Within-host Evolution of Segments Ratio for the Tripartite Genome of Alfalfa Mosaic Virus.

The existence of multipartite viruses is an intriguing mystery in evolutionary virology. Several hypotheses suggest benefits that should outweigh the costs of a reduced transmission efficiency and of segregation of coadapted genes associated with encapsidating each segment into a different particle. Advantages range from increasing genome size despite high mutation rates, faster replication, more efficient selection resulting from reassortment during mixed infections, better regulation of gene expression, or enhanced virion stability and cell-to-cell movement. However, support for these hypotheses is scarce. Here we report experiments testing whether an evolutionary stable equilibrium exists for the three genomic RNAs of Alfalfa mosaic virus (AMV). Starting infections with different segment combinations, we found that the relative abundance of each segment evolves towards a constant ratio. Population genetic analyses show that the segment ratio at this equilibrium is determined by frequency-dependent selection. Replication of RNAs 1 and 2 was coupled and collaborative, whereas the replication of RNA 3 interfered with the replication of the other two. We found that the equilibrium solution is slightly different for the total amounts of RNA produced and encapsidated, suggesting that competition exists between all RNAs during encapsidation. Finally, we found that the observed equilibrium appears to be host-species dependent.

Seed tolerance to deterioration in arabidopsis is affected by virus infection.

Seed longevity is the period during which the plant seed is able to germinate. This property is strongly influenced by environment conditions experienced by seeds during their formation and storage. In the present study we have analyzed how the biotic stress derived from the infection of Cauliflower mosaic virus (CaMV), Turnip mosaic virus (TuMV), Cucumber mosaic virus (CMV) and Alfalfa mosaic virus (AMV) affects seed tolerance to deterioration measuring germination rates after an accelerated aging treatment. Arabidopsis wild type plants infected with AMV and CMV rendered seeds with improved tolerance to deterioration when compared to the non-inoculated plants. On the other hand, CaMV infection generated seeds more sensitive to deterioration. No seeds were obtained from TuMV infected plants. Similar pattern of viral effects was observed in the double mutant athb22 athb25, which is more sensitive to accelerated seed aging than wild type. However, we observed a significant reduction of the seed germination for CMV (65% vs 55%) and healthy (50% vs 30%) plants in these mutants. The seed quality differences were overcomed using the A. thaliana athb25-1D dominant mutant, which over accumulated gibberellic acid (GA), except for TuMV which generated some siliques with low seed tolerance to deterioration. For AMV and TuMV (in athb25-1D), the seed quality correlated with the accumulation of the messengers of the gibberellin 3-oxidase family, the mucilage of the seed and the GA1. For CMV and CaMV it was not a good correlation suggesting that other factors are affecting seed viability.

The coat protein of Alfalfa mosaic virus interacts and interferes with the transcriptional activity of the bHLH transcription factor ILR3 promoting salicylic acid-dependent defence signalling response.

During virus infection, specific viral component-host factor interaction elicits the transcriptional reprogramming of diverse cellular pathways. Alfalfa mosaic virus (AMV) can establish a compatible interaction in tobacco and Arabidopsis hosts. We show that the coat protein (CP) of AMV interacts directly with transcription factor (TF) ILR3 of both species. ILR3 is a basic helix-loop-helix (bHLH) family member of TFs, previously proposed to participate in diverse metabolic pathways. ILR3 has been shown to regulate NEET in Arabidopsis, a critical protein in plant development, senescence, iron metabolism and reactive oxygen species (ROS) homeostasis. We show that the AMV CP-ILR3 interaction causes a fraction of this TF to relocate from the nucleus to the nucleolus. ROS, pathogenesis-related protein 1 (PR1) mRNAs, salicylic acid (SA) and jasmonic acid (JA) contents are increased in healthy Arabidopsis loss-of-function ILR3 mutant (ilr3.2) plants, which implicates ILR3 in the regulation of plant defence responses. In AMV-infected wild-type (wt) plants, NEET expression is reduced slightly, but is induced significantly in ilr3.2 mutant plants. Furthermore, the accumulation of SA and JA is induced in Arabidopsis wt-infected plants. AMV infection in ilr3.2 plants increases JA by over 10-fold, and SA is reduced significantly, indicating an antagonist crosstalk effect. The accumulation levels of viral RNAs are decreased significantly in ilr3.2 mutants, but the virus can still systemically invade the plant. The AMV CP-ILR3 interaction may down-regulate a host factor, NEET, leading to the activation of plant hormone responses to obtain a hormonal equilibrium state, where infection remains at a level that does not affect plant viability.

