Search

Cowpea chlorotic mottle virus (CCMV) is a bromovirus transmitted by species of chrysomelid beetles, including the spotted cucumber beetle, Diabrotica undecimpunctata howardii Barber. An experimental system was set up to identify the viral determinant(s) of the beetle transmission of CCMV. Nicotiana clevelandii was selected as an experimental plant host because it supports the replication and accumulation of both CCMV and a second member of the family Bromoviridae, cucumber mosaic virus (CMV). Using a reverse genetic system for CMV, a cDNA copy of the CCMV capsid protein (CP) gene was substituted for that of the CMV CP gene. The resulting 'CMV-hybrid' consisted of wild-type CMV RNA1, RNA2, and a chimeric CMV RNA3 expressing the CCMV structural protein. The CMV-hybrid replicated and formed virions in N. clevelandii; in electron micrographs the hybrid virus was indistinguishable from CCMV. In beetle feeding assays, both CCMV and the CMV-hybrid were transmitted by D. undecimpunctata, while beetle transmission of CMV was not observed. Conversely, only CMV was observed to be transmitted by the aphid Myzus persicae. Surprisingly, the CMV-hybrid was transmitted more efficiently than the parental CCMV, and a virus-induced alteration in beetle feeding behaviour is proposed to account for the difference. These results indicate that the CCMV CP is a viral determinant for beetle vector transmission.

Brome mosaic virus (BMV) requires encapsidation-competent coat protein (CP) for cell-to-cell movement and the 3a movement protein (MP) is involved in determining the CP requirement for BMV movement. However, these conclusions have been drawn by using BMV strain M1 (BMV-M1) and a related strain. Here, the ability of the MPs of five other natural BMV strains to mediate the movement of BMV-M1 in the absence of CP was tested. The MP of BMV M2 strain (BMV-M2) efficiently mediated the movement of CP-deficient BMV-M1 and the MPs of two other strains functioned similarly to some extent. Furthermore, BMV-M2 itself moved between cells independently of CP, demonstrating that BMV-M1 and -M2 use different movement modes. Reassortment between CP-deficient BMV-M1 and -M2 showed the involvement of RNA3 in determining the CP requirement for cell-to-cell movement and the involvement of RNAs 1 and 2 in movement efficiency and symptom induction in the absence of CP. Spontaneous BMV MP mutants generated in planta that exhibited CP-independent movement were also isolated and analysed. Comparison of the nucleotide differences of the MP genes of BMV-M1, the natural strains and mutants capable of CP-independent movement, together with further mutational analysis of BMV-M1 MP, revealed that single amino acid differences at the C terminus of MP are sufficient to alter the requirement for CP in the movement of BMV-M1. Based on these findings, a possible virus strategy in which a movement mode is selected in plant viruses to optimize viral infectivity in plants is discussed.

The 3a movement protein (MP) plays a central role in the movement of Brome mosaic virus (BMV). To identify the functional regions in BMV MP, 24 alanine-scanning (AS) MP mutants of BMV were constructed. Infectivity of the AS mutants in the host plant Chenopodium quinoa showed that the central region of BMV MP is important for viral movement and both termini of BMV MP have effects on the development of systemic symptoms. A green-fluorescent-protein-expressing RNA3-based BMV vector containing a 2A sequence from Foot-and-mouth disease virus was also constructed. Using this vector, two AS mutants that showed more efficient cell-to-cell movement than wild-type BMV were identified. The MPs of these two AS mutants, which have mutations at their C termini, mediated cell-to-cell movement independently of coat protein (CP), unlike wild-type BMV MP. Furthermore, a BMV mutant with a truncation in the C-terminal 42 amino acids of MP was also able to move from cell to cell without CP, but did not move systemically, even in the presence of CP. These results and an encapsidation analysis suggest that the C terminus of BMV MP is involved in the requirement for CP in cell-to-cell movement and plays a role in long-distance movement. Furthermore, the ability to spread locally and form virions is not sufficient for the long-distance movement of BMV. The roles of MP and CP in BMV movement are discussed.

We have developed methods for producing viral-based protein cages in high yield that are amenable to genetic modification. Expression of the structural protein ofCowpea chlorotic mottle bromovirus(CCMV) using the yeast-basedPichia pastorisheterologous 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 theP. pastorissystem 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 inP. pastorisfurther enhances the development of viral-based protein cages as biotemplates for nanotechnology and for future studies examining details of icosahedral virus assembly.

