Expression of alfalfa mosaic virus coat protein in tobacco mosaic virus (TMV) deficient in the production of its native coat protein supports long-distance movement of a chimeric TMV.
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Alfalfa mosaic virus (AlMV) coat protein is involved in systemic infection of host plants, and a specific mutation in this gene prevents the virus from moving into the upper uninoculated leaves. The coat protein also is required for different viral functions during early and late infection. To study the role of the coat protein in long-distance movement of AlMV independent of other vital functions during virus infection, we cloned the gene encoding the coat protein of AlMV into a tobacco mosaic virus (TMV)-based vector Av. This vector is deficient in long-distance movement and is limited to locally inoculated leaves because of the lack of native TMV coat protein. Expression of AlMV coat protein, directed by the subgenomic promoter of TMV coat protein in Av, supported systemic infection with the chimeric virus in Nicotiana benthamiana, Nicotiana tabacum MD609, and Spinacia oleracea. The host range of TMV was extended to include spinach as a permissive host. Here we report the alteration of a host range by incorporating genetic determinants from another virus. (+info)
Alfalfa mosaic virus RNAs serve as cap donors for tomato spotted wilt virus transcription during coinfection of Nicotiana benthamiana.
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Tomato spotted wilt virus (TSWV) was shown to use alfalfa mosaic virus (AMV) RNAs as cap donors in vivo during a mixed infection in Nicotiana benthamiana. By use of nested reverse transcription-PCR, TSWV N and NSs mRNAs provided with capped leader sequences derived from all four AMV RNAs could be cloned and sequenced. The sequence specificity of the putative TSWV endonuclease involved is discussed. (+info)
Effect of C-terminal mutations of alfalfa mosaic virus coat protein on dimer formation and assembly in vitro.
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The coat protein (CP) of alfalfa mosaic virus (AMV) strain 425 assembles to bacilliform or rod-shaped particles in the presence of nucleic acids or to T = 1 empty icosahedral particles in the absence of nucleic acids. To study the determinants of CP assembly, recombinant CPs (rCPs) that contained a (His)(6) region were expressed in Escherichia coli. Wt rCP and a mutant rCP, which lacked the last nine amino acids of the C terminus (amino acids 213-221), assembled to particles that were identical in electron micrographs. However, a mutant rCP, which lacked the last 18 amino acids of the C terminus (amino acids 204-221), did not assemble. Likewise, a mutant with alanine substitutions at W(191), F(197), and P(198) did not assemble. Furthermore rCP with a single alanine substitution at W(191) did not assemble, whereas the rCP, which had an arginine and an alanine substitution at A(196) and F(197), respectively, formed rod-shaped particles. The mutations that prevented assembly prevented dimer formation, which indicates that dimers are the minimal building blocks of particles. Our results indicate that two separate regions in the C terminus of AMV CP are critical for dimer formation and assembly and that changes in key amino acids in one of the regions affect both assembly and particle morphology. (+info)
A conformational switch at the 3' end of a plant virus RNA regulates viral replication.
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3' untranslated regions of alfamo- and ilar-virus RNAs fold into a series of stem-loop structures to which the coat protein binds with high affinity. This binding plays a role in initiation of infection ('genome activation') and has been thought to substitute for a tRNA-like structure that is found at the 3' termini of related plant viruses. We propose the existence of an alternative conformation of the 3' ends of alfamo- and ilar-virus RNAs, including a pseudoknot. Based on (i) phylogenetic comparisons, (ii) in vivo and in vitro functional analyses of mutants in which the pseudoknot has been disrupted or restored by compensatory mutations, (iii) competition experiments between coat protein and viral replicase, and (iv) investigation of the effect of magnesium, we demonstrate that this pseudoknot is required for replication of alfalfa mosaic virus. This conformation resembles the tRNA-like structure of the related bromo- and cucumo-viruses. A low but specific interaction with yeast CCA-adding enzyme was found. The existence of two mutually exclusive conformations for the 3' termini of alfamo- and ilar-virus RNAs could enable the virus to switch from translation to replication and vice versa. The role of coat protein in this modulation and in genome activation is discussed. (+info)
In vitro transcription by the turnip yellow mosaic virus RNA polymerase: a comparison with the alfalfa mosaic virus and brome mosaic virus replicases.
