Alfalfa Mosaic Virus Coat Protein Research Articles

Peptide-based candidate vaccine against respiratory syncytial virus

We engineered a 21-mer peptide representing amino acids 170–190 of the respiratory syncytial virus (RSV) G protein as a fusion with the Alfalfa mosaic virus (AlMV) coat protein (CP), produced recombinant AlMV particles presenting this peptide (VMR-RSV) on their surfaces and tested the immunogenicity in vitro in human dendritic cells and in vivo in non-human primates. Significant pathogen-specific immune responses were generated in both systems: (i) human dendritic cells armed with VMR-RSV generated vigorous CD4+ and CD8+ T cell responses; (ii) non-human primates that received these particles responded by mounting strong cellular and humoral immune responses. This approach may validate the use of a novel RSV vaccine delivery vehicle in humans.

Expression of chimeric HCV peptide in transgenic tobacco plants infected with recombinant alfalfa mosaic virus for development of a plant-derived vaccine against HCV

Hepatitis C virus (HCV) is the major etiologic agent of blood transfusion–associated and sporadic non-A non-B hepatitis affecting more than 180 million worldwide. Vaccine development for HCV has been difficult and there is no vaccine or effective therapy against this virus. In this paper, we describe the development of an experimental plant-derived subunit vaccine against HCV. Our subunit vaccine originates from a consensus HCV-HVR1 epitope (R9) that antigenically mimics many natural HVR1 variants. This HVR1 sequence was cloned into the open reading frame of a plant virus, Alfalfa Mosaic Virus (ALMV) coat protein (CP). The chimeric ALMV RNA4 containing sequence-encoding R9 epitope was introduced into full-length infectious ALMV-RNA3 that was utilized as an expression vector. The recombinant chimeric protein is expressed in transgenic tobacco plants (P12) expressing ALMV RNA1 and 2. Plant–derived HVR1/ALMV-CP reacted with HVR1 and/or ALMV-CP specific monoclonal antibodies and immune sera from individuals infected with HCV. Using plant-virus based transient expression to produce this unique chimeric antigen will facilitate the development and production of an experimental HCV vaccine. A plant derived recombinant HCV vaccine can potentially reduce expenses normally associated with production and delivery of conventional vaccine. Key Words: Hepatitis C virus (HCV), transgenic tobacco plants (P12), consensus HCV HVR1 epitope (R9), and chimeric ALMV-RNA4. African Journal of Biotechnology Vol.3(11) 2004: 588-594

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.

Efficient translation of alfamovirus RNAs requires the binding of coat protein dimers to the 3' termini of the viral RNAs.

The coat protein (CP) of Alfalfa mosaic virus (AMV) is required to initiate infection by the viral tripartite RNA genome whereas infection by the tripartite Brome mosaic virus (BMV) genome is independent of CP. AMV CP stimulates translation of AMV RNA in vivo 50- to 100-fold. The 3' untranslated region (UTR) of the AMV subgenomic CP messenger RNA 4 contains at least two CP binding sites. A CP binding site in the 3'-terminal 112 nucleotides of RNA 4 was found to be required for efficient translation of the RNA whereas an upstream binding site was not. Binding of CP to the AMV 3' UTR induces a conformational change of the RNA but this change alone was not sufficient to stimulate translation. CP mutant R17A is unable to bind to the 3' UTR and translation in vivo of RNA 4 encoding this mutant occurs at undetectable levels. Replacement of the 3' UTR of this mutant RNA 4 by the 3' UTR of BMV RNA 4 restored translation of R17A-CP to wild-type levels. Apparently, the BMV 3' UTR stimulates translation independently of CP. AMV CP mutant N199 is defective in the formation of CP dimers and did not stimulate translation of RNA 4 in vivo although the mutant CP did bind to the 3' UTR. The finding that N199-CP does not promote AMV infection corroborates the notion that the requirement of CP in the inoculum reflects its role in translation of the viral RNAs.

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, CPΔC9 and CPC-ARF, were highly active in initiating replication with 63–84% of wild-type (wt) CP activity. However, all mutants that did not form dimers, CPΔC18, CPΔC19, 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.