Structural studies on tobacco streak virus coat protein: Insights into the pleomorphic nature of ilarviruses.

Tobacco streak virus (TSV), the type member of Ilarvirus genus, is a major plant pathogen. TSV purified from infected plants consists of a ss-RNA genome encapsidated in spheroidal particles with diameters of 27, 30 and 33nm constructed from multiple copies of a single species of coat protein (CP) subunits. Apart from protecting the viral genome, CPs of ilarviruses play several key roles in the life cycle of these viruses. Unlike the related bromo and cucumoviruses, ilarvirus particles are labile and pleomorphic, which has posed difficulties in their crystallization and structure determination. In the current study, a truncated TSV-CP was crystallized in two distinct forms and their structures were determined at resolutions of 2.4Å and 2.1Å, respectively. The core of TSV CP was found to possess the canonical ß-barrel jelly roll tertiary structure observed in several other viruses. Dimers of CP with swapped C-terminal arms (C-arm) were observed in both the crystal forms. The C-arm was found to be flexible and is likely to be responsible for the polymorphic and pleomorphic nature of TSV capsids. Consistent with this observation, mutations in the hinge region of the C-arm that reduce the flexibility resulted in the formation of more uniform particles. TSV CP was found to be structurally similar to that of Alfalfa mosaic virus (AMV) accounting for similar mechanism of genome activation in alfamo and ilar viruses. This communication represents the first report on the structure of the CP from an ilarvirus.

The photosystem II oxygen-evolving complex protein PsbP interacts with the coat protein of Alfalfa mosaic virus and inhibits virus replication.

Alfalfa mosaic virus (AMV) coat protein (CP) is essential for many steps in virus replication from early infection to encapsidation. However, the identity and functional relevance of cellular factors that interact with CP remain unknown. In an unbiased yeast two-hybrid screen for CP-interacting Arabidopsis proteins, we identified several novel protein interactions that could potentially modulate AMV replication. In this report, we focus on one of the novel CP-binding partners, the Arabidopsis PsbP protein, which is a nuclear-encoded component of the oxygen-evolving complex of photosystem II. We validated the protein interaction in vitro with pull-down assays, in planta with bimolecular fluorescence complementation assays, and during virus infection by co-immunoprecipitations. CP interacted with the chloroplast-targeted PsbP in the cytosol and mutations that prevented the dimerization of CP abolished this interaction. Importantly, PsbP overexpression markedly reduced virus accumulation in infected leaves. Taken together, our findings demonstrate that AMV CP dimers interact with the chloroplast protein PsbP, suggesting a potential sequestration strategy that may preempt the generation of any PsbP-mediated antiviral state.

The movement protein (NSm) of Tomato spotted wilt virus is the avirulence determinant in the tomato Sw-5 gene-based resistance.

The avirulence determinant triggering the resistance conferred by the tomato gene Sw-5 against Tomato spotted wilt virus (TSWV) is still unresolved. Sequence comparison showed two substitutions (C118Y and T120N) in the movement protein NSm present only in TSWV resistance-breaking (RB) isolates. In this work, transient expression of NSm of three TSWV isolates [RB1 (T120N), RB2 (C118Y) and non-resistance-breaking (NRB)] in Nicotiana benthamiana expressing Sw-5 showed a hypersensitive response (HR) only with NRB. Exchange of the movement protein of Alfalfa mosaic virus (AMV) with NSm supported cell-to-cell and systemic transport of the chimeric AMV RNAs into N. tabacum with or without Sw-5, except for the constructs with NBR when Sw-5 was expressed, although RB2 showed reduced cell-to-cell transport. Mutational analysis revealed that N120 was sufficient to avoid the HR, but the substitution V130I was required for systemic transport. Finally, co-inoculation of RB and NRB AMV chimeric constructs showed different prevalence of RB or NBR depending on the presence or absence of Sw-5. These results indicate that NSm is the avirulence determinant for Sw-5 resistance, and mutations C118Y and T120N are responsible for resistance breakdown and have a fitness penalty in the context of the heterologous AMV system.

