Mutational evidence that the VPg is involved in the replication and not the movement of Pea enation mosaic virus-1.

Pea enation mosaic disease is caused by an obligatory association between the enamovirus Pea enation mosaic virus-1 (PEMV-1) and the umbravirus Pea enation mosaic virus-2 (PEMV-2). Encapsidated RNAs 1 and 2 are covalently linked to a 3138 Da VPg encoded by the RNA of PEMV-1. To determine the role of the VPg in the pathogenicity of PEMV (PEMV-1+PEMV-2), the infectivity of clones with mutations in key amino acids in the VPg was evaluated in protoplasts and in plants. Using quantitative, real-time RT-PCR, we concluded that the inability of certain mutants to infect plants was due to their replicative (and not their movement) incompetence. Mutant clones that produced delayed and less severe infections accumulated 10- to 100-fold less RNA-1 compared to WT-RNA-1 both in plants and in protoplasts. The RNAs of clones that produced WT-like infections accumulated to levels similar to those of WT-PEMV. Also, we demonstrate that the severity of symptoms produced by WT-PEMV is proportional to the amount of RNA-1 that accumulates in infected plants and seems to be independent of the amount of RNA-2. A dual role for the VPg in the pathogenicity of PEMV is proposed.

Sequencing, genomic localization and initial characterization of the VPg of pea enation mosaic enamovirus.

The amino acid sequence of the genome-linked viral protein (VPg) of pea enation mosaic enamovirus (PEMV) has been determined. The VPg is encoded by nt 1811-1894 within ORF1 of RNA1 downstream of the proteinase motif. Direct N terminus sequencing of intact and endoproteinase Asp-N-digested VPg combined with electrospray mass spectroscopy confirmed that the VPg is composed of 28 amino acids with a molecular mass of 3138 Da. The context of the N and C terminus residues as well as the position and size of the VPg suggest that the mature VPg may be generated via post-translational proteolytic processing of the polyprotein arrangement of membrane anchor-proteinase-VPg-polymerase encoded by ORFs 1 and 2. Computer comparisons did not reveal any significant similarity between the VPg of PEMV and any other sequences including those of the VPgs of related subgroup II luteoviruses.

Expression and suppression of circulative aphid transmission in pea enation mosaic virus.

Pea enation mosaic virus (PEMV) is composed of two autonomously replicating virus RNAs related to the genomic RNAs of viruses in the genera Luteovirus and Umbravirus. The transmission of PEMV resembles that of its luteovirus relatives in utilizing circulative aphid transmission. However, unlike its luteovirus counterparts, PEMV can also be mechanically transmitted. Prolonged mechanical passage of PEMV can lead to the loss of aphid transmissibility, a trait that is mirrored by specific changes in the PEMV virion composition. These changes were used to examine the virus contribution to vector transmission and the mechanisms by which it is regulated. Using a local lesion isolation technique, one aphid transmissible and two aphid non-transmissible isolates of PEMV were compared. Structural analysis of a 54 kDa minor structural subunit unique to the aphid transmissible isolate demonstrated that it was a fusion of the 21 kDa virus coat protein and a 33 kDa protein encoded immediately downstream of the 21 kDa ORF, consistent with the formation of the 54 kDa subunit by translational readthrough. Genetic analyses utilizing exchanges between infectious in vitro transcripts of each isolate demonstrated that although the 33 kDa protein was non-essential for infection, its presence was mandatory for aphid transmission, and that specific changes within the 33 kDa ORF were sufficient to confer or abolish aphid transmission. This study also demonstrates that isolates of PEMV exist as mixtures of aphid transmissible and non-transmissible genotypes, and provides insight into the mechanisms used by this virus to down-regulate aphid transmission in response to a specific selection pressure.

The satellite RNAs associated with the groundnut rosette disease complex and pea enation mosaic virus: sequence similarities and ability of each other's helper virus to support their replication.