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Recently, we showed that the main determinant in the tRNA-like structure of turnip yellow mosaic virus RNA to initiate minus-strand synthesis in vitro is the 3' ACCA end. By mutational analysis of the 3'-terminal hairpin, we show here that only a non-base-paired ACCA end is functional and that the stability of the wild-type 3'-proximal hairpin is the most favorable, in that it has the lowest DeltaG value and a high transcription efficiency. With a nested set of RNA fragments, we show that the minimum template length is 9 nucleotides and that transcription is improved with increasing the length of the template. The results also suggest that proper base stacking contributes to efficient transcription initiation. Internal initiation is shown to take place on every NPyCPu sequence of a nonstructured template. However, the position of the internal initiation site in the template is important, and competition between the different sites takes place. Internal initiation was also studied with the RNA-dependent RNA polymerase of brome mosaic virus (BMV) and alfalfa mosaic virus (AlMV). The BMV polymerase can start internally on ACCA sequences, though inefficiently. Unexpectedly, the polymerases of both AlMV and BMV can start efficiently on an internal AUGC sequence. (+info)
The complete nucleotide sequence of apple mosaic virus (ApMV) RNA 1 and RNA 2: ApMV is more closely related to alfalfa mosaic virus than to other ilarviruses.
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The complete nucleotide sequences of apple mosaic virus RNA 1 and 2 have been characterized. Apple mosaic virus RNA 1 is 3476 nucleotides in length and encodes a single large open reading frame (ORF), whereas apple mosaic virus RNA 2 is 2979 nucleotides in length and also encodes a single ORF. The amino acid sequences encoded by RNA 1 and 2 show similarity to all of the other ilarviruses for which sequence data are available, but both are more closely related to alfalfa mosaic virus (AMV) than to other ilarviruses. Points of similarity include the absence of ORF 2b, present on the RNA 2 of all previously characterized ilarviruses. The close relationship to AMV also occurs in the movement protein, encoded by RNA 3, but not with the coat protein. These data suggest that the present taxonomy should be revised, and that AMV should be considered an aphid-transmissible ilarvirus. (+info)
Genetic dissection of the multiple functions of alfalfa mosaic virus coat protein in viral RNA replication, encapsidation, and movement.
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Coat protein (CP) of alfalfa mosaic virus (AMV) binds as a dimer to the 3' termini of the three genomic RNAs and is required for initiation of infection, asymmetric plus-strand RNA accumulation, virion formation, and spread of the virus in plants. A mutational analysis of the multiple functions of AMV CP was made. Mutations that interfered with CP dimer formation in the two-hybrid system had little effect on the initiation of infection or plus-strand RNA accumulation but interfered with virion formation and reduced or abolished cell-to-cell movement of the virus in plants. Six of the 7 basic amino acids in the N-terminal arm of CP (positions 5, 6, 10, 13, 16, and 25) could be deleted or mutated into alanine without affecting any step of the replication cycle except systemic movement in plants. Mutation of Arg-17 interfered with initiation of infection (as previously shown by others) and cell-to-cell movement of the virus but not with plus-strand RNA accumulation or virion formation. The results indicate that in addition to the RNA-binding domain, different domains of AMV CP are involved in initiation of infection, plus-strand RNA accumulation, virion formation, cell-to-cell movement, and systemic spread of the virus. (+info)
RNAs 1 and 2 of Alfalfa mosaic virus, expressed in transgenic plants, start to replicate only after infection of the plants with RNA 3.
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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. (+info)