The multifunctional capsid proteins of plant RNA viruses.

This article summarizes studies of viral coat (capsid) proteins (CPs) of RNA plant viruses. In addition, we discuss and seek to interpret the knowledge accumulated to data. CPs are named for their primary function; to encapsidate viral genomic nucleic acids. However, encapsidation is only one feature of an extremely diverse array of structural, functional, and ecological roles played during viral infection and spread. Herein, we consider the evolution of viral CPs and their multitude of interactions with factors encoded by the virus, host plant, or viral vector (biological transmission agent) that influence the infection and epidemiological facets of plant disease. In addition, applications of today's understanding of CPs in the protection of crops from viral infection and use in the manufacture of valuable compounds are considered.

A single amino acid mutation in the carnation ringspot virus capsid protein allows virion formation but prevents systemic infection.

A Carnation ringspot virus (CRSV) variant (1.26) was identified that accumulates virions but is incapable of forming a systemic infection. The 1.26 capsid protein gene possesses a Ser-->Pro mutation at amino acid 282. Conversion of 1.26 amino acid 282 to Ser restored systemic infection, while the reciprocal mutation in wild-type CRSV abolished systemic infection. Similar mutations introduced into the related Red clover necrotic mosaic virus capsid protein gene failed to induce the packaging but nonsystemic movement phenotype. These results provide additional support for the theory that virion formation is necessary but not sufficient for systemic movement with the dianthoviruses.

Spontaneous deletion enhances movement of a cucumber necrosis virus based chimera expressing the red clover necrotic mosaic virus movement protein genedagger.

Summary The 35-kDa movement protein (MP) gene of red clover necrotic mosaic virus (RCNMV) and 3' flanking sequence were inserted in a cucumber necrosis virus (CNV) deletion mutant lacking a large portion of the coding region for the MP. Nicotiana benthamiana plants inoculated with chimeric synthetic transcripts of the resulting hybrid cDNA clone (M5/RM2) developed both local and systemic symptoms and accumulated high levels of chimeric viral RNA. Reverse transcriptase polymerase chain reaction (RT-PCR) and sequence analysis of viral RNA extracted from systemically infected leaves of four different plants revealed that in each plant a large portion (305, 308, 315 or 127 nts) of the 3' terminus of the inserted sequence spontaneously deleted during infection. In three of the deletion derivatives, the truncated RCNMV MP open reading frame (ORF) was fused in-frame with the remaining portion of the 3' terminal region of CNV MP ORF. The movement efficiencies of M5/RM2, a cloned copy of one of the deletion derivatives (ClM5/RM2dd1), and a stop codon mutant of ClM5/RM2dd1 (ClM5/RM2dd1stop), which prevents translational fusion to the CNV MP, were compared and it was determined that deletion of RCNMV MP sequences in conjunction with fusion to CNV MP sequences increases the movement efficiency of the chimeric virus genome. Absence of the C-terminal region of the RCNMV MP in RCNMV RNA-2 abolished RCNMV movement. However, movement could be complemented in trans if cells were coinoculated with ClM5/RM2dd1. Complementation of RCNMV movement did not occur using ClM5/RM2dd1stop, suggesting a role for appended CNV MP sequences in movement of the RCNMV genome. The ability of the CNV replicase to delete unnecessary or deleterious RCNMV sequences and to append the required CNV MP sequences reinforces the role of RNA recombination in the adaptation and evolution of viral genomes.

Sequence element required for efficient -1 ribosomal frameshifting in red clover necrotic mosaic dianthovirus.

The RNA-1 of the bipartite red clover necrotic mosaic dianthovirus (RCNMV) genome encodes the 88-kDa polymerase. The polymerase is translated from both 5' proximal and internal open reading frames by a -1 ribosomal frameshifting event. A shifty heptanucleotide conforming to the simultaneous slippage model is identified, and a downstream stem-loop structure and atypical pseudoknot are predicted. A beta-glucuronidase reporter assay identified a 118-nucleotide element containing both the shifty heptanucleotide and the predicted secondary structures that were required for efficient -1 ribosomal frameshift expression in vivo. A series of site-directed and compensatory mutations affecting the base-paired regions of the predicted secondary structure were introduced into a RCNMV RNA-1 cDNA clone from which infectious transcripts were derived. Mutations that destroyed the predicted pseudoknot had no effect on frameshifting efficiency in vitro or infectivity of the virus, whereas mutations destabilizing the stem-loop structure abolished both ribosomal frameshifting in vitro and biological activity. These results demonstrate the essential role of a predicted secondary structure that does not involve a pseudoknot in the expression of the RCNMV polymerase by ribosomal frameshifting.