Deletions within the 3' non-translated region of Alfalfa mosaic virus RNA4 do not affect replication but significantly reduce long-distance movement of chimeric Tobacco mosaic virus.

Alfalfa mosaic virus (AlMV) RNAs 1 and 2 with deletions in their 3' non­translated regions (NTRs) have been previously shown to be encapsidated into virions by coat protein (CP) expressed from RNA3, indicating that the 3' NTRs of RNAs 1 and 2 are not required for virion assembly. Here, we constructed various mutants by deleting sequences within the 3' NTR of AlMV subgenomic (sg) RNA4 (same as of RNA3) and examined the effect of these deletions on replication and translation of chimeric Tobacco mosaic virus (TMV) expressing AlMV sgRNA4 from the TMV CP sg promoter (Av/A4) in tobacco protoplasts and Nicotiana benthamiana plants. While the Av/A4 mutants were as competent as the wild-type Av/A4 in RNA replication in protoplasts, their encapsidation, long-distance movement and virus accumulation varied significantly in N. benthamiana. These data suggest that the 3' NTR of AlMV sgRNA4 contains potential elements necessary for virus encapsidation.

Systemic transport of Alfalfa mosaic virus can be mediated by the movement proteins of several viruses assigned to five genera of the 30K family.

We previously showed that the movement protein (MP) gene of Alfalfa mosaic virus (AMV) is functionally exchangeable for the cell-to-cell transport of the corresponding genes of Tobacco mosaic virus (TMV), Brome mosaic virus, Prunus necrotic ringspot virus, Cucumber mosaic virus and Cowpea mosaic virus. We have analysed the capacity of the heterologous MPs to systemically transport the corresponding chimeric AMV genome. All MPs were competent in systemic transport but required the fusion at their C terminus of the coat protein-interacting C-terminal 44 aa (A44) of the AMV MP. Except for the TMV MP, the presence of the hybrid virus in upper leaves correlated with the capacity to move locally. These results suggest that all the MPs assigned to the 30K superfamily should be exchangeable not only for local virus movement but also for systemic transport when the A44 fragment is present.

Alfalfa mosaic virus replicase proteins, P1 and P2, localize to the tonoplast in the presence of virus RNA.

To identify the virus components important for assembly of the Alfalfa mosaic virus replicase complex, we used live cell imaging of Arabidopsis thaliana protoplasts that expressed various virus cDNAs encoding native and GFP-fusion proteins of P1 and P2 replicase proteins and full-length virus RNAs. Expression of P1-GFP alone resulted in fluorescent vesicle-like bodies in the cytoplasm that colocalized with FM4-64, an endocytic marker, and RFP-AtVSR2, RabF2a/Rha1-mCherry, and RabF2b/Ara7-mCherry, all of which localize to multivesicular bodies (MVBs), which are also called prevacuolar compartments, that mediate traffic to the lytic vacuole. GFP-P2 was driven from the cytosol to MVBs when expressed with P1 indicating that P1 recruited GFP-P2. P1-GFP localized on the tonoplast, which surrounds the vacuole, in the presence of infectious virus RNA, replication competent RNA2, or P2 and replication competent RNA1 or RNA3. This suggests that a functional replication complex containing P1, P2, and a full-length AMV RNA assembles on MVBs to traffic to the tonoplast.

In vitro and in vivo studies of the RNA conformational switch in Alfalfa mosaic virus.

