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.

Conserved Structure Associated with Different 3'CITEs Is Important for Translation of Umbraviruses.

The cap-independent translation of plus-strand RNA plant viruses frequently depends on 3' structures to attract translation initiation factors that bind ribosomal subunits or bind directly to ribosomes. Umbraviruses are excellent models for studying 3' cap-independent translation enhancers (3'CITEs), as umbraviruses can have different 3'CITEs in the central region of their lengthy 3'UTRs, and most also have a particular 3'CITE (the T-shaped structure or 3'TSS) near their 3' ends. We discovered a novel hairpin just upstream of the centrally located (known or putative) 3'CITEs in all 14 umbraviruses. These CITE-associated structures (CASs) have conserved sequences in their apical loops and at the stem base and adjacent positions. In 11 umbraviruses, CASs are preceded by two small hairpins joined by a putative kissing loop interaction (KL). Converting the conserved 6-nt apical loop to a GNRA tetraloop in opium poppy mosaic virus (OPMV) and pea enation mosaic virus 2 (PEMV2) enhanced translation of genomic (g)RNA, but not subgenomic (sg)RNA reporter constructs, and significantly repressed virus accumulation in Nicotiana benthamiana. Other alterations throughout OPMV CAS also repressed virus accumulation and only enhanced sgRNA reporter translation, while mutations in the lower stem repressed gRNA reporter translation. Similar mutations in the PEMV2 CAS also repressed accumulation but did not significantly affect gRNA or sgRNA reporter translation, with the exception of deletion of the entire hairpin, which only reduced translation of the gRNA reporter. OPMV CAS mutations had little effect on the downstream BTE 3'CITE or upstream KL element, while PEMV2 CAS mutations significantly altered KL structures. These results introduce an additional element associated with different 3'CITEs that differentially affect the structure and translation of different umbraviruses.

Novel 3' Proximal Replication Elements in Umbravirus Genomes.

The 3' untranslated regions (UTRs) of positive-strand RNA plant viruses commonly contain elements that promote viral replication and translation. The ~700 nt 3'UTR of umbravirus pea enation mosaic virus 2 (PEMV2) contains three 3' cap-independent translation enhancers (3'CITEs), including one (PTE) found in members of several genera in the family Tombusviridae and another (the 3'TSS) found in numerous umbraviruses and several carmoviruses. In addition, three 3' terminal replication elements are found in nearly every umbravirus and carmovirus. For this report, we have identified a set of three hairpins and a putative pseudoknot, collectively termed "Trio", that are exclusively found in a subset of umbraviruses and are located just upstream of the 3'TSS. Modification of these elements had no impact on viral translation in wheat germ extracts or in translation of luciferase reporter constructs in vivo. In contrast, Trio hairpins were critical for viral RNA accumulation in Arabidopsis thaliana protoplasts and for replication of a non-autonomously replicating replicon using a trans-replication system in Nicotiana benthamiana leaves. Trio and other 3' terminal elements involved in viral replication are highly conserved in umbraviruses possessing different classes of upstream 3'CITEs, suggesting conservation of replication mechanisms among umbraviruses despite variation in mechanisms for translation enhancement.

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.

Coat proteins of necroviruses target 14-3-3a to subvert MAPKKKα-mediated antiviral immunity in plants.

Mitogen-activated protein kinase (MAPK) cascades play an important role in innate immunity against various pathogens in plants and animals. However, we know very little about the importance of MAPK cascades in plant defense against viral pathogens. Here, we used a positive-strand RNA necrovirus, beet black scorch virus (BBSV), as a model to investigate the relationship between MAPK signaling and virus infection. Our findings showed that BBSV infection activates MAPK signaling, whereas viral coat protein (CP) counteracts MAPKKKα-mediated antiviral defense. CP does not directly target MAPKKKα, instead it competitively interferes with the binding of 14-3-3a to MAPKKKα in a dose-dependent manner. This results in the instability of MAPKKKα and subversion of MAPKKKα-mediated antiviral defense. Considering the conservation of 14-3-3-binding sites in the CPs of diverse plant viruses, we provide evidence that 14-3-3-MAPKKKα defense signaling module is a target of viral effectors in the ongoing arms race of defense and viral counter-defense.