RNA-mediated trans-activation of transcription from a viral RNA.

The red clover necrotic mosaic virus genome is composed of two single-stranded RNA components, RNA-1 and RNA-2. The viral capsid protein is translated from a subgenomic RNA (sgRNA) that is transcribed from genomic RNA-1. Here, a 34-nucleotide sequence in RNA-2 is shown to be required for transcription of sgRNA. Mutations that prevent base-pairing between the RNA-1 subgenomic promoter and the 34-nucleotide trans-activator prevent expression of a reporter gene. A model is proposed in which direct binding of RNA-2 to RNA-1 trans-activates sgRNA synthesis. This RNA-mediated regulation of transcription is unusual among RNA viruses, which typically rely on protein regulators.

Mutations in viral movement protein alter systemic infection and identify an intercellular barrier to entry into the phloem long-distance transport system.

Viral systemic infection of a plant host involves two processes, cell-to-cell movement and long-distance transport. Molecular determinants associated with these two processes were probed by investigating the effects that alanine scanning mutations in the movement protein (MP) of red clover necrotic mosaic virus (RCNMV) had on viral infection in the plant hosts Nicotiana edwardsonii, Vigna unguiculata (cowpea), and the experimental plant Nicotiana benthamiana. Plants were inoculated with RCNMV expressing wild-type and mutant forms of the MP. Immunocytochemical studies at the light and electron microscope levels were performed on these plants, using a polyclonal antibody raised against the RCNMV capsid protein to identify the cells/tissues that RCNMV could infect. These experiments demonstrated that one cellular boundary at which the RCNMV MP functions to facilitate entry into the phloem long-distance transport system is located at the interfaces between the bundle sheath and phloem parenchyma cells and the companion cell-sieve element complex. Interestingly, in Nicotiana tabacum, a host that only allows a local infection, RCNMV cell-to-cell movement was found to be blocked at this same intercellular boundary. Four mutants that were able to systemically infect N. benthamiana were partially or completely defective for systemic infection of N. edwardsonii and cowpea, which indicated that these MP mutants exhibited host-specific defects. Thus, the roles of the RCNMV MP in cell-to-cell movement and in long-distance transport appear to be genetically distinct. These results are discussed in terms of the mechanism by which RCN MV enters the phloem to establish a systemic infection.

Characteristics of Beet Soilborne Mosaic Virus, a Furo-like Virus Infecting Sugar Beet.

Beet soilborne mosaic virus (BSBMV) is a rigid rod-shaped virus transmitted by Polymyxa betae. Particles were 19 nm wide and ranged from 50 to over 400 nm, but no consistent modal lengths could be determined. Nucleic acids extracted from virions were polyadenylated and typically separated into three or four discrete bands of variable size by agarose-formaldehyde gel electrophoresis. RNA 1 and 2, the largest of the RNAs, consistently averaged 6.7 and 4.6 kb, respectively. The sizes and number of smaller RNA species were variable. The molecular mass of the capsid protein of BSBMV was estimated to be 22.5 kDa. In Northern blots, probes specific to the 3' end of individual beet necrotic yellow vein virus (BNYVV) RNAs 1-4 hybridized strongly with the corresponding BNYVV RNA species and weakly with BSBMV RNAs 1, 2, and 4. Probes specific to the 5' end of BNYVV RNAs 1-4 hybridized with BNYVV but not with BSBMV. No cross-reaction between BNYVV and BSBMV was detected in Western blots. In greenhouse studies, root weights of BSBMV-infected plants were significantly lower than mock-inoculated controls but greater than root weights from plants infected with BNYVV. Results of serological, hybridization, and virulence experiments indicate that BSBMV is distinct from BNYVV. However, host range, capsid size, and the number, size, and polyadenylation of its RNAs indicate that BSBMV more closely resembles BNYVV than it does other members of the genus Furovirus.

Mapping of the red clover necrotic mosaic virus subgenomic RNA.

The red clover necrotic mosaic dianthovirus capsid protein is expressed in vivo from a subgenomic RNA (sgRNA) identical to the 3'-terminal 1.5 kb of RNA-1. The 5' leader sequence of the capsid protein sgRNA is 62 nucleotides, contains a 14-nucleotide putative promoter sequence homologous to the RNA-1 5' terminus, and exhibits a high level of similarity with the tobacco mosaic virus 5' leader translational enhancer element omega. Analysis of the RNA-1 secondary structure, in the region adjacent to the position where the 5' end of the sgRNA was mapped, predicts a stable stem-loop which includes the putative sgRNA promoter element. It is suggested that this structure is important for recognition of the sgRNA transcriptional initiation from the full-length negative-sense RNA-1.

