Cucumber mosaic virus mutants with altered physical properties and defective in aphid vector transmission

Two mutant strains of cucumber mosaic virus (CMV) were investigated with respect to virion stability and molecular determinants of aphid vector transmission. The mutant 2A1-MT-60x, derived from the mechanically passaged wild type 2A1-AT, is poorly transmissible by the aphid Aphis gossypii and not transmissible by the aphid Myzus persicae, whereas the wild type virus is transmissible by both aphid species. The mutant phenotype was shown to be conferred by a single encoded amino acid change of alanine to threonine at position 162 of the coat protein (CP). Modifying the mutant CP gene to encode the wild type sequence (alanine) at position 162 restored aphid transmission. To test for a correspondence between changes in the physical stability of virions and defects in aphid transmission, a urea disruption assay was developed. Virions of aphid-transmissible strains 2A1-AT and CMV-Fny were stable with treatments of up to between 3 and 4 M urea. In this assay mutant viruses 2A1-MT-60x and CMV-M were less stable, as they were completely disrupted at urea concentrations of 2 and 1 M urea, respectively. The mutant 2A1-MT-60x also accumulated at a reduced level in infected squash relative to the wild type virus. These studies suggest that a primary factor in the loss of aphid transmissibility of some strains of CMV is a reduction in virion stability.     

A peptide that binds the pea aphid gut impedes entry of Pea enation mosaic virus into the aphid hemocoel

Development of ways to block virus transmission by aphids could lead to novel and broad-spectrum means of controlling plant viruses. Viruses in the Luteoviridae enhanced are obligately transmitted by aphids in a persistent manner that requires virion accumulation in the aphid hemocoel. To enter the hemocoel, the virion must bind and traverse the aphid gut epithelium. By screening a phage display library, we identified a 12-residue gut binding peptide (GBP3.1) that binds to the midgut and hindgut of the pea aphid Acyrthosiphon pisum. Binding was confirmed by labeling the aphid gut with a GBP3.1-green fluorescent protein fusion. GBP3.1 reduced uptake of Pea enation mosaic virus (Luteoviridae) from the pea aphid gut into the hemocoel. GBP3.1 also bound to the gut epithelia of the green peach aphid and the soybean aphid. These results suggest a novel strategy for inhibiting plant virus transmission by at least three major aphid pest species.     

Location and sequence of a region of cauliflower mosaic virus gene 2 responsible for aphid transmissibility

The nucleotide sequence of the aphid nontransmissible Campbell isolate of cauliflower mosaic virus has been determined between the BstEll and XhoI sites, the region known to be associated with aphid transmission. Comparison of the DNA sequence with that of the aphid transmissible isolates revealed no gross structural alterations in any of the open reading frames (ORFs). Recombinant DNA hybrid constructs and in vitro alteration of ORF VII were used to verify that ORF VII was not involved in the regulation of the downstream ORF II. Instead, a modification within a small region of ORF II which alters only one amino acid of the potential protein product relative to the aphid-transmissible isolate was found to be responsible for the lack of aphid transmission in the Campbell isolate.     

A reinvestigation provides no evidence for sugar residues on structural proteins of poleroviruses and argues against a role for glycosylation of virus structural proteins in aphid transmission

Poleroviruses are strictly transmitted by aphids. Glycosylation of Turnip yellows virus (TuYV) was previously reported and this modification was supposed to be required for aphid transmission. Using different approaches based on (i) a lectin-binding assay, (ii) use of specific complex glycan antibodies and (iii) mass spectrometry, we found no evidence that the structural proteins of TuYV and Cucurbit aphid-borne yellow virus (CABYV) carry glycan residues. Moreover, mutation of each of the four potential N-glycosylation sites of the structural protein sequences of CABYV indicated that, unless more than one site on the structural protein is glycosylated, N-glycosylation is not involved in aphid transmission. These results did not corroborate the previous hypothesis for the role of glycosylation in aphid transmission. They, however, revealed the presence of a glycosylated plant protein in purified polerovirus suspensions, whose function in aphid transmission should be further investigated.     

Forecasting aphid outbreaks and epidemics of Cucumber mosaic virus in lupin crops in a Mediterranean-type environment

Cucumber mosaic virus (CMV) causes a serious disease of narrow-leafed lupin (Lupinus angustifolius). It is seed-borne in lupin and seed-infected plants act as the primary virus source for secondary spread by aphid vectors within crops. Infection with CMV causes yield losses of up to 60% in epidemic years, but has little impact on yield in years when spread is limited. Aphids also cause sporadic yield losses due to direct feeding damage. A simulation model was developed to forecast aphid outbreaks and epidemics of CMV in lupin crops growing in the 'grainbelt' of south-west Australia, which has a Mediterranean-type climate. The model uses rainfall during summer and early autumn to calculate an index of aphid build-up on weeds, crop volunteers and self-regenerating annual pastures in each 'grainbelt' locality before the growing season commences in late autumn. The index is used to forecast the timing of aphid immigration into crops. The subsequent aphid build-up and movement within the crop, spread of CMV from virus-infected source plants within the crop, yield losses and percentage of harvested seed-infected are then calculated. The model evaluates the effects of different sowing dates, percentages of CMV infection in seed sown and plant population densities on virus spread. The model simulations were validated with 14 years' field data from six different sites in the 'grainbelt', representing a wide range of pre-growing season rainfall scenarios, sowing dates, percentages of infection in seed sown and plant population densities. The model was incorporated into a decision support system (DSS) for use by lupin farmers and agricultural consultants in planning CMV management and targeting sprays against aphids to prevent direct feeding damage. The inputs required from the user are lupin cultivar, anticipated emergence date, percentage CMV infection in seed sown, plant density and location. The output consists of a personalised risk forecast for the user and includes predictions for date of first aphid arrival, aphid numbers, CMV spread, final virus incidence, yield loss due to infection and percentage infection in harvested seed. Predictions from the DSS are accessible via an Internet site and from other information sources. The model can serve as a template for modelling similar virus/aphid vector pathosystems in other regions of the world, especially those with Mediterranean-type climates.     