Expression in plants and immunogenicity of plant virus-based experimental rabies vaccine

A new approach to the production and delivery of vaccine antigens is the use of engineered amino virus-based vectors. A chimeric peptide containing antigenic determinants from rabies virus glycoprotein (G protein) (amino acids 253–275) and nucleoprotein (N protein) (amino acids 404–418) was PCR-amplified and cloned as a translational fusion product with the alfalfa mosaic virus (AlMV) coat protein (CP). This recombinant CP was expressed in two plant virus-based expression systems. The first one utilized transgenic Nicotiana tabacum cv. Samsun NN plants providing replicative functions in trans for full-length infectious RNA3 of AlMV (NF1-g24). The second one utilized Nicotiana benthamiana and spinach ( Spinacia oleracea) plants using autonomously replicating tobacco mosaic virus (TMV) lacking native CP (Av/A4-g24). Recombinant virus containing the chimeric rabies virus epitope was isolated from infected transgenic N. tabacum cv. Samsun NN plants and used for parenteral immunization of mice. Mice immunized with recombinant virus were protected against challenge infection. Based on the previously demonstrated efficacy of this plant virus-based experimental rabies vaccine when orally administered to mice in virus-infected unprocessed raw spinach leaves, we assessed its efficacy in human volunteers. Three of five volunteers who had previously been immunized against rabies virus with a conventional vaccine specifically responded against the peptide antigen after ingesting spinach leaves infected with the recombinant virus. When rabies virus non-immune individuals were fed the same material, 5/9 demonstrated significant antibody responses to either rabies virus or AlMV. Following a single dose of conventional rabies virus vaccine, three of these individuals showed detectable levels of rabies virus-neutralizing antibodies, whereas none of five controls revealed these antibodies. These findings provide clear indication of the potential of the plant virus-based expression systems as supplementary oral booster for rabies vaccinations.

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.

Partial Resistance of Transgenic Peas to Alfalfa Mosaic Virus under Greenhouse and Field Conditions

Transformed pea (Pisum sativum L.) lines were produced with two chimeric gene constructs encoding the coat protein (CP) of alfalfa mosaic virus (AMV) strain NZ1. To determine whether transformed lines have improved AMV resistance, progeny of independently derived transgenic lines were tested in the greenhouse and five lines with improved virus resistance were identified. Resistance was observed only in individual plants that accumulated detectable CP product from the transgene, suggesting that resistance is CP mediated. Plants that accumulated detectable amounts of transgene CP product yet were susceptible to AMV were found among the progeny of most lines, indicating that these lines are only partially resistant to the challenging AMV strains, 425 and NZ1(Lincoln). A field test was conducted with the progeny of four independently derived transgenic lines. To test for improved virus resistance under field conditions, plots of transgenic lines and nontransgenic controls were inoculated with two AMV strains, NZ1(Lincoln) and NZ34. Analysis of disease severity in seedlings in inoculated plots confirmed that partial virus resistance can be produced by genetically modifying peas with AMV CP sequences.

Human respiratory syncytial virus vaccine antigen produced in plants.

Human respiratory syncytial virus (RSV) is the primary cause of respiratory infection in infants worldwide. Currently there is no available vaccine, although studies in animal models have demonstrated protective immunity induced by an epitope of the RSV G-protein representing amino acids 174-187. Two peptides containing amino acids 174-187 of the G-protein of the human RSV A2 strain (NF1-RSV/172-187 and NF2-RSV/170-191) were separately engineered as translational fusions with the alfalfa mosaic virus coat protein and individually expressed in Nicotiana tabacum cv. Samsun NN plants through virus infection. RSV G-protein peptides were expressed in infected plant tissues at significant levels within 2 wk of inoculation and purified as part of recombinant alfalfa mosaic virions. BALB/c mice immunized intraperitoneally with three doses of the purified recombinant viruses showed high levels of serum antibody specific for RSV G-protein and were protected against infection with RSV Long strain.

Genetic Dissection of the Multiple Functions of Alfalfa Mosaic Virus Coat Protein in Viral RNA Replication, Encapsidation, and Movement

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.

Agronomic Performance of Transgenic Burley Tobaccos Expressing TVMV or AMV Coat Protein Genes with and without Virus Challenges

Eighty transgenic tobacco (Nicotiana tabacum L.) lines expressing either the tobacco vein mottling virus (TVMV) coat protein (CP) gene or the alfalfa mosaic virus (AMV) CP gene were evaluated for their agronomic performance with or without virus challenge. AH TVMV CP lines were characterized previously for their virus resistance following challenge with three different potyviruses. In the absence of virus challenge, the cured leaf yield of more than 80% of the TVMV CP lines was not significantly different from their nontransgenic counterparts; half of the poor performing lines were in one of the five genetic backgrounds. Similarly, most of the negative variation for yield among the AMV CP transgenic lines was within a single genetic background. Variation for other agronomic traits, including plant height, leaf size, and stalk diameter closely followed the results for yield. Following virus challenge, cured leaf yield of the most resistant transgenic lines was similar to the non‐challenged treatment. Among the AMV CP transgenic lines, yield loss following challenge with AMV was reduced from 22 to 30%. These results suggest that the primary selection criterion in a population of transgenic tobacco lines containing a viral CP gene should be for virus resistance, but it must be recognized that variation for agronomic performance may exist among the resistant selections, particularly in some genetic backgrounds.

