Production and use of cDNA clones from arabis mosaic virus

Arabis mosaic virus (AMV) genomic RNAs were converted to dsDNA and cloned into bacterial plasmids. Insert sizes of cDNA clones ranged from 0·2 to 3·2 kbp. Restriction enzyme mapping identified clones representing at least 90% of the RNA-2 genome. A 0·9 kbp clone specific to RNA-1 was also identified. Northern blot hybridisations of AMV RNAs with clones from either RNA-1 or RNA-2 showed no cross reactions. The sensitivity of virus detection in dot hybridisation was 15 pg of purified genomic RNA and 40 pg of purified virus particles. The possibility of using cDNA clones for the detection of AMV in strawberry sap was demonstrated. Two AMV dsRNAs corresponding to genomic RNAs in size were isolated from infected cucumber plants and reacted in hybridisation experiments.     

Peanut yellow spot virus: A distinct tospovirus species based on serology and nucleic acid hybridisation

Nucleocapsids of peanut yellow spot virus (PYSV), purified from peanut (= groundnut) plant tissue, contained a protein with a molecular mass of 29 kDa. In ELISA and immuno-blot analysis the virus did not react with tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV) and peanut bud necrosis virus (PBNV) antisera. PYSV contained three RNA species, a large (L) RNA (c.8900 nucleotides), a medium (M) RNA (c.4800 nucleotides) and a small (S) RNA (c.3000 nucleotides), similar to other tospoviruses. In addition, a fourth RNA species of approximately 1800 nucleotides was also present in purified preparations. Hybridisation analysis under high stringency conditions revealed no hybridisation between PYSV RNAs and cDNA probes representing the nucleocapsid (N) gene, the glycoprotein (GP) gene and the 3' half of the RNA polymerase gene of PBNV. PYSV genomic RNAs also failed to hybridise with cDNA probes from the GP genes of TSWV and INSV. In reciprocal tests, the cDNA clones of PYSV S and M RNAs did not hybridise with any of the PBNV RNAs. Based on the absence of serological relationships between PYSV and PBNV, TSWV and INSV and lack of nucleotide homology based on hybridisation studies between the PYSV RNAs and cDNA clones from PBNV, TSWV and INSV, PYSV should be considered as a distinct species of the genus Tospovirus under a new serogroup, putatively designated ‘V’.     

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.     

The nucleotide sequence of the S RNA of Impatiens necrotic spot virus, a novel tospovirus.

Impatiens necrotic spot virus (INSV) shares a number of properties with tomato spotted wilt virus (TSWV), the type species of the genus tospovirus within the family Bunyaviridae. INSV, however, differs from TSWV in plant host range and serology. In order to define the genomic structure and the taxonomic status of this TSWV-like virus, the nucleotide sequence of its genomic S RNA segment has been determined. The molecular data obtained demonstrate that, like TSWV, INSV has an ambisense S RNA molecule, encoding a non-structural protein in viral sense and the nucleocapsid protein in viral complementary sense. The level of nucleotide sequence homology between their S RNAs, as well as the divergence in amino acid sequence homology of their gene products, confirm previous conclusions from serological studies that INSV and TSWV represent distinct virus species within the newly created genus, tospovirus.     

Functional equivalence of common and unique sequences in the 3' untranslated regions of alfalfa mosaic virus RNAs 1, 2, and 3.

The 3' untranslated regions (UTRs) of alfalfa mosaic virus (AMV) RNAs 1, 2, and 3 consist of a common 3'-terminal sequence of 145 nucleotides (nt) and upstream sequences of 18 to 34 nt that are unique for each RNA. The common sequence can be folded into five stem-loop structures, A to E, despite the occurrence of 22 nt differences between the three RNAs in this region. Exchange of the common sequences or full-length UTRs between the three genomic RNAs did not affect the replication of these RNAs in vivo, indicating that the UTRs are functionally equivalent. Mutations that disturbed base pairing in the stem of hairpin E reduced or abolished RNA replication, whereas compensating mutations restored RNA replication. In vitro, the 3' UTRs of the three RNAs were recognized with similar efficiencies by the AMV RNA-dependent RNA polymerase (RdRp). A deletion analysis of template RNAs indicated that a 3'-terminal sequence of 127 nt in each of the three AMV RNAs was not sufficient for recognition by the RdRp. Previously, it has been shown that this 127-nt sequence is sufficient for coat protein binding. Apparently, sequences required for recognition of AMV RNAs by the RdRp are longer than sequences required for CP binding.     