The 3' termini of Alfalfa mosaic virus (AMV) RNAs adopt two mutually exclusive conformations, a coat protein binding (CPB) and a tRNA-like (TL) conformer, which consist of a linear array of stem-loop structures and a pseudoknot structure, respectively. Previously, switching between CPB and TL conformers has been proposed as a mechanism to regulate the competing processes of translation and replication of the viral RNA (R. C. L. Olsthoorn et al., EMBO J. 18:4856-4864, 1999). In the present study, the switch between CPB and TL conformers was further investigated. First, we showed that recognition of the AMV 3' untranslated region (UTR) by a tRNA-specific enzyme (CCA-adding enzyme) in vitro is more efficient when the distribution is shifted toward the TL conformation. Second, the recognition of the 3' UTR by the viral replicase was similarly dependent on the ratio of CBP and TL conformers. Furthermore, the addition of CP, which is expected to shift the distribution toward the CPB conformer, inhibited recognition by the CCA-adding enzyme and the replicase. Finally, we monitored how the binding affinity to CP is affected by this conformational switch in the yeast three-hybrid system. Here, disruption of the pseudoknot enhanced the binding affinity to CP by shifting the balance in favor of the CPB conformer, whereas stabilizing the pseudoknot did the reverse. Together, the in vitro and in vivo data clearly demonstrate the existence of the conformational switch in the 3' UTR of AMV RNAs.

Alfalfa mosaic virus coat protein bridges RNA and RNA-dependent RNA polymerase in vitro.

Alfalfa mosaic virus (AMV) RNA replication requires the viral coat protein (CP). AMV CP is an integral component of the viral replicase; moreover, it binds to the viral RNA 3'-termini and induces the formation of multiple new base pairs that organize the RNA conformation. The results described here suggest that AMV coat protein binding defines template selection by organizing the 3'-terminal RNA conformation and by positioning the RNA-dependent RNA polymerase (RdRp) at the initiation site for minus strand synthesis. RNA-protein interactions were analyzed by using a modified Northwestern blotting protocol that included both viral coat protein and labeled RNA in the probe solution ("far-Northwestern blotting"). We observed that labeled RNA alone bound the replicase proteins poorly; however, complex formation was enhanced significantly in the presence of AMV CP. The RNA-replicase bridging function of the AMV CP may represent a mechanism for accurate de novo initiation in the absence of canonical 3' transfer RNA signals.

Arabidopsis thaliana is an asymptomatic host of Alfalfa mosaic virus.

The susceptibility of Arabidopsis thaliana ecotypes to infection by Alfalfa mosaic virus (AMV) was evaluated. Thirty-nine ecotypes supported both local and systemic infection, 26 ecotypes supported only local infection, and three ecotypes could not be infected. No obvious symptoms characteristic of virus infection developed on the susceptible ecotypes under standard conditions of culture. Parameters of AMV infection were characterized in ecotype Col-0, which supported systemic infection and accumulated higher levels of AMV than the symptomatic host Nicotiana tabacum. The formation of infectious AMV particles in infected Col-0 was confirmed by infectivity assays on a hypersensitive host and by electron microscopy of purified virions. Replication and transcription of AMV was confirmed by de novo synthesis of AMV subgenomic RNA in Col-0 protoplasts transfected with AMV RNA or plasmids harboring AMV cDNAs.

Cell-to-cell movement of Alfalfa mosaic virus can be mediated by the movement proteins of Ilar-, bromo-, cucumo-, tobamo- and comoviruses and does not require virion formation.

RNA 3 of Alfalfa mosaic virus (AMV) encodes the movement protein (MP) and coat protein (CP). Chimeric RNA 3 with the AMV MP gene replaced by the corresponding MP gene of Prunus necrotic ringspot virus, Brome mosaic virus, Cucumber mosaic virus or Cowpea mosaic virus efficiently moved from cell-to-cell only when the expressed MP was extended at its C-terminus with the C-terminal 44 amino acids of AMV MP. MP of Tobacco mosaic virus supported the movement of the chimeric RNA 3 whether or not the MP was extended with the C-terminal AMV MP sequence. The replacement of the CP gene in RNA 3 by a mutant gene encoding a CP defective in virion formation did not affect cell-to-cell transport of the chimera's with a functional MP. A GST pull-down technique was used to demonstrate for the first time that the C-terminal 44 amino acids of the MP of a virus belonging to the family Bromoviridae interact specifically with AMV virus particles. Together, these results demonstrate that AMV RNA 3 can be transported from cell-to-cell by both tubule-forming and non-tubule-forming MPs if a specific MP-CP interaction occurs.