The RNA of Maize Chlorotic Mottle Virus, an Obligatory Component of Maize Lethal Necrosis Disease, Is Translated via a Variant Panicum Mosaic Virus-Like Cap-Independent Translation Element.

Maize chlorotic mottle virus (MCMV) combines with a potyvirus in maize lethal necrosis disease (MLND), a serious emerging disease worldwide. To inform resistance strategies, we characterized the translation initiation mechanism of MCMV. We report that MCMV RNA contains a cap-independent translation element (CITE) in its 3' untranslated region (UTR). The MCMV 3' CITE (MTE) was mapped to nucleotides 4164 to 4333 in the genomic RNA. 2'-Hydroxyl acylation analyzed by primer extension (SHAPE) probing revealed that the MTE is a distinct variant of the panicum mosaic virus-like 3' CITE (PTE). Like the PTE, electrophoretic mobility shift assays (EMSAs) indicated that eukaryotic translation initiation factor 4E (eIF4E) binds the MTE despite the absence of an m7GpppN cap structure, which is normally required for eIF4E to bind RNA. Using a luciferase reporter system, mutagenesis to disrupt and restore base pairing revealed that the MTE interacts with the 5' UTRs of both genomic RNA and subgenomic RNA1 via long-distance kissing stem-loop interaction to facilitate translation. The MTE stimulates a relatively low level of translation and has a weak, if any, pseudoknot, which is present in the most active PTEs, mainly because the MTE lacks the pyrimidine-rich tract that base pairs to a G-rich bulge to form the pseudoknot. However, most mutations designed to form a pseudoknot decreased translation activity. Mutations in the viral genome that reduced or restored translation prevented and restored virus replication, respectively, in maize protoplasts and in plants. In summary, the MTE differs from the canonical PTE but falls into a structurally related class of 3' CITEs.IMPORTANCE In the past decade, maize lethal necrosis disease has caused massive crop losses in East Africa. It has also emerged in China and parts of South America. Maize chlorotic mottle virus (MCMV) infection is required for this disease. While some tolerant maize lines have been identified, there are no known resistance genes that confer immunity to MCMV. In order to improve resistance strategies against MCMV, we focused on how the MCMV genome is translated, the first step of gene expression by all positive-strand RNA viruses. We identified a structure (cap-independent translation element) in the 3' untranslated region of the viral RNA genome that allows the virus to usurp a host translation initiation factor, eIF4E, in a way that differs from host mRNA interactions with the translational machinery. This difference indicates eIF4E may be a soft target for engineering of-or breeding for-resistance to MCMV.

The Multifunctional Long-Distance Movement Protein of Pea Enation Mosaic Virus 2 Protects Viral and Host Transcripts from Nonsense-Mediated Decay.

The nonsense-mediated decay (NMD) pathway presents a challenge for RNA viruses with termination codons that precede extended 3' untranslated regions (UTRs). The umbravirus Pea enation mosaic virus 2 (PEMV2) is a nonsegmented, positive-sense RNA virus with an unusually long 3' UTR that is susceptible to NMD. To establish a systemic infection, the PEMV2 long-distance movement protein p26 was previously shown to both stabilize viral RNAs and bind them for transport through the plant's vascular system. The current study demonstrated that p26 protects both viral and nonviral messenger RNAs from NMD. Although p26 localizes to both the cytoplasm and nucleolus, p26 exerts its anti-NMD effects exclusively in the cytoplasm independently of long-distance movement. Using a transcriptome-wide approach in the model plant Nicotiana benthamiana, p26 protected a subset of cellular NMD target transcripts, particularly those containing long, structured, GC-rich 3' UTRs. Furthermore, transcriptome sequencing (RNA-seq) revealed that the NMD pathway is highly dysfunctional during PEMV2 infection, with 1,820 (48%) of NMD targets increasing in abundance. Widespread changes in the host transcriptome are common during plant RNA virus infections, and these results suggest that, in at least some instances, virus-mediated NMD inhibition may be a major contributing factor.IMPORTANCE Nonsense-mediated decay (NMD) represents an RNA regulatory pathway that degrades both natural and faulty messenger RNAs with long 3' untranslated regions. NMD targets diverse families of RNA viruses, requiring that viruses counteract the NMD pathway for successful amplification in host cells. A protein required for long-distance movement of Pea enation mosaic virus 2 (PEMV2) is shown to also protect both viral and host mRNAs from NMD. RNA-seq analyses of the Nicotiana benthamiana transcriptome revealed that PEMV2 infection significantly impairs the host NMD pathway. RNA viruses routinely induce large-scale changes in host gene expression, and, like PEMV2, may use NMD inhibition to alter the host transcriptome in an effort to increase virus amplification.