Transformation of Brown Leghorn chicken embryo fibroblasts by avian myeloblastosis virus proviral DNA.

Brown Leghorn chicken embryo fibroblasts were transfected with a mixture of avian myeloblastosis virus (AMV) and myeloblastosis-associated virus type 1 (MAV1) proviral DNA purified from lambda-Charon 4A recombinant clones. A transformed cell line (T1AM) able to grow without anchorage in semisolid medium was obtained. The presence of both proviral AMV and MAV sequences was detected in T1AM DNA by hybridization with v-myb- and MAV1-specific probes. Altered AMV and MAV1 proviral genomes were found in T1AM genome. Characterization of the RNA species expressed in transformed cells showed that in addition to a 2.5-kilobase (kb) putative subgenomic v-myb-specific RNA, three other myb-containing RNAs (9.4, 8.4, and 7.0 kb) were present in T1AM cells. No AMV genomic RNA was detected. Also, a new 5.0-kb MAV1-specific RNA species was expressed in transformed cells in addition to MAV1 genomic RNA species (7.8 kb). No infectious AMV virions are released by T1AM cells. Chicken embryo fibroblasts infected by T1AM-released virions contained and expressed all MAV1 sequences detected in T1AM transformed cells but did not express any transformation parameter. These results indicated that the presence of AMV proviral sequences in T1AM cells is responsible for their transformed phenotype.     

Multifunctional roles for the N-terminal basic motif of Alfalfa mosaic virus coat protein: nucleolar/cytoplasmic shuttling, modulation of RNA-binding activity, and virion formation

In addition to virion formation, the coat protein (CP) of Alfalfa mosaic virus (AMV) is involved in the regulation of replication and translation of viral RNAs, and in cell-to-cell and systemic movement of the virus. An intriguing feature of the AMV CP is its nuclear and nucleolar accumulation. Here, we identify an N-terminal lysine-rich nucleolar localization signal (NoLS) in the AMV CP required to both enter the nucleus and accumulate in the nucleolus of infected cells, and a C-terminal leucine-rich domain which might function as a nuclear export signal. Moreover, we demonstrate that AMV CP interacts with importin-α, a component of the classical nuclear import pathway. A mutant AMV RNA 3 unable to target the nucleolus exhibited reduced plus-strand RNA synthesis and cell-to-cell spread. Moreover, virion formation and systemic movement were completely abolished in plants infected with this mutant. In vitro analysis demonstrated that specific lysine residues within the NoLS are also involved in modulating CP-RNA binding and CP dimerization, suggesting that the NoLS represents a multifunctional domain within the AMV CP. The observation that nuclear and nucleolar import signals mask RNA-binding properties of AMV CP, essential for viral replication and translation, supports a model in which viral expression is carefully modulated by a cytoplasmic/nuclear balance of CP accumulation.     

Intracellular localization and movement phenotypes of alfalfa mosaic virus movement protein mutants

Thirteen mutations were introduced in the movement protein (MP) gene of Alfalfa mosaic virus (AMV) fused to the green fluorescent protein (GFP) gene and the mutant MP-GFP fusions were expressed transiently in tobacco protoplasts, tobacco suspension cells, and epidermal cells of tobacco leaves. In addition, the mutations were introduced in the MP gene of AMV RNA 3 and the mutant RNAs were used to infect tobacco plants. Ten mutants were affected in one or more of the following functions of MP: the formation of tubular structures on the surface of protoplasts, association with the endoplasmic reticulum (ER) of suspension cells and epidermal cells, targeting to punctate structures in the cell wall of epidermis cells, movement from transfected cells to adjacent cells in epidermis tissue, cell-to-cell movement, or long-distance movement in plants. The mutations point to functional domains of the MP and support the proposed order of events in AMV transport. Studies with several inhibitors indicate that actin or microtubule components of the cytoskeleton are not involved in tubule formation by AMV MP. Evidence was obtained that tubular structures on the surface of transfected protoplasts contain ER- or plasmalemma-derived material.     

