Targeting the Highly Conserved 3' Untranslated Region of Iris Severe Mosaic Virus for Sensitive Monitoring of the Disease Prevalence in Iris Production.

Iris severe mosaic virus (ISMV, Potyviridae) can threaten the sustainability of iris production and the marketability of the plants. Effective intervention and control strategies require rapid and early detection of viral infections. The wide range of viral symptoms, from asymptomatic to severe chlorosis of the leaves, renders diagnosis solely based on visual indicators ineffective. A nested PCR-based diagnostic assay was developed for the reliable detection of ISMV in iris leaves and in rhizomes. Considering the genetic variability of ISMV, two primer pairs were designed to detect the highly conserved 3' untranslated region (UTR) of the viral genomic RNA. The specificity of the primer pairs was confirmed against four other potyviruses. The sensitivity of detection was enhanced by one order of magnitude using diluted cDNA and a nested approach. Nested PCR facilitated detecting ISMV on field-grown samples beyond the capabilities of a currently available immunological test and in iris rhizome, which would facilitate ensuring clean stock is planted. This approach dramatically improves the detection threshold of ISMV on potentially low virus titer samples. The study provides a practical, accurate, and sensitive tool for the early detection of a deleterious virus that infects a popular ornamental and landscape plant.

All eggs in one basket: How potyvirus infection is controlled at a single cap-independent translation event.

Regulation of protein synthesis is a strong determinant of potyviral pathogenicity. The Potyviridae family is the largest family of plant-infecting positive sense RNA viruses. Similar to the animal-infecting Picornaviridae family, the potyviral RNA genome lacks a 5' cap, and instead has a viral protein (VPg) linked to its 5' end. Potyviral genomes are mainly translated into one large polyprotein relying on a single translation event to express all their protein repertoire. In the absence of the 5' cap, the Potyviridae family depends on cis-acting elements in their 5' untranslated regions (UTR) to recruit the translation machinery. In this review, we summarize the diverse 5'UTR-driven, cap-independent translation mechanisms employed by the Potyviridae family including scanning-dependent mechanism, internal initiation, and the stimulatory role of the VPg. These mechanisms have direct implications on potyviral pathogenicity, including host range specificity and resistance. Finally, we discuss how these viral strategies could not only inform new avenues for engineering and/or breeding for crop resistance but would also provide opportunities for the development of biotechnological tools for large-scale protein production in plant systems.

Multiple Cis-acting Polypyrimidine Tract Elements Regulate a Cooperative Mechanism for Triticum Mosaic Virus Internal Ribosomal Entry Site Activity.

Diverse elements within the 5' untranslated region of an mRNA can influence the translation efficiency at the main AUG codon. We previously identified a core picornaviral like Y16X11-AUG motif with 16-nt polypyrimidine CU tract separated by an 11-nt spacer sequence from the 13th AUG codon, which is recognized as the preferred initiation site within the Triticum mosaic virus (TriMV) internal ribosome entry site (IRES) element. The motif is proposed to function as an internal ribosomal landing site at the designated start codon. Here, we exposed the cooperative role of multiple CU-rich segments flanking the TriMV YX-AUG motif to reach and drive internal initiation of translation at the preferred start site. We propose that these auxiliary domains may enhance the ribosome capacity and their delivery at proximity of the correct initiation site. These polypyrimidine tracts can be modulated with a cryptic AUG in a position-dependent manner to replace the native YX-AUG motif, and thus uncovering a new layer of control of start codon selection. In line with these observations, mass spectrometry analysis of proteins directly interacting with translationally impaired TriMV IRES mutants that bear these motifs indicated an enrichment in 40S and 60S ribosomal related proteins, revealing a new function of polypyrimidine tracts to regulate IRES-driven translation. Accessibility of these RNA regions for in trans interaction was validated by SHAPE analysis of the entire TriMV leader sequence and supported by the ability of anti-sense oligonucleotides designed to block the CU tracts accessibility to impair IRES activity. This is the first evidence that defines the core modular domains required for ribosomal recruitment and start codon selection in a complex, multi-AUG viral 5' UTR for translation in plants.

StPIP1, a PAMP-induced peptide in potato, elicits plant defenses and is associated with disease symptom severity in a compatible interaction with Potato virus Y.

