Genome Of Alfalfa Mosaic Virus Research Articles

An Evolved 5' Untranslated Region of Alfalfa Mosaic Virus Allows the RNA Transport of Movement-Defective Variants.

Although the coat protein (CP) has a relevant role in the long-distance movement of alfalfa mosaic virus (AMV) and brome mosaic virus (BMV), its precise function is not fully understood. Previous results showed that a specific interaction between the C termini of the movement protein (MP) and the cognate CP is required for systemic transport. Thus, we have performed a compensatory evolution experiment using an AMV RNA3 derivative defective in long-distance transport that carries a BMV MP lacking the C-terminal 48 residues and unable to interact with the AMV CP. After several passages, five independent evolution lineages were able to move long distance. The analysis of the viral RNA of these lineages showed the presence of three different modifications located exclusively at the 5' untranslated region (5' UTR). The three evolved 5' UTR variants accumulated comparable levels of viral RNA and CP but reduced the accumulation of virus particles and the affinity between the 5' UTR and the AMV CP. In addition, the evolved 5' UTR increased cell-to-cell transport for both the AMV RNA3 carrying the BMV MP and that carrying the AMV MP. Finally, the evolved 5' UTRs allowed the systemic transport of an AMV RNA3 carrying a CP mutant defective in virus particles and increased the systemic transport of several AMV RNA3 derivatives carrying different viral MPs associated with the 30K superfamily. Altogether, our findings indicate that virus particles are not required for the systemic transport of AMV but also that BMV MP is competent for the short- and long-distance transport without the interaction with the CP. IMPORTANCE The results obtained in the present work could challenge the view of the role of the virus particle in the systemic transport of plant viruses. In this sense, we show that two different MPs are competent to systemically transport the AMV genome without the requirement of the virus particles, as reported for viruses lacking a CP (e.g., Umbravirus). The incapability of the viral MP to interact with the CP triggered virus variants that evolved to reduce the formation of virus particles, probably to increase the accessibility of the MP to the viral progeny. Our results point to the idea that virus particles would not be necessary for the viral systemic transport but would be necessary for vector virus transmission. This idea is reinforced by the observation that heterologous MPs also increased the systemic transport of the AMV constructs that have reduced encapsidation capabilities.

Whole-Genome Characterization of Alfalfa Mosaic Virus Obtained from Metagenomic Analysis of Vinca minor and Wisteria sinensis in Iran: with Implications for the Genetic Structure of the Virus.

Alfalfa mosaic virus (AMV), an economically important pathogen, is present worldwide with a very wide host range. This work reports for the first time the infection of Vinca minor and Wisteria sinensis with AMV using RNA sequencing and reverse transcription polymerase chain reaction confirmation. De novo assembly and annotating of contigs revealed that RNA1, RNA2, and RNA3 genomic fragments consist of 3,690, 2,636, and 2,057 nucleotides (nt) for IR-VM and 3,690, 2,594, and 2,057 nt for IR-WS. RNA1 and RNA3 segments of IR-VM and IR-WS closely resembled those of the Chinese isolate HZ, with 99.23–99.26% and 98.04–98.09% nt identity, respectively. Their RNA2 resembled that of Canadian isolate CaM and American isolate OH-2-2017, with 97.96–98.07% nt identity. The P2 gene revealed more nucleotide diversity compared with other genes. Genes in the AMV genome were under dominant negative selection during evolution, and the P1 and coat protein (CP) proteins were subject to the strongest and weakest purifying selection, respectively. In the population genetic analysis based on the CP gene sequences, all 107 AMV isolates fell into two main clades (A, B) and isolates of clade A were further divided into three groups with significant subpopulation differentiation. The results indicated moderate genetic variation within and no clear geographic or genetic structure between the studied populations, implying moderate gene flow can play an important role in differentiation and distribution of genetic diversity among populations. Several factors have shaped the genetic structure and diversity of AMV: selection, recombination/reassortment, gene flow, and random processes such as founder effects.