Virion formation is required for the long-distance movement of red clover necrotic mosaic virus in movement protein transgenic plants.

The red clover necrotic mosaic dianthovirus (RCNMV) genome is split between two single-stranded RNA species. The polycistronic RNA-1 encodes the viral RNA polymerase and capsid protein (CP) and the monocistronic RNA-2 encodes the 35 kDa cell-to-cell movement protein (MP). Nicotiana benthamiana plants transformed with the RCNMV MP gene were generated. When inoculated onto the MP transgenic plants, cell-to-cell movement of RNA-1 occurred at a rate similar to wild-type virus. However, long-distance (leaf-to-leaf) movement of RNA-1 was not observed. Neither CP nor virions were detected in the inoculated leaves of the MP transgenic plants. When RNA-1 was coinoculated with RNA-2 mutants, which do not express a functional MP, onto MP transgenic plants, CP and virions were readily detected and a systemic infection resulted. These results demonstrate that both RNA-1 and RNA-2 are necessary for the accumulation of both CP and virions. Furthermore, CP accumulation was found to be required for long-distance movement of RCNMV. Therefore, these data provide evidence that CP, in the form of virions, is necessary for the long-distance movement of RCNMV.

Tobamovirus and dianthovirus movement proteins are functionally homologous.

The movement proteins (MPs) of tobacco mosaic tobamovirus (TMV) and red clover necrotic mosaic dianthovirus (RCNMV) enlarge plasmodesmata size exclusion limits, transport RNA from cell to cell, and bind nucleic acids in vitro. Despite these functional similarities, they have no sequence homology. However, they do appear to have similar secondary structures. We have used transgenic plants expressing either the TMV MP or the RCNMV MP, and a chimeric TMV that encodes the RCNMV MP as its only functional MP gene, to demonstrate that the MPs of TMV and RCNMV are functionally homologous. Further, both TMV and RCNMV can act as helper viruses to allow the cell-to-cell movement of the heterologous movement-defective viruses. These data support the conclusion that, despite other differences, such as particle morphology, host range, and sequence, TMV and RCNMV share a common mechanism for cell-to-cell movement.

Identification and analysis of the site of -1 ribosomal frameshifting in red clover necrotic mosaic virus.

The genomic RNA-1 of red clover necrotic mosaic dianthovirus (RCNMV) contains the heptanucleotide GGAUUUU that precedes the termination codon of the 5' proximal p27 open reading frame (ORF). This heptanucleotide is followed by a sequence with the potential to form a stable, complex secondary structure. Translation of RNA-1 is postulated to utilize a -1 ribosomal frameshifting mechanism to express the 88-kDa viral RNA polymerase. Using site-directed mutagenesis together with cell-free translation to monitor frameshifting and a biological assay of the mutants in plants, we establish the role of the GGAUUUU as the site where -1 ribosomal frameshifting occurs. The frameshifting signal sequence conforms to the simultaneous slippage model. Stop codons flanking the shifty signal are not required for frameshifting but the p27 ORF termination codon is necessary for maintaining optimal infectivity of the virus. Mutations abolishing the RCNMV RNA-1 internal p57 ORF initiation codon did not affect infectivity of the virus, suggesting that this cistron is only expressed in vivo as an 88-kDa ribosomal frameshifting product. Shifty heptanucleotide signals from a number of animal retroviruses and RNA plant viruses facilitate RCNMV frameshifting in vitro. However, only a limited number of the heterologous shifty heptanucleotides were functional in plant cells. We suggest that specific shifty tRNA populations in the cell facilitate viral -1 ribosomal frameshifting. This analysis also suggests that the slippery sequence requirements are not identical in mammalian and in plant systems.

Nucleotide sequence of carnation ringspot dianthovirus RNA-1.

