Cowpea Chlorotic Mottle Bromovirus Research Articles

Cowpea chlorotic mottle bromovirus replication proteins support template-selective RNA replication in Saccharomyces cerevisiae.

Positive-strand RNA viruses generally assemble RNA replication complexes on rearranged host membranes. Alphaviruses, other members of the alpha-like virus superfamily, and many other positive-strand RNA viruses invaginate host membrane into vesicular RNA replication compartments, known as spherules, whose interior is connected to the cytoplasm. Brome mosaic virus (BMV) and its close relative, cowpea chlorotic mottle virus (CCMV), form spherules along the endoplasmic reticulum. BMV spherule formation and RNA replication can be fully reconstituted in S. cerevisiae, enabling many studies identifying host factors and viral interactions essential for these processes. To better define and understand the conserved, core pathways of bromovirus RNA replication, we tested the ability of CCMV to similarly support spherule formation and RNA replication in yeast. Paralleling BMV, we found that CCMV RNA replication protein 1a was the only viral factor necessary to induce spherule membrane rearrangements and to recruit the viral 2a polymerase (2apol) to the endoplasmic reticulum. CCMV 1a and 2apol also replicated CCMV and BMV genomic RNA2, demonstrating core functionality of CCMV 1a and 2apol in yeast. However, while BMV and CCMV 1a/2apol strongly replicate each others’ genomic RNA3 in plants, neither supported detectable CCMV RNA3 replication in yeast. Moreover, in contrast to plant cells, in yeast CCMV 1a/2apol supported only limited replication of BMV RNA3 (<5% of that by BMV 1a/2apol). In keeping with this, we found that in yeast CCMV 1a was significantly impaired in recruiting BMV or CCMV RNA3 to the replication complex. Overall, we show that many 1a and 2apol functions essential for replication complex assembly, and their ability to be reconstituted in yeast, are conserved between BMV and CCMV. However, restrictions of CCMV RNA replication in yeast reveal previously unknown 1a-linked, RNA-selective host contributions to the essential early process of recruiting viral RNA templates to the replication complex.

Synthesis of minus-strand copies of a viral transgene during viral infections of transgenic plants

Viral transgenes designed to provide resistance to specific plant viruses frequently consist of the coat protein gene and a contiguous 3′ untranslated region (3′UTR) of viral origin. In many RNA viruses the viral 3′UTR establishes a recognition and initiation site for viral RNA replication. Thus the transgenic transcript may contain a functional virus replication site. Experiments were designed to determine if a challenging virus would recognize this replication site on a nuclear derived transcript and synthesize the complementary RNA. These data demonstrate that upon infection by a virus that recognizes the viral replication site, a full-length complement of the transgenic transcript is produced. In these experiments the replication complex of Brome Mosaic bromovirus recognized the transgenic transcript derived from a Cowpea Chlorotic Mottle bromovirus transgene. The resulting RNA may contribute to RNA recombination events.

Capsid protein gene and the type of host plant differentially modulate cell-to-cell movement of cowpea chlorotic mottle virus

A study was undertaken to measure the rate of coat protein (CP) independent cell-to-cell movement of cowpea chlorotic mottle bromovirus (CCMV) in three different host plants. A CCMV RNA3 variant in which the CP gene was substituted with enhanced green fluorescent protein (C3/DeltaCP-EGFP) was coinoculated to three different host plants with transcripts of wild type RNAs 1 and 2. Comparative analysis of cell-to-cell movement monitored by the EGFP expression at various days post inoculation revealed that the rate of spread varied with the type of host species inoculated: fastest movement was observed in Nicotiana benthamiana while the rate spread was significantly slower in the natural host cowpea. When CP was expressed as EGFP fusion (C3/CP:EGFP) the rate of spread in N. benthamiana and C. quinoa was slower than that was observed in the absence of CP and remained subliminal in cowpea. Analysis of infection foci by confocal laser scanning microscope revealed that localization of CP:EGFP fusion was distinct in N. benthamiana and C.quinoa and accumulated as fluorescent inclusions at the cell periphery. Additional experiments involving coinoculation of either C3/DeltaCP-EGFP or C3/CP:EGFP with heterologous brome mosaic bromovirus (BMV) genomic RNAs 1 and 2 revealed that, in addition to movement protein and CP, viral replicase also influences cell-to-cell spread. The significance of these results in relation to the mechanism of bromovirus movement is discussed.