Coat protein activation of alfalfa mosaic virus replication is concentration dependent.

Alfalfa mosaic virus (AMV) and ilarvirus RNAs are infectious only in the presence of the viral coat protein; therefore, an understanding of coat protein's function is important for defining viral replication mechanisms. Based on in vitro replication experiments, the conformational switch model states that AMV coat protein blocks minus-strand RNA synthesis (R. C. Olsthoorn, S. Mertens, F. T. Brederode, and J. F. Bol, EMBO J. 18:4856-4864, 1999), while another report states that coat protein present in an inoculum is required to permit minus-strand synthesis (L. Neeleman and J. F. Bol, Virology 254:324-333, 1999). Here, we report on experiments that address these contrasting results with a goal of defining coat protein's function in the earliest stages of AMV replication. To detect coat-protein-activated AMV RNA replication, we designed and characterized a subgenomic luciferase reporter construct. We demonstrate that activation of viral RNA replication by coat protein is concentration dependent; that is, replication was strongly stimulated at low coat protein concentrations but decreased progressively at higher concentrations. Genomic RNA3 mutations preventing coat protein mRNA translation or disrupting coat protein's RNA binding domain diminished replication. The data indicate that RNA binding and an ongoing supply of coat protein are required to initiate replication on progeny genomic RNA transcripts. The data do not support the conformational switch model's claim that coat protein inhibits the initial stages of viral RNA replication. Replication activation may correlate with low local coat protein concentrations and low coat protein occupancy on the multiple binding sites present in the 3' untranslated regions of the viral RNAs.

Cofolding organizes alfalfa mosaic virus RNA and coat protein for replication.

Alfalfa mosaic virus genomic RNAs are infectious only when the viral coat protein binds to the RNA 3' termini. The crystal structure of an alfalfa mosaic virus RNA-peptide complex reveals that conserved AUGC repeats and Pro-Thr-x-Arg-Ser-x-x-Tyr coat protein amino acids cofold upon interacting. Alternating AUGC residues have opposite orientation, and they base pair in different adjacent duplexes. Localized RNA backbone reversals stabilized by arginine-guanine interactions place the adenosines and guanines in reverse order in the duplex. The results suggest that a uniform, organized 3' conformation, similar to that found on viral RNAs with transfer RNA-like ends, may be essential for replication.

A plant virus replication system to assay the formation of RNA pseudotriloop motifs in RNA-protein interactions.

A pseudotriloop is formed by transloop base pairing between the first (5') and the fifth nucleotide in a hexanucleotide RNA loop ("hexaloop") to subtend a triloop of nucleotides 2-4. This structure has been found in hairpins involved in the regulation of iron metabolism in mammalian cells and in transcription of plant virus subgenomic RNA. Several hexaloop hairpins, including HIV-transactivation-responsive element and hepatitis B virus , potentially adopt a pseudotriloop conformation. Here we show that an RNA plant virus whose replication depends on a conventional triloop hairpin can be used to verify the existence of pseudotriloop structures in vivo. Our data suggest that the pseudotriloop may represent a common motif in RNA-protein recognition.

Coordinate replication of alfalfa mosaic virus RNAs 1 and 2 involves cis- and trans-acting functions of the encoded helicase-like and polymerase-like domains.