The nucleotide sequence of carnation ringspot virus (CRSV) RNA-1, the type member of the dianthovirus genus, has been determined. The 3756 nucleotide genomic RNA-1 contains three large open reading frames (ORFs), capable of encoding 27K, 54K and 38K polypeptides. In addition, a small ORF encoding a 10K polypeptide at the 3' terminus of the RNA has been identified. The gene organization of CRSV RNA-1 is similar to those of red clover necrotic mosaic (RCNMV) and sweet clover necrotic mosaic (SCNMV) dianthoviruses with the exception that CRSV RNA-1 contains the additional 3'-terminal ORF. The 27K and 54K proteins possess significant sequence similarity to corresponding polypeptides of the other dianthoviruses. The 54K protein also contains the conserved RNA-dependent RNA polymerase motif. The identification of a shifty heptanucleotide preceding the p27 ORF termination codon and a predicted secondary structure following the terminator suggest that a translational frameshifting event allows translation to continue past the p27 ORF into the p54 ORF, which is in the -1 frame, generating an 88K fusion protein. Amino acid sequence alignment of the 38K protein with the corresponding RCNMV and SCNMV polypeptides indicate that this is the viral capsid protein.

Cell-to-Cell Trafficking of Macromolecules through Plasmodesmata Potentiated by the Red Clover Necrotic Mosaic Virus Movement Protein.

Direct evidence is presented for cell-to-cell trafficking of macromolecules via plasmodesmata in higher plants. The fluorescently labeled 35-kD movement protein of red clover necrotic mosaic virus (RCNMV) trafficked rapidly from cell to cell when microinjected into cowpea leaf mesophyll cells. Furthermore, this protein potentiated rapid cell-to-cell trafficking of RCNMV RNA, but not DNA. Electron microscopic studies demonstrated that the 35-kD movement protein does not unfold the RCNMV RNA molecules. Thus, if unfolding of RNA is necessary for cell-to-cell trafficking, it may well involve participation of endogenous cellular factors. These findings support the hypothesis that trafficking of macromolecules is a normal plasmodesmal function, which has been usurped by plant viruses for their cell-to-cell spread.

Alanine scanning mutagenesis of a plant virus movement protein identifies three functional domains.

Alanine scanning mutagenesis was performed on the red clover necrotic mosaic virus (RCNMV) movement protein (MP), and 12 mutants were assayed in vitro for RNA binding characteristics and in vivo for their ability to potentiate RCNMV cell-to-cell movement. The mutant phenotypes that were identified in vitro and in vivo suggest both that cooperative RNA binding is not necessary for cell-to-cell movement in vivo and that only a fraction of the wild-type RNA binding may be required. The MP mutants defined at least three distinct functional regions in the MP: an RNA binding domain, a cooperative RNA binding domain, and a third domain that is necessary for cell-to-cell movement in vivo. This third domain may be required for targeting the MP to cell walls and plasmodesmata, interacting with host proteins, folding, or possibly binding RNA into a functional ribonucleoprotein complex capable of cell-to-cell movement.

Synthesis of the putative red clover necrotic mosaic virus RNA polymerase by ribosomal frameshifting in vitro.

The red clover necrotic mosaic virus (RCNMV) genome is split between two single-stranded RNA species termed RNA-1 and RNA-2. RNA-1 directs the synthesis of 88-kDa (p88), 57-kDa (p57), 37-kDa (p37), and 27-kDa (p27) polypeptides and RNA-2 a 35-kDa (p35) polypeptide in vitro. The coding order of the RNA-1 products was determined to be 5'-p27-p57-p37-3'. Antibodies to synthetic peptides representing the carboxyl terminal portions of p27 and p57 immunoprecipitated their respective polypeptides in addition to p88, suggesting that p88 is a fusion protein. A frameshift heptanucleotide sequence element has been identified in RCNMV RNA-1. In addition, a stable stem-loop secondary structure adjacent to the heptanucleotide sequence is predicted. Together, these sequence elements suggest that a ribosomal frameshifting event occurs which allows translational readthrough of the p27 open reading frame into the p57 open reading frame, generating the observed p88 product. An RNA-1 expression construct fusing the p57 and the CP open reading frame was engineered to investigate the ribosomal frameshifting event. CP antibodies immunoprecipitated a fusion protein of the predicted size containing the carboxyl portion of CP. Site-directed mutagenesis of the frameshift element indicates that in vitro, p88 can also be expressed alternatively by suppression of an amber termination codon. Based on these data, we propose that the putative RCNMV RNA polymerase is an 88-kDa polypeptide expressed by a ribosomal frameshifting mechanism similar to those utilized by retroviruses.

The roles of the red clover necrotic mosaic virus capsid and cell-to-cell movement proteins in systemic infection.