Heterologous expression of the modified coat protein of Cowpea chlorotic mottle bromovirus results in the assembly of protein cages with altered architectures and function.

We have developed methods for producing viral-based protein cages in high yield that are amenable to genetic modification. Expression of the structural protein of Cowpea chlorotic mottle bromovirus (CCMV) using the yeast-based Pichia pastoris heterologous expression system resulted in the assembly of particles that were visibly indistinguishable from virus particles produced in the natural host. We have shown that a collection of non-infectious CCMV coat protein mutants expressed in the P. pastoris system assemble into viral protein cages with altered architectures and function. This provides an alternative to other heterologous expression systems for production of viral structural proteins in which expression has resulted in unassembled cages. Heterologous expression in P. pastoris further enhances the development of viral-based protein cages as biotemplates for nanotechnology and for future studies examining details of icosahedral virus assembly.

Effects of the Cowpea chlorotic mottle bromovirus β-hexamer structure on virion assembly

The X-ray crystal structure of Cowpea chlorotic mottle bromovirus (CCMV) revealed a unique tubular structure formed by the interaction of the N-termini from six coat protein subunits at each three-fold axis of the assembled virion. This structure, termed the β-hexamer, consists of six short β-strands. The β-hexamer was postulated to play a critical role in the assembly and stability of the virion by stabilizing hexameric capsomers (Speir et al., 1995). Mutational analyses of the β-hexamer structure, utilizing both in vitro and in vivo assembly assays, demonstrate that this structure is not required for virion formation devoid of nucleic acids in vitro or for RNA-containing virions in vivo. However, the β-hexamer structure does contribute to virion stability in vitro and modulates disease expression in vivo. These results support a model for CCMV assembly through pentamer intermediates.

Expression and characterization of the 3a movement protein of cowpea chlorotic mottle bromovirus.

Cowpea chlorotic mottle bromovirus (CCMV) 3a protein is required for cell-to-cell movement of the virus in host plants. The 3a protein was produced in Escherichia coli using an expression vector. Gel retardation analysis and UV cross-linking experiments demonstrated that the CCMV 3a protein (CC3a) bound single-stranded (ss) RNA cooperatively without sequence specificity. Binding competition analysis showed that CC3a bound ss-nucleic acids more strongly than double-stranded nucleic acids.

Effect of C-terminal deletions in the movement protein of cowpea chlorotic mottle virus on cell-to-cell and long-distance movement.

In order to elucidate the function of the C-terminal region of cowpea chlorotic mottle bromovirus (CCMV) movement protein (MP) in cell-to-cell movement, a set of deletions ranging from 10 to 80 amino acids (deltaMP10, deltaMP20, deltaMP33, deltaMP43, deltaMP60 and deltaMP80) was engineered into the MP gene encoded by the biologically active clone C3/deltaCP-EGFP, a variant of CCMV RNA3 that contained wild-type (wt) MP and the enhanced green fluorescent protein (EGFP) gene in place of the coat protein (CP). The effect of each MP deletion on cell-to-cell movement was examined in three susceptible host plants: Chenopodium quinoa, Nicotiana benthamiana and cowpea (Vigno sinensis cv. Black Eye). The results indicate that, except for mutant deltaMP43, infections resulting from the deletion mutants remained subliminal. Interestingly, infections resulting from inoculating mutant deltaMP43, which lacked the 43 most C-terminal amino acids, spread rapidly between cells and the number of infected cells expressing EGFP approached that of control inoculations made with C3/deltaCP-EGFP. To verify whether the presence of wt CP altered the movement behaviour of these mutants, each MP deletion was also incorporated into the genetic background of wt CCMV RNA3 (pCC3) and inoculated independently to all three hosts. The results suggest that the overall movement process exhibited by each MP mutant is influenced profoundly by the presence of CP and the particular host plant tested.