Nitric Oxide as a Downstream Signaling Molecule in Brassinosteroid-Mediated Virus Susceptibility to Maize Chlorotic Mottle Virus in Maize.

Maize chlorotic mottle virus (MCMV) infection causes growth abnormalities in maize. Transcriptome sequencing was conducted to compare the global gene expression of MCMV-inoculated plants with that of mock-inoculated plants. Data analyses showed that brassinosteroid (BR)-associated genes were upregulated after MCMV infection. Exogenous 2,4-epibrassinolide (BL) or brassinazole (BRZ) applications indicated that BR pathway was involved in the susceptibility to MCMV infection. In addition, treatment of BL on maize induced the accumulation of nitric oxide (NO), and the changes of NO content played positive roles in the disease incidence of MCMV. Moreover, MCMV infection was delayed when the BL-treated plants were applied with NO scavenger, which suggested that BR induced the susceptibility of maize to MCMV infection in a NO-dependent manner. Further investigation showed the maize plants with knock-down of DWARF4 (ZmDWF4, a key gene of BR synthesis) and nitrate reductase (ZmNR, a key gene of NO synthesis) by virus-induced gene silencing displayed higher resistance to MCMV than control plants. Taken together, our results suggest that BR pathway promotes the susceptibility of maize to MCMV in a NO-dependent manner.

Hsc70-2 is required for Beet black scorch virus infection through interaction with replication and capsid proteins.

Dissecting the complex molecular interplay between the host plant and invading virus improves our understanding of the mechanisms underlying viral pathogenesis. In this study, immunoprecipitation together with the mass spectrometry analysis revealed that the heat shock protein 70 (Hsp70) family homolog, Hsc70-2, was co-purified with beet black scorch virus (BBSV) replication protein p23 and coat protein (CP), respectively. Further experiments demonstrated that Hsc70-2 interacts directly with both p23 and CP, whereas there is no interaction between p23 and CP. Hsc70-2 expression is induced slightly during BBSV infection of Nicotiana benthamiana, and overexpression of Hsc70-2 promotes BBSV accumulation, while knockdown of Hsc70-2 in N. benthamiana leads to drastic reduction of BBSV accumulation. Infection experiments revealed that CP negatively regulates BBSV replication, which can be mitigated by overexpression of Hsc70-2. Further experiments indicate that CP impairs the interaction between Hsc70-2 and p23 in a dose-dependent manner. Altogether, we provide evidence that besides specific functions of Hsp70 family proteins in certain aspects of viral infection, they can serve as a mediator for the orchestration of virus infection by interacting with different viral components. Our results provide new insight into the role of Hsp70 family proteins in virus infection.

Analysis of the interacting partners eIF4F and 3'-CITE required for Melon necrotic spot virus cap-independent translation.

We have shown previously that the translation of Melon necrotic spot virus (MNSV, family Tombusviridae, genus Carmovirus) RNAs is controlled by a 3'-cap-independent translation enhancer (CITE), which is genetically and functionally dependent on the eukaryotic translation initiation factor (eIF) 4E. Here, we describe structural and functional analyses of the MNSV-Mα5 3'-CITE and its translation initiation factor partner. We first mapped the minimal 3'-CITE (Ma5TE) to a 45-nucleotide sequence, which consists of a stem-loop structure with two internal loops, similar to other I-shaped 3'-CITEs. UV crosslinking, followed by gel retardation assays, indicated that Ma5TE interacts in vitro with the complex formed by eIF4E + eIF4G980-1159 (eIF4Fp20 ), but not with each subunit alone or with eIF4E + eIF4G1003-1092 , suggesting binding either through interaction with eIF4E following a conformational change induced by its binding to eIF4G980-1159 , or through a double interaction with eIF4E and eIF4G980-1159 . Critical residues for this interaction reside in an internal bulge of Ma5TE, so that their mutation abolished binding to eIF4E + eIF4G1003-1092 and cap-independent translation. We also developed an in vivo system to test the effect of mutations in eIF4E in Ma5TE-driven cap-independent translation, showing that conserved amino acids in a positively charged RNA-binding motif around amino acid position 228, implicated in eIF4E-eIF4G binding or belonging to the cap-recognition pocket, are essential for cap-independent translation controlled by Ma5TE, and thus for the multiplication of MNSV.