Midgut infection by tomato spotted wilt virus and vector incompetence of Frankliniella tritici

Abstract:  The mechanism leading to vector competence of thrips species to transmit tomato spotted wilt virus (TSWV) is not well characterized. We investigated the interaction of TSWV and the non-vector species Frankliniella tritici . A monoclonal antibody to the non-structural protein (NSs) of the TSWV was used to detect TSWV replication within the thrips by immunofluorescence microscopy and enzyme-linked immonosorbent assay (ELISA). TSWV was acquired by F. tritici , replicated and moved within the alimentary canal of F. tritici similar to a known vector of TSWV, Frankliniella occidentalis . However, virus was not found in the salivary glands of F. tritici , which is a prerequisite to virus transmission. Thus, movement to the salivary glands may determine vector incompetence of F. tritici .     

Effectiveness of tomato-spotted wilt virus management tactics

Tomato-spotted wilt (TSW) is caused by the tomato-spotted wilt virus (TSWV) and is a major disease affecting the production of tomato and pepper in the Southeastern United States. Before initiating a multistate, regional project addressing this issue, a survey was conducted in North Carolina, South Carolina, Georgia, and Florida to assess the need for improved management of TSWV in these crops. We investigated farmer's stated effectiveness of four common TSWV management tactics (reflective mulch, resistant cultivar, imidacloprid, and Actiguard) in pepper and tomato production using logistic regression. We found that the odds that farmers were satisfied with the use of reflective mulch alone in controlling TSWV is 9-1, about one and a half times the amount obtained from using Actiguard alone. Moreover, the odds that farmers were satisfied with a practice that combines reflective mulch and Actiguard was far greater than that obtained from using each of the technique separately. We found some similarities between farmer's stated effectiveness and revealed effectiveness from experiments.     

A plant viral coat protein RNA binding consensus sequence contains a crucial arginine.

A defining feature of alfalfa mosaic virus (AMV) and ilarviruses [type virus: tobacco streak virus (TSV)] is that, in addition to genomic RNAs, viral coat protein is required to establish infection in plants. AMV and TSV coat proteins, which share little primary amino acid sequence identity, are functionally interchangeable in RNA binding and initiation of infection. The lysine-rich amino-terminal RNA binding domain of the AMV coat protein lacks previously identified RNA binding motifs. Here, the AMV coat protein RNA binding domain is shown to contain a single arginine whose specific side chain and position are crucial for RNA binding. In addition, the putative RNA binding domain of two ilarvirus coat proteins, TSV and citrus variegation virus, is identified and also shown to contain a crucial arginine. AMV and ilarvirus coat protein sequence alignment centering on the key arginine revealed a new RNA binding consensus sequence. This consensus may explain in part why heterologous viral RNA-coat protein mixtures are infectious.     

Monocistronic translation of alfalfa mosaic virus RNAs.

The four alfalfa mosaic virus RNAs (respectively 24 S, 20 S, 17 S and 12 S) have been used separately as messengers in two in vitro protein synthesizing systems: wheat germ and rabbit reticulocyte lysate. In both systems a polypeptide corresponding to the translation of the entire length of the RNA can be found for RNAs 24 S, 20 S and 12 S, but not for 17 S RNA, the translation product of which is only 35,000 daltons. The number of initiation sites has been determined for each RNA by analyzing the initiation peptides synthesized in the presence of spasomycin and show that there is only one initiation or binding site perRNA. We thus conclude that each AMV RNA behaves as a monocistronic messenger in in vitro translating systems.     