Molecular Characterization of the Coat Protein Genome of Alfalfa Mosaic Virus (AMV) Isolates from Alfalfa in Van Province

Virus-like symptoms were observed in alfalfa plants (Medicago sativa L.) grown in Van province of Turkey in 2019. The symptoms observed were dwarfing, mosaic pattern and yellowing of leaves, rosetting, and decreasing in leaf sizes. Genomic RNA of 19 symptomatic and non-symptomatic alfalfa leaves were extracted for the detection of virus RNA using virus-specific primer pair by RT-PCR. Of the 19 alfalfa leaf specimens, the six yielded the expected 700 bp DNA band in severely symptomatic alfalfa specimens. Two of them randomly selected were inserted into a plasmid vector. Obtained recombinant plasmids were sequenced in both directions. According to the results of the sequence analysis, it was revealed that the virus in infected alfalfa was the Alfalfa mosaic virus. The sequence data were recorded into the GenBank with access numbers MT210179 and MT210178 and denominated as Alakoy Y9 and Alakoy Y1 isolates, respectively. According to the phylogenetic tree created with 16 AMV sequences registered in the GeneBank for both sequences, the AMV CP gene sequence indicated the highest similarity with USA (JN256023), Brazil (FJ858265), and Puglia (Y09110) isolate and the lowest with Korea isolate (KY348844), at the nucleotide level. It was also revealed that both sequences show 98.45% nucleotide similarity with 7 nucleotide exchanges. According to the literature research results, this is the first report and molecular analysis of Alfalfa mosaic virus (AMV) in Medicago sativa L. in Van province of Turkey.

Arabidopsis m6A demethylase activity modulates viral infection of a plant virus and the m6A abundance in its genomic RNAs

N6-methyladenosine (m6A) is an internal, reversible nucleotide modification that constitutes an important regulatory mechanism in RNA biology. Unlike mammals and yeast, no component of the m6A cellular machinery has been described in plants at present. m6A has been identified in the genomic RNAs of diverse mammalian viruses and, additionally, viral infection was found to be modulated by the abundance of m6A in viral RNAs. Here we show that the Arabidopsis thaliana protein atALKBH9B (At2g17970) is a demethylase that removes m6A from single-stranded RNA molecules in vitro. atALKBH9B accumulates in cytoplasmic granules, which colocalize with siRNA bodies and associate with P bodies, suggesting that atALKBH9B m6A demethylase activity could be linked to mRNA silencing and/or mRNA decay processes. Moreover, we identified the presence of m6A in the genomes of two members of the Bromoviridae family, alfalfa mosaic virus (AMV) and cucumber mosaic virus (CMV). The demethylation activity of atALKBH9B affected the infectivity of AMV but not of CMV, correlating with the ability of atALKBH9B to interact (or not) with their coat proteins. Suppression of atALKBH9B increased the relative abundance of m6A in the AMV genome, impairing the systemic invasion of the plant, while not having any effect on CMV infection. Our findings suggest that, as recently found in animal viruses, m6A modification may represent a plant regulatory strategy to control cytoplasmic-replicating RNA viruses.

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.

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.

Role of the alfalfa mosaic virus methyltransferase-like domain in negative-strand RNA synthesis.

RNAs 1 and 2 of the tripartite genome of alfalfa mosaic virus (AMV) encode the replicase proteins P1 and P2, respectively. P1 contains a methyltransferase-like domain in its N-terminal half, which has a putative role in capping the viral RNAs. Six residues in this domain that are highly conserved in the methyltransferase domains of alphavirus-like viruses were mutated individually in AMV P1. None of the mutants was infectious to plants. Mutant RNA 1 was coexpressed with wild-type (wt) RNAs 2 and 3 from transferred DNA vectors in Nicotiana benthamiana by agroinfiltration. Mutation of His-100 or Cys-189 in P1 reduced accumulation of negative- and positive-strand RNA in the infiltrated leaves to virtually undetectable levels. Mutation of Asp-154, Arg-157, Cys-182, or Tyr-266 in P1 reduced negative-strand RNA accumulation to levels ranging from 2 to 38% of those for the wt control, whereas positive-strand RNA accumulation by these mutants was 2% or less. The (transiently) expressed replicases of the six mutants were purified from the agroinfiltrated leaves. Polymerase activities of these preparations in vitro ranged from undetectable to wt levels. The data indicate that, in addition to its putative role in RNA capping, the methyltransferase-like domain of P1 has distinct roles in replication-associated functions required for negative-strand RNA synthesis. The defect in negative-strand RNA synthesis of the His-100 and Cys-189 mutants could be complemented in trans by coexpression of wt P1.

RNAs 1 and 2 of Alfalfa mosaic virus, expressed in transgenic plants, start to replicate only after infection of the plants with RNA 3.