Genotypic and phenotypic analysis of bromovirus adaptive mutants derived from a single plant.

Eight adaptive mutant clones have been made from the total RNA extracted from uninoculated upper leaves of a single cowpea plant exhibiting systemic infection after inoculation with a hybrid cowpea chlorotic mottle bromovirus (CCMV) with the 3a movement protein gene of CCMV replaced by that of cowpea-nonadapted brome mosaic bromovirus (BMV). Sequence and mutational analyses of these clones showed genotypic and phenotypic diversity of the cloned virus population, but all examined clones had the adaptive mutation, A to C at position 776 within the BMV 3a gene, required for the systemic infection of cowpea. The data support the quasispecies model for RNA virus population, and suggest that the maintenance of the adaptive mutation may be due to powerful selection pressure in an infection process.

Defective Movement of Viruses in the Family Bromoviridae Is Differentially Complemented in Nicotiana benthamiana Expressing Tobamovirus or Dianthovirus Movement Proteins

ABSTRACT Taxonomically distinct tobacco mosaic tobamovirus (TMV), red clover necrotic mosaic dianthovirus (RCNMV), cucumber mosaic cucumovirus (CMV), brome mosaic bromovirus (BMV), and cowpea chlorotic mottle bromovirus (CCMV) exhibit differences in their host range. Each of these viruses encodes a functionally similar nonstructural movement protein (MP) that is essential for cell-to-cell movement of a progeny virus. Despite the lack of significant amino acid identity among the MPs of CMV, TMV, and RCNMV, movement-defective CMV (CMVFnyDeltaMP-DeltaKPN) was able to move locally and systemically in transgenic Nicotiana benthamiana expressing either TMV MP (NB-TMV-MP(+)) or RCNMV MP (NB-RCNMV-MP(+)). These observations contrast with those of previous studies in which transgenic N. tabacum cv. Xanthi plants expressing TMV MP supported only the cell-to-cell movement of CMVFnyDeltaMP-DeltaKPN. To verify whether similar complementation could be observed for movement-defective bromoviruses, NB-TMV-MP(+) and NB-RCNMV-MP(+) plants were inoculated independently with movement-defective variants of BMV (B3DeltaMP) and CCMV (CC3DeltaMP). Neither NB-TMV-MP(+) nor NB-RCNMV-MP(+) was able to rescue the defective cell-to-cell and long-distance movement of B3DeltaMP. In contrast, NB-RCNMV-MP(+) complemented the cell-to-cell, but not the long-distance, movement of CC3DeltaMP. Taken together, these studies suggest that virus movement is a complex process and that, in some cases, the host species plays a major role in determining the long-distance movement function of a virus.

First Report of Cowpea Aphid-Borne Mosaic Potyvirus from Cowpeas Grown Commercially in the U.S.