Role of the alfalfa mosaic virus movement protein and coat protein in virus transport

The movement protein (MP) and coat protein (CP) encoded by Alfalfa mosaic virus (AMV) RNA 3 are both required for virus transport. RNA 3 vectors that expressed nonfused green fluorescent protein (GFP), MP:GPF fusions, or GFP:CP fusions were used to study the functioning of mutant MP and CP in protoplasts and plants. C-terminal deletions of up to 21 amino acids did not interfere with the function of the CP in cell-to-cell movement, although some of these mutations interfered with virion assembly. Deletion of the N-terminal 11 or C-terminal 45 amino acids did not interfere with the ability of MP to assemble into tubular structures on the protoplast surface. Additionally, N- or C-terminal deletions disrupted tubule formation. A GFP:CP fusion was targeted specifically into tubules consisting of a wild-type MP. All MP deletion mutants that showed cell-to-cell and systemic movement in plants were able to form tubular structures on the surface of protoplasts. Brome mosaic virus (BMV) MP did not support AMV transport. When the C-terminal 48 amino acids were replaced by the C-terminal 44 amino acids of the AMV MP, however, the BMV/AMV chimeric protein permitted wild-type levels of AMV transport. Apparently, the C terminus of the AMV MP, although dispensable for cell-to-cell movement, confers specificity to the transport process.     

Expression and characterization of a soluble form of tomato spotted wilt virus glycoprotein GN

Tomato spotted wilt virus (TSWV), a member of the Tospovirus genus within the Bunyaviridae, is an economically important plant pathogen with a worldwide distribution. TSWV is transmitted to plants via thrips (Thysanoptera: Thripidae), which transmit the virus in a persistent propagative manner. The envelope glycoproteins, G(N) and G(C), are critical for the infection of thrips, but they are not required for the initial infection of plants. Thus, it is assumed that the envelope glycoproteins play important roles in the entry of TSWV into the insect midgut, the first site of infection. To directly test the hypothesis that G(N) plays a role in TSWV acquisition by thrips, we expressed and purified a soluble, recombinant form of the G(N) protein (G(N)-S). The expression of G(N)-S allowed us to examine the function of G(N) in the absence of other viral proteins. We detected specific binding to thrips midguts when purified G(N)-S was fed to thrips in an in vivo binding assay. The TSWV nucleocapsid protein and human cytomegalovirus glycoprotein B did not bind to thrips midguts, indicating that the G(N)-S-thrips midgut interaction is specific. TSWV acquisition inhibition assays revealed that thrips that were concomitantly fed purified TSWV and G(N)-S had reduced amounts of virus in their midguts compared to thrips that were fed TSWV only. Our findings that G(N)-S binds to larval thrips guts and decreases TSWV acquisition provide evidence that G(N) may serve as a viral ligand that mediates the attachment of TSWV to receptors displayed on the epithelial cells of the thrips midgut.     

Analysis of the pancreatic-ribonuclease-digestion products of alfalfa-mosaic-virus ribonucleic acid. Sequence homologies between the different RNAs.

Alfalfa mosaic virus (AMV) genome consists of three pieces of RNA (24-S, 20-S and 17-s RNA). For infectivity these three RNAs and the coat protein are required. In the absence of coat protein, infectivity is obtained by adding the 12-S RNA also normally present in the virus. This 12-S RNA represents the message for coat protein. Thus a redundancy of the gene for coat protein exists between 12-S RNA and one of the other RNAs. Sequence analysis of the oligonucleotides resulting from pancreatic ribonuclease digestion of the AMV RNAs indicates that the nucleotide sequence of 12-S RNA occurs in 17-S RNA. Analysis of the pancreatic ribonuclease digestion products of the two larger alfalfa mosaic virus RNAs (20-S and 24-S RNA) shows some oligonucleotides containing seven, eight and nine nucleotides with the same structure present in both RNAs. The possibility of a limited nucleotide sequence homology between these two RNAs is discussed. The comparison of the RNase digestion products of 20-S and 24-S RNA with those of 12-S or 17-S RNA revealed no homologous oligonucleotides, thus the origin of 12-S RNA appears to be 17-S RNA.