The role of small secreted peptides in plant defense responses to viruses has seldom been investigated. Here, we report a role for potato (Solanum tuberosum) PIP1, a gene predicted to encode a member of the pathogen-associated molecular pattern (PAMP)-induced peptide (PIP) family, in the response of potato to Potato virus Y (PVY) infection. We show that exogenous application of synthetic StPIP1 to potato leaves and nodes increased the production of reactive oxygen species and the expression of plant defense-related genes, revealing that StPIP1 triggers early defense responses. In support of this hypothesis, transgenic potato plants that constitutively overexpress StPIP1 had higher levels of leaf callose deposition and, based on measurements of viral RNA titers, were less susceptible to infection by a compatible PVY strain. Interestingly, systemic infection of StPIP1-overexpressing lines with PVY resulted in clear rugose mosaic symptoms that were absent or very mild in infected non-transgenic plants. A transcriptomics analysis revealed that marker genes associated with both pattern-triggered immunity and effector-triggered immunity were induced in infected StPIP1 overexpressors but not in non-transgenic plants. Together, our results reveal a role for StPIP1 in eliciting plant defense responses and in regulating plant antiviral immunity.

Overexpression of a modified eIF4E regulates potato virus Y resistance at the transcriptional level in potato.

BACKGROUND: Potato virus Y (PVY) is a major pathogen of potatoes with major impact on global agricultural production. Resistance to PVY can be achieved by engineering potatoes to express a recessive, resistant allele of eukaryotic translation initiation factor eIF4E, a host dependency factor essential to PVY replication. Here we analyzed transcriptome changes in eIF4E over-expressing potatoes to shed light on the mechanism underpinning eIF4E-mediated recessive PVY resistance. RESULTS: As anticipated, modified eIF4E-expressing potatoes demonstrated a high level of resistance, eIF4E expression, and an unexpected suppression of the susceptible allele transcript, likely explaining the bulk of the potent antiviral phenotype. In resistant plants, we also detected marked upregulation of genes involved in cell stress responses. CONCLUSIONS: Our results reveal a previously unanticipated second layer of signaling attributable to eIF4E regulatory control, and potentially relevant to establishment of a broader, more systematic antiviral host defense.

HCPro Suppression of Callose Deposition Contributes to Strain-Specific Resistance Against Potato Virus Y.

Potato virus Y (PVY; Potyviridae) is a continuing challenge for potato production owing to the increasing popularity of strain-specific resistant cultivars. Hypersensitive resistance (HR) is one type of plant defense responses to restrict virus spread. In many potato cultivars, such as cultivar Premier Russet (PR), local necrosis at the site of infection protects against the most common PVYO strain, but the HR often fails to restrain necrotic strains, which spread systemically. Here, we established the role of callose accumulation in the strain-specific resistance responses to PVY infection. We first uncovered that PVY, independent of the strain, is naturally capable of suppressing pathogenesis-related callose formation in a susceptible host. Such activity can be dissociated from viral replication by the transient expression of the viral-encoded helper component proteinase (HCPro) protein, identifying it as the pathogen elicitor. However, unlike the necrotic strain, PVYO and its corresponding HCPro are unable to block callose accumulation in resistant PR potatoes, in which we observed an abundance of callose deposition and the inability of the virus to spread. The substitution of eight amino acid residues within the HCPro C-terminal region that differ between PVYO and PVYN strains and were previously shown to be responsible for eliciting the HR response, are sufficient to restore the ability of HCProO to suppress callose accumulation, despite the resistant host background, in line with a new viral function in pathogenicity.

The 3' Untranslated Region of a Plant Viral RNA Directs Efficient Cap-Independent Translation in Plant and Mammalian Systems.

Many plant viral RNA genomes lack a 5' cap, and instead are translated via a cap-independent translation element (CITE) in the 3' untranslated region (UTR). The panicum mosaic virus-like CITE (PTE), found in many plant viral RNAs, binds and requires the cap-binding translation initiation factor eIF4E to facilitate translation. eIF4E is structurally conserved between plants and animals, so we tested cap-independent translation efficiency of PTEs of nine plant viruses in plant and mammalian systems. The PTE from thin paspalum asymptomatic virus (TPAV) facilitated efficient cap-independent translation in wheat germ extract, rabbit reticulocyte lysate, HeLa cell lysate, and in oat and mammalian (BHK) cells. Human eIF4E bound the TPAV PTE but not a PTE that did not stimulate cap-independent translation in mammalian extracts or cells. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) footprinting revealed that both human and wheat eIF4E protected the conserved guanosine (G)-rich domain in the TPAV PTE pseudoknot. The central G plays a key role, as it was found to be required for translation and protection from SHAPE modification by eIF4E. These results provide insight on how plant viruses gain access to the host's translational machinery, an essential step in infection, and raise the possibility that similar PTE-like mechanisms may exist in mRNAs of mammals or their viruses.