Cowpea aphid-borne mosaic potyvirus (CABMV) is one of several seed-borne viruses known to limit cowpea (Vigna unguiculata (L.) Walp. subsp. unguiculata) production in Africa, Europe, and Asia, but CABMV has not been reported on commercially grown cowpeas in the United States (1). However, a sesame (Sesamum indicum L.)-infecting isolate of CABMV was recently characterized from plants growing near cowpea introduction plots in Georgia (2). In February 1997, we received samples of three seed lots of cowpea cv. Chinese Red that had been harvested in southern Texas during 1996. Approximately 28% of the plants grown from these seed lots expressed strong mosaic symptoms on primary and trifoliate leaves. Viruslike symptoms were reproduced following mechanical transmission to plants of Chinese Red cowpea, Nicotiana benthamiana, and soybean (Glycine max L.) cv. Lee. When Coronet and Pinkeye Purple Hull-BVR cowpeas were inoculated with sap extracts from symptomatic Chinese Red plants, chlorotic lesions developed on inoculated leaves, but only Coronet plants supported symptomless systemic infections. Similarly inoculated plants of Chenopodium quinoa (L.) and common bean (Phaseolus vulgaris L.) cvs. Pinto and Black Valentine developed localized chlorotic lesions. In Ouchterlony gel diffusion assays, extracts from symptomatic cowpea plants did not react with antisera to blackeye cowpea mosaic potyvirus (BlCMV), cucumber mosaic cucu-movirus (CMV), southern bean mosaic sobemovirus, cowpea mosaic comovirus, cowpea severe mosaic comovirus, or cowpea chlorotic mottle bromovirus. In the indirect enzyme-linked immunosorbent assay, sap extracts from symptomatic plants reacted with antiserum to CABMV, giving OD values at A405 of 0.10 to 0.25, and reacted weakly with antiserum to BlCMV, with OD values at A405 less than 0.035. Extracts from healthy control plants gave OD values at A405 less than 0.010. No positive reactions were obtained with antisera to bean yellow mosaic potyvirus, peanut mottle potyvirus, soybean mosaic potyvirus, or CMV. To our knowledge, this is the first report of CABMV in commercially grown cowpea from the U.S.

Molecular Studies on Bromovirus Capsid Protein

To determine whether the role of coat protein (CP) in cell-to-cell movement of dicot-adapted cowpea chlorotic mottle bromovirus (CCMV) is distinct from that of monocot-adapted brome mosaic bromovirus (BMV), two reporter genes, β-glucuronidase (GUS) and enhanced green fluorescent protein (EGFP), were substituted for the CP in a biologically active clone of CCMV RNA3 (C3). Primary leaves ofNicotiana benthamiana, Chenopodium quinoa,and cowpea were co-inoculated with wild-type (wt) CCMV RNA 1 and -2 and either C3/ΔCP-GUS or C3/ΔCP-EGFP and analyzed for GUS activity or the presence of green fluorescence. The visual appearance of infections caused by GUS or EGFP variants indicated that, in CCMV, epidermal cell-to-cell movement can occur without a functional CP. By contrast, inoculation of MP defective variants of C3/ΔCP-GUS or C3/ΔCP-EGFP resulted in subliminal infections. Additional experiments examining the infectivity of wt BMV RNA 1 and -2 and a BMV RNA3 variant bearing the EGFP in the place of CP (B3/ΔCP-EGFP) confirmed previous observations that, unlike CCMV, epidermal cell-to-cell movement of BMV is dependent on the expression of a functional CP. Taken together, the results demonstrate that BMV and CCMV use different mechanisms for initial epidermal cell-to-cell spread, and the individual role played by the respective CP genes in this active process is discussed.

The carboxy-terminal two-thirds of the cowpea chlorotic mottle bromovirus capsid protein is incapable of virion formation yet supports systemic movement.

Previous investigations into recombination in cowpea chlorotic mottle bromovirus (CCMV) resulted in the recovery of an unusual recombinant virus, 3-57, which caused a symptomless infection of cowpeas but formed no detectable virions. Sequence analysis of cDNA clones derived from 3-57 determined that mutations near the 5' terminus of the capsid protein gene introduced an early translational termination codon. Further mutations introduced a new in-frame start codon that allowed translation of the 3' two-thirds of the capsid protein gene. Based on the mutations observed in 3-57, wild-type CCMV clones were modified to determine if the carboxyl two-thirds of the capsid protein functions independently of the complete protein in long-distance movement. Analysis of these mutants determined that while virion formation is not required for systemic infection, the carboxy-terminal two-thirds of the capsid protein is both required and sufficient for systemic movement of viral RNA. This indicates that the CCMV capsid protein is multifunctional, with a distinct long-distance movement function in addition to its role in virion formation.