Nicotiana benthamiana plants asymptomatically infected by Pelargonium line pattern virus show unusually high accumulation of viral small RNAs that is neither associated with DCL induction nor RDR6 activity.

Pelargonium line pattern virus (PLPV, Tombusviridae) normally establishes systemic, low-titered and asymptomatic infections in its hosts. This type of interaction may be largely determined by events related to RNA silencing, a major antiviral mechanism in plants. This mechanism is triggered by double or quasi double-stranded (ds) viral RNAs which are cut by DCL ribonucleases into virus small RNAs (vsRNAs). Such vsRNAs are at the core of the silencing process as they guide sequence-specific RNA degradation Host RNA dependent-RNA polymerases (RDRs), and particularly RDR6, strengthen antiviral silencing by promoting biosynthesis of secondary vsRNAs. To approach PLPV-host relationship, here we have characterized the vsRNAs that accumulate in PLPV-infected Nicotiana benthamiana. Such accumulation was found unprecedented high despite DCLs were not induced in infected tissue and neither vsRNA generation nor PLPV infection was apparently affected by RDR6 impairment. From the obtained data, triggers and host factors likely involved in anti-PLPV silencing are proposed.

Efficient Translation of Pelargonium line pattern virus RNAs Relies on a TED-Like 3´-Translational Enhancer that Communicates with the Corresponding 5´-Region through a Long-Distance RNA-RNA Interaction.

Cap-independent translational enhancers (CITEs) have been identified at the 3´-terminal regions of distinct plant positive-strand RNA viruses belonging to families Tombusviridae and Luteoviridae. On the bases of their structural and/or functional requirements, at least six classes of CITEs have been defined whose distribution does not correlate with taxonomy. The so-called TED class has been relatively under-studied and its functionality only confirmed in the case of Satellite tobacco necrosis virus, a parasitic subviral agent. The 3´-untranslated region of the monopartite genome of Pelargonium line pattern virus (PLPV), the recommended type member of a tentative new genus (Pelarspovirus) in the family Tombusviridae, was predicted to contain a TED-like CITE. Similar CITEs can be anticipated in some other related viruses though none has been experimentally verified. Here, in the first place, we have performed a reassessment of the structure of the putative PLPV-TED through in silico predictions and in vitro SHAPE analysis with the full-length PLPV genome, which has indicated that the presumed TED element is larger than previously proposed. The extended conformation of the TED is strongly supported by the pattern of natural sequence variation, thus providing comparative structural evidence in support of the structural data obtained by in silico and in vitro approaches. Next, we have obtained experimental evidence demonstrating the in vivo activity of the PLPV-TED in the genomic (g) RNA, and also in the subgenomic (sg) RNA that the virus produces to express 3´-proximal genes. Besides other structural features, the results have highlighted the key role of long-distance kissing-loop interactions between the 3´-CITE and 5´-proximal hairpins for gRNA and sgRNA translation. Bioassays of CITE mutants have confirmed the importance of the identified 5´-3´ RNA communication for viral infectivity and, moreover, have underlined the strong evolutionary constraints that may operate on genome stretches with both regulatory and coding functions.

Protein expression strategies in Tobacco necrosis virus-D.

Tobacco necrosis virus (TNV-D) has a plus-strand RNA genome that is neither 5' capped nor 3' poly-adenylated. Instead, it utilizes a 3' cap-independent translational enhancer (3'CITE) located in its 3' untranslated region (UTR) for translation of its proteins. We have examined the protein expression strategies used by TNV-D and our results indicate that: (i) a base pairing interaction between conserved ACCA and UGGU motifs in the genomic 5'UTR and 3'CITE, respectively, is not required for efficient plant cell infection, (ii) similar potential 5'UTR-3'CITE interactions in the two viral subgenomic mRNAs are not needed for efficient translation of viral proteins in vitro, (iii) a small amount of capsid protein is translated from the viral genome by a largely 3'CITE-independent mechanism, (iv) the larger of two possible forms of capsid protein is efficiently translated, and (v) p7b is translated from subgenomic mRNA1 by a leaky scanning mechanism.