Optimization of an isothermal recombinase polymerase amplification method for real-time detection of Potato virus Y O and N types in potato.

Potato virus Y (PVY) is a global challenge for potato production and the leading cause of seed crop downgrading and rejection for certification. Accurate and timely diagnosis is key to effective control of PVY. Here we optimized the isothermal recombinase polymerase amplification (RPA) for accurate detection of different PVY O and N types that were tested, present in different tissues of potato plants including tubers with a primer set that specifically targets the highly conserved pipo region within the viral genome. Combined with a simplified preparation of the template by tissue homogenization, we established a rapid RPA procedure, which can allow real time detection in less than 10 min with a fluorescent probe. Specificity of the reaction was determined by the lack of cross-reactivity with other common potato viruses. Although RPA reagents remain more expensive than PCR reagents, RPA technology is equivalent in that results can be visualized by gel electrophoresis or with a fluorescent probe with greater sensitivity; and it is superior to the common PCR-based assay in its versatility, speed, and lack of need for a highly purified RNA template.

The Triticum Mosaic Virus Internal Ribosome Entry Site Relies on a Picornavirus-Like YX-AUG Motif To Designate the Preferred Translation Initiation Site and To Likely Target the 18S rRNA.

Several viruses encode an internal ribosome entry site (IRES) at the 5' end of their RNA, which, unlike most cellular mRNAs, initiates translation in the absence of a 5' m7GpppG cap. Here, we report a uniquely regulated translation enhancer found in the 739-nucelotide (nt) sequence of the Triticum mosaic virus (TriMV) leader sequence that distinguishes the preferred initiation site from a plethora of IRES-encoded AUG triplets. Through deletion mutations of the TriMV 5' untranslated region (UTR), we show that the TriMV 5' UTR encodes a cis-acting picornaviral Y16-X11-AUG-like motif with a 16-nt polypyrimidine CU-tract (Y16), at a precise, 11-nt distance (X11) from the preferred 13th AUG. Phylogenetic analyses indicate that this motif is conserved among potyviral leader sequences with multiple AUGs. Consistent with a broadly conserved mechanism, the motif could be functionally replaced with known picornavirus YX-AUG motifs and is predicted to function as target sites for the 18S rRNA by direct base pairing. Accordingly, mutations that disrupted overall complementarity to the 18S rRNA markedly reduced TriMV IRES activity, as did the delivery of antisense oligonucleotides designed to block YX-AUG accessibility. To our knowledge, this is the first report of a plant viral IRES YX-AUG motif, and our findings suggest that a conserved mechanism regulates translation for multiple economically important plant and animal positive single-stranded RNA viruses.IMPORTANCE Uncapped viral RNAs often rely on their 5' leader sequences to initiate translation, and the Triticum mosaic virus (TriMV) devotes an astonishing 7% of its genome to directing ribosomes to the correct AUG. Here we uncover a novel mechanism by which a TriMV cis-regulatory element controls cap-independent translation. The upstream region of the functional AUG contains a 16-nt polypyrimidine tract located 11 nt from the initiation site. Based on functional redundancy with similar motifs derived from human picornaviruses, the motif is likely to operate by directing ribosome targeting through base pairing with 18S rRNA. Our results provide the first report of a broad-spectrum mechanism regulating translation initiation for both plant- and animal-hosted picornaviruses.

Optimization of a magnetic capture RT-LAMP assay for fast and real-time detection of potato virus Y and differentiation of N and O serotypes.