RNAs 1 and 2 of the tripartite genome of alfalfa mosaic virus encode the replicase proteins P1 and P2, respectively, whereas RNA 3 encodes the movement protein and coat protein. Transient expression of wild-type (wt) and mutant viral RNAs and proteins by agroinfiltration of plant leaves was used to study cis- and trans-acting functions of the helicase-like domain in P1 and the polymerase-like domain in P2. Three mutations in conserved motifs of the helicase-like domain of P1 affected one or more steps leading to synthesis of minus-strand RNAs 1, 2, and 3. In leaves containing transiently expressed P1 and P2, replication of wt but not mutant RNA 1 was observed. Apparently, the transiently expressed P1 could not complement the defect in replication of the RNA 1 mutant. Moreover, the transiently expressed wt replicase supported replication of RNA 2, but this replication was blocked in trans by coexpression of mutant RNA 1. However, expression of mutant RNA 1 did not interfere with the replication of RNA 3 by the wt replicase. Similarly, a mutation in the GDD motif encoded by RNA 2 could not be complemented in trans and affected the replication of RNA 1 by a wt replicase, while replication of RNA 3 remained unaffected. In competition assays, the transient wt replicase preferentially replicated RNA 3 over RNAs 1 and 2. The results indicate that one or more functions of P1 and P2 act in cis and point to the existence of a mechanism that coordinates the replication of RNAs 1 and 2.

The importance of alfalfa mosaic virus coat protein dimers in the initiation of replication.

Deletion and substitution mutations affecting the oligomerization of alfalfa mosaic virus (AMV) coat protein (CP) were studied in protoplasts to determine their effect on genome activation, an early step in AMV replication. The CP mutants that formed dimers, CPDeltaC9 and CPC-A(R)F, were highly active in initiating replication with 63-84% of wild-type (wt) CP activity. However, all mutants that did not form dimers, CPDeltaC18, CPDeltaC19, CPC-WFP, and CPC-W, were much less active with 19-33% of wt CP activity. The accumulation and solubility of mutant CPs expressed from a virus-based vector in Nicotiana benthamiana were similar to that of wt CP. Analysis of CP-RNA interactions indicated that CP dimers and CP monomers interacted very differently with AMV RNA 3' ends. These results suggest that CP dimers are more efficient for replication than CP monomers because of differences in RNA binding rather than differences in expression and accumulation of the mutant CPs in infected cells.

A genome-activating N-terminal coat protein peptide of alfalfa mosaic virus is able to activate infection by RNAs 1, 2 and 3 but not by RNAs 1 and 2. Further support for the messenger release hypothesis.

An N-terminal genome-activating peptide of 25 amino acid residues of alfalfa mosaic virus coat protein was unable to activate the incomplete viral genome consisting of RNAs 1 and 2. The messenger release hypothesis predicts that RNA 3 must complement such an inoculum in order to produce RNA 4 that will trigger the process. This is shown indeed to be the case.

Translation of a nonpolyadenylated viral RNA is enhanced by binding of viral coat protein or polyadenylation of the RNA.

On entering a host cell, positive-strand RNA virus genomes have to serve as messenger for the translation of viral proteins. Efficient translation of cellular messengers requires interactions between initiation factors bound to the 5'-cap structure and the poly(A) binding protein bound to the 3'-poly(A) tail. Initiation of infection with the tripartite RNA genomes of alfalfa mosaic virus (AMV) and viruses from the genus Ilarvirus requires binding of a few molecules of coat protein (CP) to the 3' end of the nonpolyadenylated viral RNAs. Moreover, infection with the genomic RNAs can be initiated by addition of the subgenomic messenger for CP, RNA 4. We report here that extension of the AMV RNAs with a poly(A) tail of 40 to 80 A-residues permitted initiation of infection independently of CP or RNA 4 in the inoculum. Specifically, polyadenylation of RNA 1 relieved an apparent bottleneck in the translation of the viral RNAs. Translation of RNA 4 in plant protoplasts was autocatalytically stimulated by its encoded CP. Mutations that interfered with CP binding to the 3' end of viral RNAs reduced translation of RNA 4 to undetectable levels. Possibly, CP of AMV and ilarviruses stimulates translation of viral RNAs by acting as a functional analogue of poly(A) binding protein or other cellular proteins.