Viral RNA-dependent RNA polymerase (RdRp) plays crucial roles in the genomic replication and subgenomic transcription of Brome mosaic virus (BMV), a positive-stranded RNA plant virus. BMV RdRp is a complex of virus-encoded 1a and 2a proteins and some cellular factors, and associates with the endoplasmic reticulum at an infection-specific structure in the cytoplasm of host cells. In this study, we investigate the gross structure of the active BMV RdRp complex using monoclonal antibodies raised against the 1a and 2a proteins. Immunoprecipitation experiments showed that the intermediate region between the N-terminal methyltransferase-like domain and the C-terminal helicase-like domain of 1a protein, and the N terminus region of 2a protein are exposed on the surface of the solubilized RdRp complex. Inhibition assays for membrane-bound RdRp suggested that the intermediate region between the methyltransferase-like and the helicase-like domains of 1a protein is located at the border of the region buried within a membrane structure or with membrane-associated material.

In yeast expressing the RNA replication proteins encoded by brome mosaic virus (BMV), B3URA3, a BMV RNA3 derivative that harbours the 3a cell-to-cell movement protein gene and the yeast uracil biosynthesis gene URA3, was replicated and maintained in 85–95% of progeny at each cell division. Transmission of the B3URA3 RNA replicon from mother to daughter yeast did not require the 3a gene. Nevertheless, even after passaging for 165 cycles of RNA replication and yeast cell division, each of 40 independent Ura+ colonies tested retained B3URA3 RNAs whose electrophoretic mobilities and accumulation levels were indistinguishable from those of the original B3URA3. These and other results suggest that unselected genes in many positive-strand RNA virus replicons can be stably retained if the presence of the gene does not confer a selective disadvantage in RNA replication.

Microprojectile bombardment was used to examine the transport function of the 25 kDa movement protein (MP) encoded in the triple gene block of potato virus X (PVX). A 25 kDa MP-defective full-length cloned PVX genome carrying a beta-glucuronidase (GUS) reporter gene was co-bombarded with 35S promoter constructs containing either the 25 kDa MP gene of wild-type PVX, the MP gene of either of two tobamoviruses (tomato mosaic virus or crucifer tobamovirus), red clover necrotic mosaic dianthovirus (RCNMV) or brome mosaic bromovirus (BMV). When inoculated alone, the MP-defective PVX was unable to move out of the inoculated cell, as visualized by in situ staining for GUS activity. However, cell-to-cell movement of the mutant PVX genome was restored by co-inoculation with 35S constructs containing the MP cDNA of PVX, either tobamovirus or RCNMV. The BMV MP construct did not complement movement of the defective PVX. These results show that co-bombardment of cDNA of an MP-defective virus with plasmids designed to express MP of other viruses could be used as a fast and simple method for transcomplementation experiments.

Transgenic tobacco plants (V123 plants) expressing a set of full-length brome mosaic virus (BMV) genomic RNAs from the cauliflower mosaic virus 35S promoter were produced. The accumulation level of BMV RNAs in V123 plant cells was approximately 1% of that in non-transgenic tobacco protoplasts inoculated with BMV RNAs. The level of BMV RNA in V123 protoplasts did not increase after inoculating the protoplasts with BMV RNAs, whereas V123 protoplasts supported the accumulation of cucumber mosaic virus (CMV) RNAs to a level similar to that in non-transgenic tobacco protoplasts after inoculation with CMV RNA. Such BMV-specific resistance was also observed in protoplasts from V12 plants expressing full-length BMV RNA1 and RNA2, both of which are required and sufficient for BMV RNA replication. On the other hand, protoplasts from M12 plants, expressing truncated BMV RNA1 and RNA2 in which the 3′ 200 nucleotides required for BMV RNA replication were deleted, exhibited weaker resistance to infection with BMV RNA than V12 protoplasts, although the accumulation level of truncated BMV RNA1 and RNA2 in M12 protoplasts was higher than that of BMV RNA1 and RNA2 and V12 protoplasts. These results suggest that expression of BMV RNA replicons is involved in the induction of resistance, rather than high-level accumulation of BMV RNAs and/or their encoded proteins.