Potato virus Y (PVY) infection has been a global challenge for potato production and the leading cause of downgrading and rejection of seed crops for certification. Accurate and timely diagnosis is a key for effective disease control. Here, we have optimized a reverse transcription loop-mediated amplification (RT-LAMP) assay to differentiate the PVY O and N serotypes. The RT-LAMP assay is based on isothermal autocyclic strand displacement during DNA synthesis. The high specificity of this method relies heavily on the primer sets designed for the amplification of the targeted regions. We designed specific primer sets targeting a region within the coat protein gene that contains nucleotide signatures typical for O and N coat protein types, and these primers differ in their annealing temperature. Combining this assay with total RNA extraction by magnetic capture, we have established a highly sensitive, simplified and shortened RT-LAMP procedure as an alternative to conventional nucleic acid assays for diagnosis. This optimized procedure for virus detection may be used as a preliminary test for identifying the viral serotype prior to investing time and effort in multiplex RT-PCR tests when a specific strain is needed.

Manipulation of Oat Protoplasts for Transient Expression Assays.

Oat protoplasts are a useful and convenient system to study transient expression using whole cells. Nucleic acid can rapidly be introduced into live cells, and, depending on the assay, results can be collected the same day. Compared to plant tissue, oat cell suspension cultures provide a simple, high yielding, and consistent means to isolate protoplasts. Here, we describe how to generate an oat cell suspension culture from callus grown on solidified medium, and how to maintain the oat cells in cell suspension culture for protoplast preparation. Following the culturing procedure, we describe how to isolate oat protoplasts from cell suspension culture by enzymatic digestion of the cell walls and to transiently express nucleic acid (DNA or RNA) into the cells by electroporation.

The Triticum Mosaic Virus 5' Leader Binds to Both eIF4G and eIFiso4G for Translation.

We recently identified a remarkably strong (739 nt-long) IRES-like element in the 5' untranslated region (UTR) of Triticum mosaic virus (TriMV, Potyviridae). Here, we define the components of the cap-binding translation initiation complex that are required for TriMV translation. Using bio-layer interferometry and affinity capture of the native translation apparatus, we reveal that the viral translation element has a ten-fold greater affinity for the large subunit eIF4G/eIFiso4G than to the cap binding protein eIF4E/eIFiso4E. This data supports a translation mechanism that is largely dependent on eIF4G and its isoform. The binding of both scaffold isoforms requires an eight base-pair-long hairpin structure located 270 nucleotides upstream of the translation initiation site, which we have previously shown to be crucial for IRES activity. Despite a weak binding affinity to the mRNA, eIFiso4G alone or in combination with eIFiso4E supports TriMV translation in a cap-binding factor-depleted wheat germ extract. Notably, TriMV 5' UTR-mediated translation is dependent upon eIF4A helicase activity, as the addition of the eIF4A inhibitor hippuristanol inhibits 5' UTR-mediated translation. This inhibition is reversible with the addition of recombinant wheat eIF4A. These results and previous observations demonstrate a key role of eIF4G and eIF4A in this unique mechanism of cap-independent-translation. This work provides new insights into the lesser studied translation mechanisms of plant virus-mediated internal translation initiation.

A Unique 5' Translation Element Discovered in Triticum Mosaic Virus.

UNLABELLED: Several plant viruses encode elements at the 5' end of their RNAs, which, unlike most cellular mRNAs, can initiate translation in the absence of a 5' m7GpppG cap. Here, we describe an exceptionally long (739-nucleotide [nt]) leader sequence in triticum mosaic virus (TriMV), a recently emerged wheat pathogen that belongs to the Potyviridae family of positive-strand RNA viruses. We demonstrate that the TriMV 5' leader drives strong cap-independent translation in both wheat germ extract and oat protoplasts through a novel, noncanonical translation mechanism. Translation preferentially initiates at the 13th start codon within the leader sequence independently of eIF4E but involves eIF4G. We truncated the 5' leader to a 300-nucleotide sequence that drives cap-independent translation from the 5' end. We show that within this sequence, translation activity relies on a stem-loop structure identified at nucleotide positions 469 to 490. The disruption of the stem significantly impairs the function of the 5' untranslated region (UTR) in driving translation and competing against a capped RNA. Additionally, the TriMV 5' UTR can direct translation from an internal position of a bicistronic mRNA, and unlike cap-driven translation, it is unimpaired when the 5' end is blocked by a strong hairpin in a monocistronic reporter. However, the disruption of the identified stem structure eliminates such a translational advantage. Our results reveal a potent and uniquely controlled translation enhancer that may provide new insights into mechanisms of plant virus translational regulation. IMPORTANCE: Many members of the Potyviridae family rely on their 5' end for translation. Here, we show that the 739-nucleotide-long triticum mosaic virus 5' leader bears a powerful translation element with features distinct from those described for other plant viruses. Despite the presence of 12 AUG start codons within the TriMV 5' UTR, translation initiates primarily at the 13th AUG codon. The TriMV 5' UTR is capable of driving cap-independent translation in vitro and in vivo, is independent of eIF4E, and can drive internal translation initiation. A hairpin structure at nucleotide positions 469 to 490 is required for the cap-independent translation and internal translation initiation abilities of the element and plays a role in the ability of the TriMV UTR to compete against a capped RNA in vitro. Our results reveal a novel translation enhancer that may provide new insights into the large diversity of plant virus translation mechanisms.