RNAs 1 and 2 of the tripartite genome of Alfalfa mosaic virus (AMV) encode the two viral replicase subunits. Full-length DNA copies of RNAs 1 and 2 were used to transform tobacco plants (R12 lines). None of the transgenic lines showed resistance to AMV infection. In healthy R12 plants, the transcripts of the viral cDNAs were copied by the transgenic viral replicase into minus-strand RNAs but subsequent steps in replication were blocked. When the R12 plants were inoculated with AMV RNA 3, this block was lifted and the transgenic RNAs 1 and 2 were amplified by the transgenic replicase together with RNA 3. The transgenic expression of RNAs 1 and 2 largely circumvented the role of coat protein (CP) in the inoculum that is required for infection of nontransgenic plants. The results for the first time demonstrate the role of CP in AMV plus-strand RNA synthesis at the whole plant level.

Activation of the alfalfa mosaic virus genome by viral coat protein in non-transgenic plants and protoplasts. The protection model biochemically tested.

In non-transgenic host plants and protoplasts alfalfa mosaic virus displays a strong need for coat protein when starting an infection cycle. The "protection model" states that the three viral RNAs must have a few coat protein subunits at their 3' termini in order to protect them in the host cell against degradation by 3'- to- 5' exoribonucleases [Neeleman L, Van der Vossen EAG, Bol JF (1993) Virology 196: 883-887]. We demonstrated that the naked genome RNAs are slightly infectious, if the inoculation is done at very high concentrations, or if it is preceded by an additional inoculation with the RNAs 1 and 2 (encoding subunits for the viral RNA polymerase). This could mean that the necessity for protection by coat protein is lost if the RNAs in large quantities can overcome the activity of the degrading enzymes, or are protected by association with the RNA polymerase, respectively. However, after having tested in protoplasts the survival of separately preinoculated naked RNA 1 during several hours before RNA 2 was inoculated, on the one hand, or of simultaneously inoculated RNAs 1 and 2, with cycloheximide in the medium during the first hours after inoculation, on the other hand, we had to conclude that the viral genome RNAs are quite stable in the cell in the absence of coat protein or RNA polymerase, respectively. This invalidates the protection model. Accommodation of the above findings by our published "messenger release model" for genome activation [Houwing CJ, Jaspars EMJ (1993) Biochimie 75: 617-621] is discussed.

Accumulation of alfalfa mosaic virus RNAs 1 and 2 requires the encoded proteins in cis.

RNAs 1 and 2 of the tripartite genome of alfalfa mosaic virus (A1MV) encode the replicase proteins P1 and P2, respectively. P1 expressed in transgenic plants (P1 plants) can be used in trans to support replication of A1MV RNAs 2 and 3, and P2 expressed in transgenic plants (P2 plants) can be used in trans to support replication of A1MV RNAs 1 and 3. Wild-type RNA 1 was able to coreplicate with RNAs 2 and 3 in P1 plants, but this ability was abolished by frameshifts or deletions in the P1 gene of RNA 1. Similarly, wild-type RNA 2 coreplicated with RNAs 1 and 3 in P2 plants, but frameshifts or deletions in the P2 gene of RNA 2 interfered with this replication. Apparently, the P1 and P2 genes are required in cis for the accumulation of RNAs 1 and 2, respectively. Point mutations in the GDD motif of the P2 gene in RNA 2 interfered with accumulation of RNA 2 in P2 plants, indicating that replication of RNA 2 is linked to its translation into a functional protein. Plants transformed with both the P1 and P2 genes (P12 plants) accumulate replicase activity that is able to replicate RNA 3 in trans. An analysis of the time course of the accumulation of RNAs 1, 2, and 3 in protoplasts of P12 plants supported the conclusion that translation and replication are tightly coupled for A1MV RNAs 1 and 2 but not for RNA 3.

Preparation of soluble, biologically active alfalfa mosaic virus coat protein and its CaCl 2-induced degradation

A method for the preparation of soluble protein from five biologically distinct alfalfa mosaic virus (AMV) isolates is described. Highly purified AMV was dissociated with 1 M CaCl 2 in 10 mM sodium acetate, pH 6.0, and the precipitated RNA was removed by centrifugation. The protein was dialysed against 10 mM sodium acetate, pH 6.0, containing 0.1 M CaCl 2. If the salt concentration was reduced further, proteins from some AMV isolates precipitated. Proteins prepared by this method were shown to be immunoreactive and to activate the infectivity of the AMV genome. However, during prolonged exposure of the protein to buffers containing 0.1 M CaCl 2 it undergoes slow proteolysis thereby losing its ability to activate the AMV genome but not its immunoreactivity.