The red clover necrotic mosaic dianthovirus (RCNMV) genome is split between two single-stranded RNA species termed RNA-1 and RNA-2. RNA-2 is required for infection of whole plants but is dispensable for infection and virion formation in protoplasts. We have used full-length cDNA clones of RNA-1 and -2 from which infectious in vitro transcripts can be derived to construct a number of mutations in the RNA-1 encoded capsid protein and the RNA-2 encoded cell-to-cell movement protein genes. The capsid protein and the RNA sequence encoding the capsid protein were dispensable for infection of the inoculated leaves of Nicotiana benthamiana and N. clevelandii at both 15 and 25 degrees. In addition, capsid protein was not necessary for systemic infection of N. benthamiana at 15 degrees. As many as 39 amino acid residues could be deleted from the carboxyl-terminus of the RNA-2 encoded 35-kDa cell-to-cell movement protein without loss of or reduction in the rate of cell-to-cell movement or systemic infection. However, larger deletions within the cell-to-cell movement protein gene prevented cell-to-cell movement and systemic infection of N. benthamiana. These data suggest that the spread of RCNMV in a systemic host is a combination of two distinct events: cell-to-cell movement and long distance transport. We conclude that the RCNMV 35-kDa movement protein is required for cell-to-cell movement, whereas the capsid protein is not necessary for cell-to-cell movement and, depending on host genotype and environmental factors, may or may not be required for long distance transport.

Nucleotide sequence of the capsid protein cistrons from six potato virus Y (PVY) isolates infecting tobacco.

Complementary DNA libraries representing the capsid protein cistron of the potato virus Y (PVY) isolate 'Chilean', 'Hungarian', MsNr, NsNr, O, and 'Potato US' were synthesized and used as template for polymerase chain reaction (PCR) amplification. An AUG codon for initiating a discrete capsid protein (CP) open reading frame was embedded upstream of the first codon of the CP cistrons. PCR-amplified products of the expected size of 0.8 kilo bases were cloned into the transcription vector pBS(+). The fidelity of each PCR-amplified PVY CP cistron was tested by transcribing recombinant plasmids in vitro and translating the transcripts in two cell free translation systems. Translation analysis of in vitro transcribed PVY CP cistrons consistently yielded a polypeptide co-migrating with authentic CP that was immunoprecipitated by anti PVY 'Chilean' antibodies. The nucleotide sequence of each capsid protein gene was determined by dideoxy sequence analysis. Each capsid protein gene was determined to be 801 nucleotides in length, encoding a deduced protein of 267 amino acids with calculated M(r) ranging from 29,799 to 29,980. The nucleic acid sequence similarity between the six isolates ranged between 89 to 97% and the amino acid similarity between 91 to 99%. The high level of amino acid sequence similarity confirms the classification of these viruses as isolates of PVY.

Nucleotide sequence of carnation ringspot dianthovirus RNA-2.

RNA-2 of carnation ringspot virus (CRSV), the type member of the dianthovirus group, has been cDNA cloned and sequenced. CRSV RNA-2 is 1394 nucleotides in length and contains a single open reading frame encoding a 304 amino acid polypeptide of 33.8K. Amino acid sequence alignment of this polypeptide with the cell-to-cell movement proteins encoded by RNA-2 of red clover necrotic mosaic virus (RCNMV) Australian (Aus) and Czechoslovakian (TpM-34) isolates indicates 59.6% and 55.7% sequence identity, respectively. The N-terminal 230 amino acids are more highly conserved, with 64.3% and 62.6% sequence identity, respectively. The cell-to-cell movement proteins of the two RCNMV isolates are themselves 82.5% and 91.7% identical when the amino-terminal 230 amino acids are compared. Structural prediction comparison of the RCNMV-Aus, RCNMV-TpM-34 and tobacco mosaic virus cell-to-cell movement proteins to the putative CRSV RNA-2-encoded movement protein suggests that even though no primary amino acid sequence similarity exists, the movement protein polypeptides are possibly similar in structure and function.

Potyviridae: genus Rymovirus.

The genus Rymovirus of the family Potyviridae is comprised of seven rod-shaped viruses with the shared characteristic of being transmitted by mites. Aside from this distinguishing feature, rymoviruses are similar to aphid-transmitted potyviruses in that they share a similar particle morphology, some similar antigenic determinants, similar physico-chemical properties, the ability to induce the formation of cytoplasmic cylindrical inclusions, and the ability to infect only graminaceous hosts. In vitro translation studies with wheat streak mosaic virus (WSMV) suggest that this rymovirus uses a potyviral proteolytic processing strategy to express the 3' terminal capsid protein. At the molecular level, limited nucleotide sequence data for WSMV show similarities with aphid-transmitted potyviruses in the potyviral capsid protein, large nuclear inclusion and cylindrical inclusion regions. Thus, given the similarities between the rymoviruses and the potyviruses, it is appropriate to include this genus within the family Potyviridae.