Discovery of a Small Non-AUG-Initiated ORF in Poleroviruses and Luteoviruses That Is Required for Long-Distance Movement.

Viruses in the family Luteoviridae have positive-sense RNA genomes of around 5.2 to 6.3 kb, and they are limited to the phloem in infected plants. The Luteovirus and Polerovirus genera include all but one virus in the Luteoviridae. They share a common gene block, which encodes the coat protein (ORF3), a movement protein (ORF4), and a carboxy-terminal extension to the coat protein (ORF5). These three proteins all have been reported to participate in the phloem-specific movement of the virus in plants. All three are translated from one subgenomic RNA, sgRNA1. Here, we report the discovery of a novel short ORF, termed ORF3a, encoded near the 5' end of sgRNA1. Initially, this ORF was predicted by statistical analysis of sequence variation in large sets of aligned viral sequences. ORF3a is positioned upstream of ORF3 and its translation initiates at a non-AUG codon. Functional analysis of the ORF3a protein, P3a, was conducted with Turnip yellows virus (TuYV), a polerovirus, for which translation of ORF3a begins at an ACG codon. ORF3a was translated from a transcript corresponding to sgRNA1 in vitro, and immunodetection assays confirmed expression of P3a in infected protoplasts and in agroinoculated plants. Mutations that prevent expression of P3a, or which overexpress P3a, did not affect TuYV replication in protoplasts or inoculated Arabidopsis thaliana leaves, but prevented virus systemic infection (long-distance movement) in plants. Expression of P3a from a separate viral or plasmid vector complemented movement of a TuYV mutant lacking ORF3a. Subcellular localization studies with fluorescent protein fusions revealed that P3a is targeted to the Golgi apparatus and plasmodesmata, supporting an essential role for P3a in viral movement.

The role of the 5' untranslated regions of Potyviridae in translation.

The Potyviridae family relies on a cap-independent translation mechanism to facilitate protein expression. The genomic architecture of the viral RNAs of the Potyviridae family resembles those of the animal picornaviruses. The viral genomes lack a 5' cap structure. Instead, they have the viral protein VPg covalently linked to the 5' end of the RNA. The viral RNAs code for a single large polyprotein, which is then cleaved into several functional subunits. With their common genome organization with the Picornaviridae, it has been largely assumed that the members of the plant Potyviridae family share similar translation mechanism. We will describe the remarkably diverse translational enhancers identified within the family and their unique mechanisms of translation, from internal recruitment of the ribosomes to ribosomal scanning from the 5' end and the recruitment of the VPg in translation. The divergence among the potyviral translation enhancers is heightened with the recent discovery of Triticum mosaic virus, an atypical member of the Potyviridae family, for which its 5' leader by far exceeds the typical length of plant viral leaders and contains features typically found in animal viruses. Much remains to be learned on how these highly divergent elements enable potyviruses, which include some of the most damaging plant viruses, to take over the host translation apparatus. While no clear consensus sequence, structure or mechanism has been reported yet among the potyviral elements, more thorough studies are needed to fill in the gap of knowledge.

An efficient factor-depleted mammalian in vitro translation system.

Much of the regulation of gene expression occurs at the level of protein synthesis. In addition to the canonical translation factors, a multitude of proteins and microRNAs (miRNAs) act as regulatory trans-acting factors. Mechanistic analysis of translational control benefits from functional cell-free systems that can be depleted of the responsible regulatory factors. Although antisense oligonucleotides facilitate the functional sequestration of the regulatory RNAs, immunodepletion of protein factors is technically challenging. Here we describe a simple and robust alternative protocol for the preparation of factor-depleted in vitro translation system derived from HeLa cells. The procedure relies on RNA interference-mediated knockdown of the factor of interest prior to extract preparation, and it overcomes problems with the availability and specificity of antibodies, as well as with the co-depletion of proteins associated with the factor under study. The complete procedure can normally be conducted within 1 week and carried out in parallel for multiple (candidate) factors.