Effect of C-Terminal Mutations of Alfalfa Mosaic Virus Coat Protein on Dimer Formation and Assembly in Vitro

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 W191, F197, and P198 did not assemble. Furthermore rCP with a single alanine substitution at W191 did not assemble, whereas the rCP, which had an arginine and an alanine substitution at A196 and F197, 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.

Amino acids of alfalfa mosaic virus coat protein that direct formation of unusually long virus particles.

In contrast to most alfalfa mosaic virus (AMV) strains (YSMV, S, M and 425), AMV strains VRU and 1 5/64 can form abnormally long virus particles, an ability which has been linked to the coat protein (CP). In order to study this phenomenon, the CP-encoding RNAs 3 of AMV strains VRU and 1 5/64 were cloned and fully sequenced. Comparative sequence analyses of AMV RNA 3 sequences derived from different strains revealed two non-conservative amino acid substitutions, Ser65 and Leu175, which occur exclusively in the closely related VRU- and 15/64-CPs. When these amino acid alterations were introduced into the CP of AMV strain 425 unusually long virus particles were assembled. This confirms that amino acids Ser66 and Leu175 of the CPs of AMV strains VRU and 15/64 are involved in the formation of tubular virus particles.

Resistance to Alfalfa Mosaic Virus in Transgenic Burley Tobaccos Expressing the AMV Coat Protein Gene

There are no known genetic sources of resistance to alfalfa mosaic virus (AMV) in the genus Nicotiana. In this communication, we describe how we genetically engineered an AMV (strain 425) coat protein (CP) gene into three commercial burley tobacco (N. tabacum L.) genotypes, BY 21, TN 86, and KY 8959, and the evaluation of the transgenic lines in greenhouse and field experiments. In a replicated greenhouse trial, 11 AMV‐CP transgenic lines in a BY 21 background were resistant to two AMV isolates, AMV‐425 and AMV‐KY, but the lines were less resistant to another isolate, AMV‐NC. In a 1994 field experiment, we found that nine BY 21 AMV‐CP transgenic lines were resistant to AMV‐KY when inoculated both before and after transplanting. The level of resistance varied among the nine lines. In 1995, 10 randomly selected AMV‐CP lines in each of the three genetic backgrounds were evaluated in a replicated field trial. Mean infection scores of each set of lines were significantly less than those of their non‐transgenic counterparts, but there were no significant differences among these three mean infection scores. However, there were significant differences among lines within a genetic background for their response to AMV infection. High sensitivity to AMV infection, found in genotypes TN 86 and KY 8959 that possess an endogenous gene for resistance to potyviruses, was not evident in their AMV‐CtrPa nsgenic lines. The CP mediated resistance to AMV was effective in reducing viral symptoms in the field and it may provide a valuable source for AMV resistance in tobacco.

RNA determinants of a specific RNA-Coat protein peptide interaction in alfalfa mosaic virus: conservation of homologous features in ilarvirus RNAs

Alfalfa mosaic virus (AMV) coat protein and tobacco streak virus (TSV) coat protein bind specifically to the 3′ untranslated regions of the viral RNAs and are required with the genomic RNAs to initiate virus replication. A combination of nucleotide substitutions, hydroxyl radical footprinting, and ethylation and chemical modification interference analysis has been used to define the RNA determinants important for the specific binding of the 3′-terminal 39 nucleotides of AMV RNA 3/4 (AMV 843–881) to an amino-terminal coat protein peptide (CP26). The results demonstrate that potential phosphate and base-specific contacts as well as ribose moieties protected upon peptide binding cluster in lower hairpin stems and flanking AUGC sequences of the viral RNA, without direct involvement of loop nucleotides. Nucleotides identified in the modification-interference analyses as important for RNA-protein interactions are highly conserved among AMV and the ilarvirus RNAs. This RNA sequence homology, coupled with the recent identification of an RNA binding consensus sequence for AMV and ilarvirus coat proteins, provides a framework for understanding the functional equivalence of AMV and TSV coat proteins in binding RNA and activating virus replication and may explain why heterologous AMV and ilarvirus coat protein-RNA mixtures are infectious.