The RNA3 of prunus necrotic ringspot ilarvirus (PNRSV) has been cloned and its entire sequence determined. The RNA3 consists of 1943 nucleotides (nt) and possesses two large open reading frames (ORFs) separated by an intergenic region of 74 nt. The 5' proximal ORF is 855 nt in length and codes for a protein of molecular mass 31.4 kDa which has homologies with the putative movement protein of other members of the Bromoviridae. The 3' proximal ORF of 675 nt is the cistron for the coat protein (CP) and has a predicted molecular mass of 24.9 kDa. The sequence of the 3' non-coding region (NCR) of PNRSV RNA3 showed a high degree of similarity with those of tobacco streak virus (TSV), prune dwarf virus (PDV), apple mosaic virus (ApMV) and also alfalfa mosaic virus (AIMV). In addition it contained potential stem-loop structures with interspersed AUGC motifs characteristic for ilar- and alfamoviruses. This conserved primary and secondary structure in all 3' NCRs may be responsible for the interaction with homologous and heterologous CPs and subsequent activation of genome replication. The CP gene of an ApMV isolate (ApMV-G) of 657 nt has also been cloned and sequenced. Although ApMV and PNRSV have a distant serological relationship, the deduced amino acid sequences of their CPs have an identity of only 51.8%. The N termini of PNRSV and ApMV CPs have in common a zinc-finger motif and the potential to form an amphipathic helix.

The nucleotide sequence of citrus leaf rugose ilarvirus (CiLRV) RNA-2 consists of 2990 nucleotides and contains one open reading frame (ORF) which encodes a deduced translation product of 832 amino acids with a calculated M r of 95 501 (95K). The 5′ terminus of the RNA has a m7Gppp cap. Both the nucleotide sequence of CiLRV RNA-2 and its translated polypeptide share homologies with the nucleotide sequence and translated polypeptide, respectively, of RNA-2 of alfalfa mosaic virus (AIMV). The homology occurs in the central region of both the nucleic acid sequence and the polypeptide. Homologies between either CiLRV or AlMV and other Bromoviridae (cucumber mosaic virus - CMV, brome mosaic virus - BMV and cowpea chlorotic mottle virus - CCMV) were far less. Alignment of the 104 amino acid region (polymerase signature) of the 95K protein against 10 other ‘alpha-like’ plant viral polymerase signatures showed that CiLRV and A1MV are more closely related to each other than to CMV, BMV or CCMV. This is the first report of the full-length sequence of RNA-2 of an ilarvirus.

Summary Freshly prepared tobacco mesophyll protoplasts behave as if they contain two classes, one of which is resistant to infection but the other is susceptible and can readily be doubly infected with brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV). BMV dominates in all double infections and although both types of virus particles are produced, only those of BMV are infectious. The defective CCMV particles contain normal amounts of RNA but only RNA 3 could be detected in them. No CCMV RNA 2 could be detected in doubly infected protoplasts, showing that there was only partial replication of the genome of CCMV. The proteins encoded by CCMV RNA 3 were produced in mixed infections. It is possible that BMV may exploit the gene products of CCMV while suppressing synthesis of infectious CCMV particles. There was no evidence that BMV RNA was encapsidated in CCMV coat protein.

Summary Cassia yellow blotch virus (CYBV) is a previously undescribed bromovirus infecting the Australian indigenous legume, Cassia pleurocarpa, in western Queensland. Although CYBV infected test plants in several families, symptoms were usually restricted to local lesions on the inoculated leaves. Systemic infection occurred in only eight leguminous species and in the non-legume, Nicotiana clevelandii. Chenopodium amaranticolor was a suitable host for local lesion assays and CYBV was readily purified from N. clevelandii. Purified CYBV preparations, when negatively stained, contained isometric particles 25 to 27 nm in diameter with a penetrated central core of approx. 10 nm. The virions sedimented as a single component with a sedimentation coefficient (s 20,w) of 85S and had an isoelectric point of 3.6. At pH 7.0, the virions sedimented more slowly than at pH 5.0 and 6.5 probably because they swelled. Purified virions contained a single coat protein species with a molecular weight of 20800 comprising 196 amino acid residues as estimated by FITMOL analysis of the amino acid composition. The virion genome consisted of four single-stranded RNA molecules of which only the three largest were required for infectivity. CYBV appeared to be serologically unrelated to three other bromoviruses and this lack of relationship correlated with differences in their coat protein amino acid compositions.