Pea enation mosaic virus (PEMV) and the causal agents of groundnut rosette disease are diverse examples of disease complexes involving two RNA species, one of which is related to the genomes of luteoviruses and the other to those of umbraviruses. In both complexes, these viral RNA components may be supplemented with satellite RNAs that are dependent on the umbravirus component for replication and systemic movement, and on the luteovirus component for encapsidation and vector transmission. Sequence analysis identified regions of similarity between the satellites of groundnut rosette virus (GRV) and PEMV, particularly at the 5' and 3' termini and around duplicate sequence repeats present in each satellite RNA. The umbravirus GRV and the umbravirus-like PEMV RNA-2 were each able to support the replication and systemic spread of homologous and heterologous satellites. The presence of the PEMV satellite in infections with GRV had no effect on symptom expression in Nicotiana spp. or in Arachis hypogaea. Likewise, in Pisum sativum, the GRV satellite had no effect on the symptoms induced by PEMV. However, the intense yellow blotch symptoms induced in Nicotiana benthamiana by the YB3 GRV satellite in conjunction with GRV were also manifested when PEMV was the helper. Although PEMV RNA-1 was capable of supporting the encapsidation and aphid transmission of the GRV satellite, no evidence was obtained that the essential role of the GRV satellite in the aphid transmission of GRV could be supplied by the PEMV satellite. These data further strengthen the hypothesis of an evolutionary relationship between PEMV and the luteovirus-umbravirus complexes.

Replication of the satellite RNA of pea enation mosaic virus is controlled by RNA 2-encoded functions.

The helper virus mediating replication of the satellite RNA (RNA 3) of pea enation mosaic virus (PEMV) consists of two autonomously replicating, taxonomically unrelated viral RNAs with ties to the luteovirus (RNA 1) and the newly proposed umbravirus (RNA 2) genera. The following study dissects the relative contribution of each of the genomic RNAs of PEMV to the subsistence and dissemination of this satellite RNA. Infectivity assays in a pea protoplast system demonstrate that RNA 2 alone is responsible for the replication of RNA 3, an observation that is supported in part by shared regions of sequence homology at the 5' and 3' termini of both RNAs. In pea seedlings, infectivity assays also demonstrated that the presence of RNA 2 alone is necessary for the systemic invasion of RNA 3. In contrast, the luteovirus-like phase of PEMV (RNA 1) is solely responsible for the encapsidation and aphid transmission of both RNA 2 and the satellite RNA. In a manner comparable to several other virus-satellite systems, the satellite of PEMV also displays a differential response in its capacity to attenuate symptom expression in selected host species. Thus, the satellite RNA of PEMV exists in a trilateral arrangement with its host and two viral RNAs, comparable in many respects to the satellite-virus-host interaction occurring with groundnut rosette disease.

Assessment of the autonomy of replicative and structural functions encoded by the luteo-phase of pea enation mosaic virus.

The genome of pea enation mosaic virus (PEMV) is composed of two taxonomically unrelated RNAs, interacting to create what has traditionally been considered a bipartite virus. The cohesiveness of this interaction was assessed by examining the autonomy of each RNA in viral replication, coat protein expression and systemic invasion. Using a pea protoplast system, in vitro transcripts of RNA1 were found to be capable of initiating RNA2-independent replication, including the formation of the distinctive nuclear membrane-based replication complex associated with wild-type PEMV infection. Western blotting and electron microscopic analysis demonstrated that the synthesis of the RNA1-encoded coat protein, as well as virion assembly, was also independent of RNA2-directed functions. Mechanical inoculations with transcripts of RNA1 failed to establish a systemic RNA1 infection, whereas inoculations with RNA2 were able to establish a largely asymptomatic systemic infection. Combined inoculum containing RNA1 and RNA2 transcripts were able to recreate wild-type PEMV symptomatology, demonstrating the dependence of RNA1 on RNA2 for mechanical passage. With the notable exception of the adaptation of PEMV to establish a true systemic invasion, these data further strengthen the analogy between PEMV and the helper-dependent complexes associated with members of the luteovirus group.