The role of ABCE1 in eukaryotic posttermination ribosomal recycling.

After termination, eukaryotic 80S ribosomes remain associated with mRNA, P-site deacylated tRNA, and release factor eRF1 and must be recycled by dissociating these ligands and separating ribosomes into subunits. Although recycling of eukaryotic posttermination complexes (post-TCs) can be mediated by initiation factors eIF3, eIF1, and eIF1A (Pisarev et al., 2007), this energy-free mechanism can function only in a narrow range of low Mg(2+) concentrations. Here, we report that ABCE1, a conserved and essential member of the ATP-binding cassette (ABC) family of proteins, promotes eukaryotic ribosomal recycling over a wide range of Mg(2+) concentrations. ABCE1 dissociates post-TCs into free 60S subunits and mRNA- and tRNA-bound 40S subunits. It can hydrolyze ATP, GTP, UTP, and CTP. NTP hydrolysis by ABCE1 is stimulated by post-TCs and is required for its recycling activity. Importantly, ABCE1 dissociates only post-TCs obtained with eRF1/eRF3 (or eRF1 alone), but not post-TCs obtained with puromycin in eRF1's absence.

In vitro analysis of translation enhancers.

The genomes of many plant viruses contain translation-enhancing sequences that allow them to compete successfully with host messenger RNAs for the translation machinery. Identification of translation enhancer elements is valuable, both to gain understanding of virus gene expression control and to apply them as tools for engineering gene expression in plant cells. Here, we describe experiments designed to detect viral elements that enhance translation, focusing on cap-independent translation activity, using a high fidelity cell-free wheat germ translation extract.

Preparation and electroporation of oat protoplasts from cell suspension culture.

Protoplasts provide a convenient system for introduction of nucleic acids into plant cells. Protoplasts allow rapid assays of gene expression and virus replication, with advantages for plant biology similar to those of cultured animal cells for the study of animal systems. Traditionally, preparation and handling of protoplasts has been as much art as science, requiring a special touch by the user. The purpose of this unit is to lay out in clear detail all the methods and nuances involved in protoplast preparation using a robust, reliable system that does not require skills beyond those expected of an unspecialized molecular biologist. Because dicots and monocots differ in many biological properties, and because different procedures may work better for different plants, separate units in this chapter are devoted to protoplast preparation from dicots (Arabidopsis, tobacco; refer to UNITS 16D.1 & 16D.4) and from a monocot (oat). This unit describes methods for preparation and transfection by electroporation of protoplasts derived from an oat suspension culture.

trans regulation of cap-independent translation by a viral subgenomic RNA.

Many positive-strand RNA viruses generate 3'-coterminal subgenomic mRNAs to allow translation of 5'-distal open reading frames. It is unclear how viral genomic and subgenomic mRNAs compete with each other for the cellular translation machinery. Translation of the uncapped Barley yellow dwarf virus genomic RNA (gRNA) and subgenomic RNA1 (sgRNA1) is driven by the powerful cap-independent translation element (BTE) in their 3' untranslated regions (UTRs). The BTE forms a kissing stem-loop interaction with the 5' UTR to mediate translation initiation at the 5' end. Here, using reporter mRNAs that mimic gRNA and sgRNA1, we show that the abundant sgRNA2 inhibits translation of gRNA, but not sgRNA1, in vitro and in vivo. This trans inhibition requires the functional BTE in the 5' UTR of sgRNA2, but no translation of sgRNA2 itself is detectable. The efficiency of translation of the viral mRNAs in the presence of sgRNA2 is determined by proximity to the mRNA 5' end of the stem-loop that kisses the 3' BTE. Thus, the gRNA and sgRNA1 have "tuned" their expression efficiencies via the site in the 5' UTR to which the 3' BTE base pairs. We conclude that sgRNA2 is a riboregulator that switches off translation of replication genes from gRNA while permitting translation of structural genes from sgRNA1. These results reveal (i) a new level of control of subgenomic-RNA gene expression, (ii) a new role for a viral subgenomic RNA, and (iii) a new mechanism for RNA-mediated regulation of translation.