Evidence that alfalfa mosaic virus infection starts with three RNA-protein complexes

The tripartite genome of alfalfa mosaic virus (AMV) needs to be activated by its coat protein. To establish whether coat protein exerts its role by interacting structurally with one, two, or all three AMV-RNA species, the infectivity of mixtures of RNA-protein complexes with free RNAs were studied ( Smit and Jaspars, Virology 104, 454–461, 1980 ). These studies were not fully conclusive since some redistribution of coat protein does occur as soon as RNAs are brought into contact with RNA-protein complexes. This problem was overcome by the use of ts mutants of AMV. Free ts coat protein subunits were not able to activate the wt genomic RNAs at 30° in tobacco. Once complexed to the genomic RNAs at 0°, the biological activity of the ts coat protein remained when assayed at the nonpermissive temperature. Apparently, the ts coat protein is inactivated during attempted redistribution at 30°. Studies of mixtures of RNA-protein complexes and free RNA show that coat protein has to be present on at least two of the three RNA species. This result in combination with previous results ( Smit and Jaspars, 1980) warrants the conclusion that the alfalfa mosaic virus infection starts with three RNA-protein complexes.

Activation of the genome of alfalfa mosaic virus is enhanced by the presence of the coat protein on all three genome parts

The same amount of coat protein stimulates the infectivity of alfalfa mosaic virus RNA more when added to the three genome RNAs at once than when preincubated with one or two genome RNAs separately before the inoculum is completed. This suggests that the coat protein activates the genome by interacting with all three parts of it. It could not be demonstrated that infectivity is absolutely dependent on this multiple activation because of the possible exchange of protein between RNA molecules in the inoculum. However, factors that are likely to influence this exchange also have an effect on infectivity. Experiments showed that complex formation of coat protein with only the smallest genome RNA (that contains the coat protein gene) does not enhance infectivity as compared with other individual RNAs and protein combinations. Apparently the expression of the coat protein gene is not stimulated in this manner.

Description and Complementation Analysis of 13 Temperaturesensitive Mutants of Alfalfa Mosaic Virus

SUMMARY Thirteen thermosensitive (ts) mutants of alfalfa mosaic virus (AMV), a virus with a tripartite genome, are described. Eight of these mutants were spontaneous, one was induced with HNO2 and four were induced by u.v. irradiation of one purified component. Using supplementation tests six ts mutations were located on top component b (Tb), three on middle component (M) and four on bottom component (B). Complementation tests with mutants with ts defects on the same component showed that none of the ts mutants on Tb could complement each other; the three ts mutants on M could be subdivided into two complementation groups, while only one pair of mutants showed complementation from the four ts mutants on B. It was demonstrated that the coat proteins from the six ts mutants on Tb were not able to activate the AMV genome at 30 °C in tobacco.

Influence of a few coat protein subunits on the base-paired structure of the RNA species of alfalfa mosaic virus

Differentiated thermal melting profiles were made of all the three RNA species (RNAs 1, 2 and 3), constituting the genome of alfalfa mosaic virus and of the subgenomic coat protein messenger RNA (RNA 4) of this virus. Whereas all profiles showed multiphasicity the profile of RNA 4 was most clearly subdivided showing three clear transitions with t m values of 25.5, 35.5 and 53°C. The first transition disappeared upon addition of 6 coat protein molecules per molecule of RNA 4. The effect of coat protein on the melting profiles of the genome RNAs was much less clear.

Coat protein binds to the 3'-terminal part of RNA 4 of alfalfa mosaic virus.

All four RNAs of alfalfa mosaic virus contain a limited number of sites with a high affinity for coat protein [Van Boxsel, J. A. M. (1976), Ph.D. Thesis, University of Leiden]. In order to localize these sites in the viral RNAs, RNA 4 Tthe subgenomic messenger for coat protein) was subjected to a very mild digestion with ribonucleast T1. The ten major fragments, apparently resulting from five preferential hits, were separated and tested for messenger activity in a wheat germ cell-free system, as well as for the capacity to withdraw coat protein from intact particles. Fragments which stimulated amino acid incorporation were assumed to contain the 5 terminus. Strong evidence was obtained for the location of sites with a high affinity for coat protein near the 3' terminus. The smallest fragment which has the 3'-terminal cytosine comprises only 10% of the length of intact RNA 4 but still possesses these sites. Evidence is presented that the complete coat protein cistron is in the complementing 90% fragment. Possibly, the high-affinity sites are entirely located in the 3'-terminal extracistronic part of RNA 4. They will have the same position in RNA 3 and, possibly, also in the other parts of the genome of alfalfa mosaic virus. The need of this genome for coat protein in order to become infectious may therefore find its explanation in the fact that a conformational change at the 3' ends of the genome parts brought about by the coat protein is required for recognition by the viral replicase.