The chimeric nature of the genome of pea enation mosaic virus: the independent replication of RNA 2.

The genome of pea enation mosaic virus (PEMV) consists of two plus-sense RNAs, both of which are required for mechanical transmission. RNA 1 (5706 nucleotides) has strong sequence similarity with members of the luteovirus group, a similarity that is also manifested in the symptomatology, cytopathology and vector transmission of this virus. RNA 2 (4253 nucleotides) is hypothesized to facilitate systemic invasion and mechanical transmission, attributes that distinguish PEMV from the phloem-limited luteoviruses. Sequence analysis of RNA 2 has demonstrated that PEMV is unique among multicomponent viruses in that it lacks 3'- and 5'-terminal homology between its genomic RNAs. Sequence analysis of RNA 2 has identified an open reading frame encoding a putative product of 65K that contains a series of polymerase-like motifs typical of viral RNA-dependent RNA polymerases. This protein sequence lacks homology with the polymerase encoded on RNA 1 of PEMV, instead being more closely affiliated with the polymerases of viruses related to the carmo- and tombusvirus groups. Inoculation of pea protoplasts with RNA transcripts derived from a full-length cDNA clone of RNA 2 has demonstrated that RNA 2 replicates autonomously in the absence of RNA 1, although comparable inoculation of whole plants failed to establish a systemic infection. There is no evidence that RNA 2 encodes structural proteins, suggesting that encapsidation functions are supplied in trans by RNA 1, comparable to the helper-dependent complexes occurring within the luteovirus group. These data suggest that the PEMV genome can be characterized as a symbiotic association of two taxonomically distinct viral RNAs cooperatively interacting in the establishment of a systemic virus infection.

The nucleotide sequence and luteovirus-like nature of RNA 1 of an aphid non-transmissible strain of pea enation mosaic virus.

An examination of the genomic strategy of pea enation mosaic virus (PEMV) RNA 1 has verified strong organizational and sequence relationships between PEMV and the beet western yellows-potato leafroll luteovirus subgroup. Sequence analysis of RNA 1 demonstrated five predominant open reading frames (ORFs). The extreme 5' ORF encodes a 34K product of unknown function. The second ORF encodes an 84K product which overlaps 90% of ORF 1 (in a unique reading frame) and is expressed by internal initiation beginning at the second start codon from the 5' terminus. This protein contains a protease-like motif characteristic of serine- and cysteine-based proteases, suggesting involvement in post-translational processing of viral translation products. The third ORF is characterized by a number of RNA polymerase motifs and a helicase-like motif typical of RNA-dependent RNA polymerases. It overlaps (out of frame) the ORF 2 product and is proposed to be expressed by a frameshift fusion of the ORF 2 and ORF 3 products. The fourth ORF encodes the viral coat protein, and is immediately followed in frame by a 33K ORF thought to represent the aphid transmission subunit of the PEMV virion. Northern blot analysis of polysome-associated RNA suggests that both products are expressed from an 1800 nucleotide subgenomic mRNA, with the 33K product expressed as a read-through fusion with the coat protein monomer.

The expression, localization, and effect of a human interferon in plants.

The ORF II of Cauliflower mosaic virus (CaMV) DNA was replaced with the human IFN alpha D coding sequence to yield a stable CaMV strain designated Ca524i. Inoculation of turnip (Brassica rapa cv "Just Right") with strain Ca524i DNA excised from plasmid pCa524i resulted in the production of biologically active IFN alpha D in infected plants. This was also true for its mutant (Ca562i) where one of the CYS codons was deleted. IFN alpha D produced in planta did not hamper superinfection with a single-stranded (+) sense RNA plant virus, turnip yellow mosaic virus (TYMV). Antibody gold labeling techniques and electron microscopy of infected plants showed that IFN was localized in the CaMV inclusion bodies.