The Movement Protein and Coat Protein of Alfalfa Mosaic Virus Accumulate in Structurally Modified Plasmodesmata

In systemically infected tissues ofNicotiana benthamiana, alfalfa mosaic virus (AMV) coat protein (CP) and movement protein (MP) are detected in plasmodesmata in a layer of three to four cells at the progressing front of infection. Besides the presence of these viral proteins, the plasmodesmata are structurally modified in that the desmotubule is absent and the diameter has increased drastically (almost twofold) when compared to plasmodesmata in uninfected cells or cells in which AMV infection had been fully established. Previously reported observations on virion-containing tubule formation at the surface of AMV-infected protoplasts suggest that AMV employs a tubule-guided mechanism for intercellular movement. Although CP and MP localization to plasmodesmata is consistent with such a mechanism, no tubules were found in plasmodesmata of AMV-infected tissues. The significance of these observations is discussed.

Viral coat protein peptides with limited sequence homology bind similar domains of alfalfa mosaic virus and tobacco streak virus RNAs.

An unusual and distinguishing feature of alfalfa mosaic virus (AMV) and ilarviruses such as tobacco streak virus (TSV) is that the viral coat protein is required to activate the early stages of viral RNA replication, a phenomenon known as genome activation. AMV-TSV coat protein homology is limited; however, they are functionally interchangeable in activating virus replication. For example, TSV coat protein will activate AMV RNA replication and vice versa. Although AMV and TSV coat proteins have little obvious amino acid homology, we recently reported that they share an N-terminal RNA binding consensus sequence (Ansel-McKinney et al., EMBO J. 15:5077-5084, 1996). Here, we biochemically compare the binding of chemically synthesized peptides that include the consensus RNA binding sequence and lysine-rich (AMV) or arginine-rich (TSV) environment to 3'-terminal TSV and AMV RNA fragments. The arginine-rich TSV coat protein peptide binds viral RNA with lower affinity than the lysine-rich AMV coat protein peptides; however, the ribose moieties protected from hydroxyl radical attack by the two different peptides are localized in the same area of the predicted RNA structures. When included in an infectious inoculum, both AMV and TSV 3'-terminal RNA fragments inhibited AMV RNA replication, while variant RNAs unable to bind coat protein did not affect replication significantly. The data suggest that RNA binding and genome activation functions may reside in the consensus RNA binding sequence that is apparently unique to AMV and ilarvirus coat proteins.

Functional Significance of Three Basic N-Terminal Amino Acids of Alfalfa Mosaic Virus Coat Protein

Infection of tobacco protoplasts with mutant alfalfa mosaic virus (AMV) RNAs indicated that three basic amino acids in the N-terminus of AMV coat protein are important for the biological activity of the coat protein in the beginning of infection. Substitution of alanines for lysines at position 14 or 17 in the coat protein resulted in a 5- or 10-fold reduction in the activity of the protein, respectively. However, substitution of alanine for arginine at position 18 entirely abolished activity. Arginine 18 was also required for the coat protein to bind to the 3′ noncoding region of the virus RNAin vitro, whereas lysine 14 or 17 was not required. Thus, these results indicate that arginine 18 is essential for the activity of the coat protein in early infection and that binding of the coat protein to AMV RNA correlates with activity.

Identification of a Competitive Translation Determinant in the 3′ Untranslated Region of Alfalfa Mosaic Virus Coat Protein mRNA

We report that the competitive translational activity of alfalfa mosaic virus coat protein mRNA (CP RNA), a nonadenylated mRNA, is determined in part by the 3' untranslated region (UTR). Competitive translation was characterized both in vitro, with cotranslation assays, and in vivo, with microinjected Xenopus laevis oocytes. In wheat germ extracts, coat protein synthesis was constant when a fixed amount of full-length CP RNA was cotranslated with increasing concentrations of competitor globin mRNA. However, translation of CP RNA lacking the 3' UTR decreased significantly under competitive conditions. RNA stabilities were equivalent. In X. laevis oocytes, which are translationally saturated and are an inherently competitive translational environment, full-length CP RNA assembled into large polysomes and coat protein synthesis was readily detectable. Alternatively, CP RNA lacking the 3' UTR sedimented as small polysomes, and little coat protein was detected. Again, RNA stabilities were equivalent. Site-directed mutagenesis was used to localize RNA sequences or structures required for competitive translation. Since the CP RNA 3' UTR has an unusually large number of AUG nucleotide triplets, two AUG-containing sites were altered in full-length RNA prior to oocyte injections. Nucleotide substitutions at the sequence GAUG, 20 nucleotides downstream of the coat protein termination codon, specifically reduced full-length CP RNA translation, while similar substitutions at the next AUG triplet had little effect on translation. The competitive influence of the 3' UTR could be explained by RNA-protein interactions that affect translation initiation or by ribosome reinitiation at downstream AUG codons, which would increase the number of ribosomes committed to coat protein synthesis.