying viruses capable of infecting crops due to overlapping insect vectors and viral host-range to protect food security.

A rapid and simple quantitative method for specific detection of smaller coterminal RNA by PCR (DeSCo-PCR): application to the detection of viral subgenomic RNAs.

RNAs that are 5'-truncated versions of a longer RNA but share the same 3' terminus can be generated by alternative promoters in transcription of cellular mRNAs or by replicating RNA viruses. These truncated RNAs cannot be distinguished from the longer RNA by a simple two-primer RT-PCR because primers that anneal to the cDNA from the smaller RNA also anneal to-and amplify-the longer RNA-derived cDNA. Thus, laborious methods, such as northern blot hybridization, are used to distinguish shorter from longer RNAs. For rapid, low-cost, and specific detection of these truncated RNAs, we report detection of smaller coterminal RNA by PCR (DeSCo-PCR). DeSCo-PCR uses a nonextendable blocking primer (BP), which outcompetes a forward primer (FP) for annealing to longer RNA-derived cDNA, while FP outcompetes BP for annealing to shorter RNA-derived cDNA. In the presence of BP, FP, and the reverse primer, only cDNA from the shorter RNA is amplified in a single-tube reaction containing both RNAs. Many positive strand RNA viruses generate 5'-truncated forms of the genomic RNA (gRNA) called subgenomic RNAs (sgRNA), which play key roles in viral gene expression and pathogenicity. We demonstrate that DeSCo-PCR is easily optimized to selectively detect relative quantities of sgRNAs of red clover necrotic mosaic virus from plants and Zika virus from human cells, each infected with viral strains that generate different amounts of sgRNA. This technique should be readily adaptable to other sgRNA-producing viruses, and for quantitative detection of any truncated or alternatively spliced RNA.

Opium Poppy Mosaic Virus Has an Xrn-Resistant, Translated Subgenomic RNA and a BTE 3' CITE.

Opium poppy mosaic virus (OPMV) is a recently discovered umbravirus in the family Tombusviridae OPMV has a plus-sense genomic RNA (gRNA) of 4,241 nucleotides (nt) from which replication protein p35 and p35 extension product p98, the RNA-dependent RNA polymerase (RdRp), are expressed. Movement proteins p27 (long distance) and p28 (cell to cell) are expressed from a 1,440-nt subgenomic RNA (sgRNA2). A highly conserved structure was identified just upstream from the sgRNA2 transcription start site in all umbraviruses, which includes a carmovirus consensus sequence, denoting generation by an RdRp-mediated mechanism. OPMV also has a second sgRNA of 1,554 nt (sgRNA1) that starts just downstream of a canonical exoribonuclease-resistant sequence (xrRNAD). sgRNA1 codes for a 30-kDa protein in vitro that is in frame with p28 and cannot be synthesized in other umbraviruses. Eliminating sgRNA1 or truncating the p30 open reading frame (ORF) without affecting p28 substantially reduced accumulation of OPMV gRNA, suggesting a functional role for the protein. The 652-nt 3' untranslated region of OPMV contains two 3' cap-independent translation enhancers (3' CITEs), a T-shaped structure (TSS) near its 3' end, and a Barley yellow dwarf virus-like translation element (BTE) in the central region. Only the BTE is functional in luciferase reporter constructs containing gRNA or sgRNA2 5' sequences in vivo, which differs from how umbravirus 3' CITEs were used in a previous study. Similarly to most 3' CITEs, the OPMV BTE links to the 5' end via a long-distance RNA-RNA interaction. Analysis of 14 BTEs revealed additional conserved sequences and structural features beyond the previously identified 17-nt conserved sequence.IMPORTANCEOpium poppy mosaic virus (OPMV) is an umbravirus in the family Tombusviridae We determined that OPMV accumulates two similarly sized subgenomic RNAs (sgRNAs), with the smaller known to code for proteins expressed from overlapping open reading frames. The slightly larger sgRNA1 has a 5' end just upstream from a previously predicted xrRNAD site, identifying this sgRNA as an unusually long product produced by exoribonuclease trimming. Although four umbraviruses have similar predicted xrRNAD sites, only sgRNA1 of OPMV can code for a protein that is an extension product of umbravirus ORF4. Inability to generate the sgRNA or translate this protein was associated with reduced gRNA accumulation in vivo We also characterized the OPMV BTE structure, a 3' cap-independent translation enhancer (3' CITE). Comparisons of 13 BTEs with the OPMV BTE revealed additional stretches of sequence similarity beyond the 17-nt signature sequence, as well as conserved structural features not previously recognized in these 3' CITEs.

A trans-activator-like structure in RCNMV RNA1 evokes the origin of the trans-activator in RNA2.

The Red clover necrotic mosaic virus (RCNMV) genome consists of two plus-strand RNA genome segments, RNA1 and RNA2. RNA2 contains a multifunctional RNA structure known as the trans-activator (TA) that (i) promotes subgenomic mRNA transcription from RNA1, (ii) facilitates replication of RNA2, and (iii) mediates particle assembly and copackaging of genome segments. The TA has long been considered a unique RNA element in RCNMV. However, by examining results from RCNMV genome analyses in the ViRAD virus (re-)annotation database, a putative functional RNA element in the polymerase-coding region of RNA1 was identified. Structural and functional analyses revealed that the novel RNA element adopts a TA-like structure (TALS) and, similar to the requirement of the TA for RNA2 replication, the TALS is necessary for the replication of RNA1. Both the TA and TALS possess near-identical asymmetrical internal loops that are critical for efficient replication of their corresponding genome segments, and these structural motifs were found to be functionally interchangeable. Moreover, replacement of the TA in RNA2 with a stabilized form of the TALS directed both RNA2 replication and packaging of both genome segments. Based on their comparable properties and considering evolutionary factors, we propose that the TALS appeared de novo in RNA1 first and, subsequently, the TA arose de novo in RNA2 as a functional mimic of the TALS. This and other related information were used to formulate a plausible evolutionary pathway to describe the genesis of the bi-segmented RCNMV genome. The resulting scenario provides an evolutionary framework to further explore and test possible origins of this segmented RNA plant virus.