The in vitro translation of Pea enation mosaic virus RNA.

Pea enation mosaic virus (PEMV) RNA was isolated from virions and then translated in rabbit reticulocyte lysates and in wheat germ extracts. RNA 1 (apparent molecular weight, Mr 1.77 x 106) was shown to code for two major translation products, vp2 (Mr 88,000) and vp4 (Mr 36,000) plus a high-molecular-weight minor product vp1 (Mr 147,000). Tryptic peptide comparisons of vp2 and vp4 revealed unique amino acid sequences for each product indicating that vp2 was not a read through protein of vp4. The in vitro translation products were synthesized in the order of their molecular weight, with vp4 appearing first. Therefore, vp4 and vp2 were not produced from a slowly processed precursor. RNA 2 (Mr 1.2 x 106) was shown to code for one product, vp3 (Mr 45,000). Northern analysis of total virion RNA, which was hybridized to cDNA transcribed from total virion RNA, did not detect additional RNA species. Therefore, gene products, vp2, vp3, and vp4, appear to be translated from the two genomic RNAs rather than from encapsidated subgenomic RNAs. PEMV antiserum specifically immunoprecipitated vp2, indicating that vp2 has amino acid sequences that are related to those of capsid protein.

Mechanisms underlying systemic invasion of pea plants by pea enation mosaic virus.

Leaves of Pisum sativum L.cv. 'Perfected Wales' were inoculated with pea enation mosaic virus, at the 3rd and 4th nodes as early as size permitted. Systemic virus spread was then measured over a period of approximately 9 days by assaying the infectivity of 1-cm stem pieces above the 4th and below the 3rd node. The presence of virus antigen and double-stranded (ds) RNA in these stem pieces was measured by radioimmunoassay over the same period. Relative infectivity and amounts of dsRNA in infected tissue were lowest approximately 3 days after inoculation and sharply increased thereafter. A transient plasmodesmatal abnormality was observed 4-6 days after inoculation. The transient nature of the abnormality, which was preceded (24 h) by low infectivity and dsRNA concentrations, may indicate a plant defense reaction that is subsequently overcome. The rise in dsRNA concentration, starting the 4th day after inoculation, coincided with the increase of vesicular material (cytopathological structures) in the phloem of infected plants. Vesicular material was found in sieve elements that were mature at the time of inoculation. Their presence in this tissue can only be explained by transport of the vesicles from infected sites.

Plant virus uncoating as a result of virus-cell wall interactions.

The fate of tobacco rattle virus (TRV) particles was studied after leaf panel infiltration. It was found that end-on virus attachment to cell walls of hosts (Nicotiana tobacum L. var. Xanthi-nc) and nonhosts (Zea mays L.) occurred, and that a virus degradative phase commenced immediately after attachment. Length of TRV particles changed drastically following infiltration. The normal length of the particles became smaller than that of the particle carrying the coat protein gene (108 nm). Five days after infiltration, no particles could be detected on the walls of cells bordering intercellular spaces. Virus attachment and degradation was thus shown to be nonspecific. Autoradiographic studies showed that iodinated viral coat protein or a breakdown product of this protein ((125I-label in coat protein of complete virus) and not virus particles are transported to the vascular bundle after infiltration. Tobacco mosaic virus (TMV) and TRV were infiltrated into their respective local lesion hosts. There was an initial time interval early after the infiltration during which wounding (pin pricking) the infiltrated leaf panels resulted in local lesion formation. The results are discussed with regard to the phenomenon of viral genome release in the plant virus infection process.

Pea enation mosaic virus genoma RNA contains no polyadenylate sequences and cannot be aminoacylated.

An active synthetase enzyme preparation from peas (Pisum sativum L.) did not catalyze the aminoacylation of pea enation mosaic virus RNA. The viral RNA was shown not to contain polyadenylic acid sequences.