Composition and biosynthetic activity of polyribosomes associated with alfalfa mosaic virus infections

To investigate the mechanism of expression of genetic information in the tripartite RNA genome of alfalfa mosaic virus, the composition of polyribosomes extracted from infected tobacco plants was analyzed. By gel electrophoresis and annealing to virus-pecific double-stranded RNA it was established that 3 to 4 days after infection [ 3H]uridine-label associated with polyribosomes is almost exclusively incorporated in virus-specific RNA. Six species of polysome-associated RNA were identified: the three genome fragments, B-RNA, M-RNA, and Tb-RNA, and three subgenomic RNA, Xa-NA, Xb-RNA, and Ta-RNA. By chromatography on oligo(dT)-cellulose columns it was shown that neither the virion RNA nor the polysome-associated RNA contain a 3′-poly(A) segment. The hypothesis is put forward that the three genome fragments each contain two cistrons, one of which is translated directly from the genomic RNA, whereas the second cistron is expressed through a subgenomic messenger RNA. Upon dissociation of polyribosomes with EDTA the polysome-associated RNA are found in a ribonucleoprotein structure. When the polyribosomes are isolated with a high-salt buffer (containing 0.4 M KCI), this presumptive messenger ribonucleoprotein (mRNP) sediments in the 10 to 50 S region of a sucrose gradient and has a buoyant density of about 1.60 g/cm 3. When the polyribosomes are isolated with a low-salt buffer (containing 0.06 M KCI), the mRNP sediments in the 40 to 70 S region of a sucrose gradient and has a buoyant density of 1.45 g/cm 3. This demonstrates that under high-salt conditions a considerable amount of protein is dissociated from the mRNP-structure or is prevented from association with it. Moreover, a negligible amount of [ 3H]leucine is incorporated in mRNP from polyribosomes prepared with high-salt buffer, whereas the mRNP from polyribosomes isolated with low-salt buffer is extensively labeled. The majority of this label appears to be incorporated in viral coat protein. The dissociation of coat protein from polyribosomes in high-salt buffer is not surprising, since virions also fall apart at this ionic strength. Upon addition of a postribosomal supernatant from wheat germ and other requirements for protein synthesis, polyribosomes prepared with high-salt or low-salt buffer are equally efficient in completing nascent peptide chains. In both cases the only detectable virus-specific polypeptide labeled in vitro is viral coat protein. The mechanism controlling in vitro and in vivo translation and the possible role of the coat protein in these processes are discussed.

Replication of alfalfa mosaic virus RNAs

The RNAs constituting the genome of alfalfa mosaic virus (AMV) have been separated by electrophoresis on polyacrylamide gels and inoculated alone or in mixture upon susceptible plants. The isolation and analysis of replicative forms (RF) found in these plants revealed that the mixtures which induce the formation of RF are the same as those which give rise to local lesions on hypersensitive plants: that is, only the mixture of 4 species of AMV RNA, or the mixture of the 3 largest RNAs in addition to coat protein is able to induce the formation of RF, whereas no single RNA alone or with the addition of small amounts of coat protein gives rise to the corresponding replicative form. The problem of the existence of a replicative form of 12 S RNA was also examined. It was never possible to show any replicative form of 12 S RNA even when a strain producing much 12 S RNA (AMV 425) was used. Some possibilities of 12 S RNA biosynthesis were examined.

Three nucleoprotein components of alfalfa mosaic virus necessary for infectivity

All five nucleoprotein components of alfalfa mosaic virus 425 have been purified by several cycles of density gradient centrifugation in a zonal rotor. The purified components were homogeneous in the analytical ultracentrifuge, had characteristic particle lengths, and were noninfectious at concentrations normally used for biological assay of the virus. Infectivity could be restored to usual values by combining three of the components provided that ethylenediamine tetraacetate was added during the purification procedure. These components were: bottom component, middle component, and top component b. None of these components could be omitted from the mixture without reducing the infectivity to very low values. On the other hand top components a and o were inactive in any combination whatsoever. Activity was not lost when one or two of the active components were exchanged with similar components from the virus isolates Caldy and 15 64 . These results together with those of earlier work strongly indicate that three components contribute unique and essential pieces of information to the genome of alfalfa mosaic virus. The fact that in earlier work with purified RNA species the middle component RNA was overlooked and that top component a RNA was thought to be active instead of top component b RNA is discussed.