Occurrence of Cowpea Stunt Disease Causing Viruses on Wild Bean in Arkansas.

Cucumber mosaic cucumovirus (CMV) and blackeye cowpea mosaic potyvirus (BlCMV) interact synergistically in dually infected plants to cause cowpea stunt disease (1,2). During a July 1996 survey of cowpea stunt-affected fields in the Arkansas River valley, several wild bean (Strophostyles helvola L. Elliott) plants expressing mosaic symptoms were observed and collected. Sap was extracted from symptomatic leaves and used as inoculum to conduct a host range study. Virus symptoms diagnostic for BlCMV, CMV, or cowpea stunt were observed 6 to 8 days post-inoculation on Vigna unguiculata (L.) Walp. subsp. unguiculata cv. Coronet, while chlorotic lesions and mild systemic mosaic symptoms developed on Nicotiana benthamiana. Enzyme-linked immunosorbent assays (ELISAs) conducted on extracts from symptomatic cowpea and N. benthamiana 14 days after inoculation verified the presence of CMV and BlCMV in single and mixed infections. Additional symptomatic wild bean plants were collected from the same site in October 1996. ELISAs and Ouchterlony gel diffusion assays confirmed the presence of CMV and BlCMV, as well as soybean mosaic potyvirus (SMV), cowpea chlorotic mottle bromovirus, and alfalfa mosaic virus (AlMV) in symptomatic wild bean plants. While some of the collected plants were infected with only one virus, others had combined infections of CMV and BlCMV or these two viruses with AlMV. Interestingly, AlMV isolated from wild bean caused symptoms on greenhouse-grown cowpeas that were as severe or more severe than those caused by the cowpea stunt synergy, often resulting in plant death within 8 to 10 days after inoculation. The SMV from wild bean did not induce symptoms on cowpea and was not detected in extracts from inoculated cowpea plants by Ouchterlony gel diffusion assays, but was able to systemically infect soybean (Glycine max L.) cvs. Bragg and Lee. Another virus known to infect cowpea, southern bean mosaic sobemovirus, was not detected in any of the samples tested. To our knowledge, this is the first report that wild bean can serve as an alternate host for cowpea stunt-causing viruses in single and mixed infections. In addition, these data suggest that AlMV may pose a threat to Arkansas-grown cowpeas.

Host-specificity restriction by bromovirus cell-to-cell movement protein occurs after initial cell-to-cell spread of infection in nonhost plants

The nonstructural 3a protein of the positive-strand RNA bromovinuses is required for infection spread in plants and is acrucial determinant of host specificity in systemic infection. To determine the paths of wild-type (wt) bromovirus infection spread, the step at which 3a mutants are arrested, and the nature of the host specificity associated with the 3a gene, we used in situ hybridization to examine infection spread by cowpea chlorotic mottle bromovirus (CCMV) and its derivatives at the level of individual cells in cowpea leaf epidermis. From 1 to 3 days post inoculation (dpi), wt CCMV spread from initially infected cells to adjacent cells, creating expanding infection foci whose radii grew by one additional epidermal cell diameter every 5 hr. By 3 to 4 dpi, vascular elements contacting such foci acted as conduits for further infection spread. By contrast, a 3a frameshift derivative multiplied in initially infected epidermal cells but failed to move into neighboring cells even by 4 dpi, showing that the 3a gene is essential for cell-to-cell spread. Most interestingly, a CCMV derivative with the 3a gene replaced by that of a bromovirus not adapted to cowpea, brome mosaic virus (BMV), initially spread from cell to cell in cowpea plants, but stopped spreading between 1 and 2 dpi, when most infection foci encompassed 40–80 epidermal cells. Thus, the host-specificity restriction imposed by BMV 3a protein did not result from an inability to direct the spread of infection out of initially infected cowpea cells, but from a much later block. The apparent absence of any preexisting anatomical boundary at the limit of infection spread and localized tissue changes at the infection foci suggested that induced host responses might have contributed to this block.