Position of the kissing-loop interaction associated with PTE-type 3'CITEs can affect enhancement of cap-independent translation.

The Panicum mosaic virus-like translation enhancer (PTE) functions as a cap-independent translation enhancer (3'CITE) in members of several Tombusviridae genera including 7/19 carmoviruses. For nearly all PTE, a kissing-loop connects the element with a hairpin found in several conserved locations in the genomic RNA (5' terminal hairpin or ~100 nt from the 5' end) and small subgenomic RNA (~63 nt from the 5' end). Moving the interaction closer to the 5' end in reporter mRNAs using Saguaro cactus virus (SCV) sequences had either a minimal or substantial negative effect on translation. Movement of the kissing loop from position 104 to the SCV 5' terminal hairpin also reduced translation by 4-fold. These results suggest that relocating the PTE kissing loop closer to the 5' end reduces PTE efficiency, in contrast to results for the Barley yellow dwarf BTE and Tomato bushy stunt virus Y-shaped 3'CITEs, suggesting that different 3'CITEs have different bridging requirements.

Translational readthrough in Tobacco necrosis virus-D.

The plus-strand RNA genome of Tobacco necrosis virus-D (TNV-D) expresses its polymerase via translational readthrough. The RNA signals involved in this readthrough process were characterized in vitro using a wheat germ extract translation system and in vivo via protoplast infections. The results indicate that (i) TNV-D requires a long-range RNA-RNA interaction between an extended stem-loop (SL) structure proximal to the readthrough site and a sequence in the 3'-untranslated region of its genome; (ii) stability of the extended SL structure is important for its function; (iii) TNV-D readthrough elements are compatible with UAG and UGA, but not UAA; (iv) a readthrough defect can be rescued by a heterologous readthrough element in vitro, but not in vivo; and (v) readthrough elements can also mediate translational frameshifting. These results provide new information on determinants of readthrough in TNV-D and further support the concept of a common general mechanism for readthrough in Tombusviridae.

Investigations on the Tobacco Necrosis Virus D p60 replicase protein.

Tobacco Necrosis Virus D (TNV-D), in the genus Betanecrovirus (family Tombusviridae), possesses a single-stranded, positive-sense RNA genome containing six open reading frames (ORFs). Two 5'-proximal ORFs (1 and 2) encode overlapping polypeptides of 22 and 82 kDa (p22 and p82, respectively) which are both required for replication. The p22 auxiliary protein contains no replication motifs but the C-terminal region, downstream of a leaky stop codon, encodes a 60 kDa polypeptide (p60) which contains conserved RNA-dependent RNA polymerase (RdRP) motifs. Here we have expressed and purified recombinant p60 and show that in vitro it binds and efficiently synthesises both TNV-D RNA and Satellite tobacco necrosis virus C RNA. Alanine scanning mutagenesis of conserved amino acids in characteristic motifs in p60 revealed that some mutations significantly reduced RNA synthesis but mutating the second asparagine residue in the conserved GDD box was lethal. The effects of mutating identical amino acids in p60 on virus replication in vivo were examined in Nicotiana benthamiana plants following infection with RNA transcribed from wild type (wt) and mutant constructs. In inoculated leaves the behaviour of the mutants paralleled the in vitro data but systemic infection was precluded in all but one mutant which had reverted to wt. This study is the first to demonstrate the nucleic acid-binding and synthetic capabilities of a betanecrovirus polymerase.

Differential roles of Hsp70 and Hsp90 in the assembly of the replicase complex of a positive-strand RNA plant virus.

Assembly of viral replicase complexes of eukaryotic positive-strand RNA viruses is a regulated process: multiple viral and host components must be assembled on intracellular membranes and ordered into quaternary complexes capable of synthesizing viral RNAs. However, the molecular mechanisms underlying this process are poorly understood. In this study, we used a model virus, Red clover necrotic mosaic virus (RCNMV), whose replicase complex can be detected readily as the 480-kDa functional protein complex. We found that host heat shock proteins Hsp70 and Hsp90 are required for RCNMV RNA replication and that they interact with p27, a virus-encoded component of the 480-kDa replicase complex, on the endoplasmic reticulum membrane. Using a cell-free viral translation/replication system in combination with specific inhibitors of Hsp70 and Hsp90, we found that inhibition of p27-Hsp70 interaction inhibits the formation of the 480-kDa complex but instead induces the accumulation of large complexes that are nonfunctional in viral RNA synthesis. In contrast, inhibition of p27-Hsp90 interaction did not induce such large complexes but rendered p27 incapable of binding to a specific viral RNA element, which is a critical step for the assembly of the 480-kDa replicase complex and viral RNA replication. Together, our results suggest that Hsp70 and Hsp90 regulate different steps in the assembly of the RCNMV replicase complex.

The Red clover necrotic mosaic virus capsid protein N-terminal lysine-rich motif is a determinant of symptomatology and virion accumulation.

The interaction between viral capsid protein (CP) and its cognate viral RNA modulates many steps in the virus infection cycle, such as replication, translation and assembly. The N-terminal 50 amino acids of the Red clover necrotic mosaic virus (RCNMV) CP are rich in basic residues (especially lysine) and are essential for the core functions of the CP, namely RNA binding and virion assembly. To further elucidate additional biological roles for these basic residues, a series of alanine substitution mutations was introduced into infectious clones of RCNMV RNA-1 and assayed for symptomatology, virion formation and systemic infection. Infectivity assays conducted in Nicotiana benthamiana revealed that all nine alanine substitution mutants (ASMs) were competent for systemic infection. Two ASMs (K4A and K7A/K8A) induced severe symptoms and delayed the systemic spread of viral genomes when compared with wild-type RCNMV. However, these ASMs were still competent for virion formation. Three other ASMs (K25A, K33A and K38A) displayed milder symptoms and significant reductions in virion accumulation when compared with wild-type RCNMV, but retained the ability to spread systemically. Evidence from these last three ASMs, as well as a CP null mutant, showed that RCNMV is able to move systemically in N. benthamiana as a nonvirion form. These observations reaffirm the necessity of the N-terminal lysine-rich residues of the RCNMV CP for efficient virion accumulation. They also reveal additional roles for the CP in the modulation of host symptomatology, independent of its role in virion assembly and the rate of systemic viral movement in N. benthamiana.

A long-distance RNA-RNA interaction plays an important role in programmed -1 ribosomal frameshifting in the translation of p88 replicase protein of Red clover necrotic mosaic virus.

Programmed -1 ribosomal frameshifting (-1 PRF) is one viral translation strategy to express overlapping genes in positive-strand RNA viruses. Red clover necrotic mosaic virus (RCNMV) uses this strategy to express its replicase component protein p88. In this study, we used a cell-free translation system to map cis-acting RNA elements required for -1 PRF. Our results show that a small stem-loop structure adjacent to the cap-independent translation element in the 3' untranslated region (UTR) of RCNMV RNA1 is required for -1 PRF. Site-directed mutagenesis experiments suggested that this stem-loop regulates -1 PRF via base-pairing with complementary sequences in a bulged stem-loop adjacent to the shifty site. The existence of RNA elements responsible for -1 PRF and the cap-independent translation of replicase proteins in the 3' UTR of RNA1 might be important for switching translation to replication and for regulating the ratio of p88 to p27.

Viral cell-to-cell movement requires formation of cortical punctate structures containing Red clover necrotic mosaic virus movement protein.

Movement protein (MP) of Red clover necrotic mosaic virus (RCNMV) forms punctate structures on the cortical endoplasmic reticulum (ER) of Nicotiana benthamiana cells, which are associated with viral RNA1 replication (Kaido et al., Virology 395, 232-242. 2009). We investigated the significance of ER-targeting by MP during virus movement from cell to cell, by analyzing the function of a series of MPs with varying length deletions at their C-terminus, either fused or not fused with green fluorescent protein (GFP). The C-terminal 70 amino acids were crucial to ER-localization of MP-GFP and cell-to-cell movement of the recombinant virus encoding it. However, C-terminal deletion did not affect MP functions, such as increasing the size exclusion limit of plasmodesmata, single-stranded RNA binding in vitro, and MP interacting in vivo. We discuss the possible role of this MP region in virus movement from cell to cell.