A single amino acid substitution at N-terminal region of coat protein of turnip mosaic virus alters antigenicity and aphid transmissibility

Summary The antigenic activity of the N-terminal region of coat protein of turnip mosaic virus (TuMV) aphid transmissible strain 1 and non-transmissible strain 31 was examined by using a panel of monoclonal antibodies (MAbs) raised against the two virus strains as well as antisera raised against several synthetic peptides from the N-terminal region of the protein. The reactivity of these antibodies was tested in ELISA and in a biosensor system (BIAcore Pharmacia) using virus particles, dissociated coat protein and synthetic peptides as antigens. Substitution of a single amino acid at position 8 in the coat protein of TuMV strain 1 abolished any cross-reactivity between MAbs to strain 1 and the substituted peptide (strain 31) in ELISA although some cross-reactivity was apparent in BIAcore inhibition experiments. In reciprocal tests with MAbs to strain 31 no cross-reactivity with the heterologous peptide was detected in either type of assay. The amino acid residue present at position 8 appears to play a critical role in the binding capacity of MAbs specific for the N-terminal region of TuMV. Antiserum to a synthetic peptide corresponding to residues 1–14 of the protein of TuMV strain 1 was found to react strongly with dissociated coat protein and intact virus particles and was able to inhibit the aphid transmission of the virus. Antiserum to the corresponding peptide of strain 31 did not have this capacity.     

A map of the diversity of RNA3 recombinants appearing in plants infected with Cucumber mosaic virus and Tomato aspermy virus

In order to better understand the role of recombination in creating the diversity of viral genomes that is acted on by selection, we have studied in detail the population of recombinant RNA3 molecules occurring in tobacco plants coinfected with wild-type strains of cucumber mosaic virus (CMV) and tomato aspermy virus (TAV) under conditions of minimal selection pressure. Recombinant RNA3s were observed in 9.6% of the samples. Precise homologous recombination predominated since it was observed at 28 different sites, primarily in six hot spots. Imprecise homologous recombination was observed at two sites, particularly within a GU repeat in the 5' noncoding region. Seven of the eight aberrant homologous recombination sites observed were clustered in the 3' noncoding region. These results have implications on the role of recombination in host adaptation and virus evolution. They also provide essential baseline information for understanding the potential epidemiological impact of recombination in transgenic plants expressing viral sequences.     

The cysteine-histidine-rich region of the movement protein of Cucumber mosaic virus contributes to plasmodesmal targeting, zinc binding and pathogenesis

Viral movement proteins (MPs) are central to the establishment of viral pathogenesis, and yet relatively little is understood about the structural and functional aspects of MPs or about the host factors on which they depend. Through chemical mutagenesis of transgenic Arabidopsis expressing Cucumber mosaic virus (CMV) MP fused with the green fluorescent protein, we have studied the function of a central region of the MP, defined by a number of conserved cysteine and histidine residues (Cys-His-rich region), which potentially functions as a zinc-binding domain. Transient expression of mutant MPs identified through an in planta screen for altered MP function or constructed with altered putative zinc ligands through site-directed mutagenesis showed that mutations in the Cys-His-rich region affected localization to and trafficking through plasmodesmata. In vitro zinc-binding analysis revealed that wild type (wt) CMV MP had the ability to bind zinc and that movement-defective mutants bound zinc with less affinity than wt MP. Furthermore, a correlation between the association of the MP with plasmodesmata and virus pathogenesis was shown. We discuss roles of the Cys-His region in biochemical and biological functions of the MP during virus movement.     

Nested deletion analysis of Wheat streak mosaic virus HC-Pro: Mapping of domains affecting polyprotein processing and eriophyid mite transmission

A series of in-frame and nested deletion mutations which progressively removed 5'-proximal sequences of the Wheat streak mosaic virus (WSMV) HC-Pro coding region (1152 nucleotides) was constructed and evaluated for pathogenicity to wheat. WSMV HC-Pro mutants with 5'-proximal deletions of 12 to 720 nucleotides systemically infected wheat. Boundary sequences flanking the deletions were stable and unaltered by passage through plants for all deletion mutants except HCD12 (lacking HC-Pro codons 3-6) that exhibited strong bias for G to A substitution at nucleotide 1190 in HC-Pro codon 2 (aspartic acid to asparagine). HC-Pro mutants with 5'-proximal deletions of up to 720 nucleotides retained autoproteolytic activity in vitro. In contrast, 5'-proximal deletion of 852 nucleotides of the HC-Pro coding region (HCD852) abolished both infectivity and in vitro proteolytic activity, confirming that the proteolytic domain of WSMV HC-Pro resides within the carboxy-terminal third of the protein and includes the cysteine proteinase motif (GYCY) conserved among four genera of the family Potyviridae. Inoculation of wheat with HC-Pro deletion mutants also bearing the GUS reporter gene revealed that HCD852 was unable to establish primary infection foci in inoculated leaves, indicating that processing of the P3 amino-terminus was essential. Deletion of as few as 24 nucleotides of HC-Pro (codons 3-10) eliminated transmission by the eriophyid mite vector Aceria tosichella Keifer. Collectively, these results demonstrated similar organization of proteinase and vector transmission functional domains among divergent HC-Pro homologues encoded by potyviruses and tritimoviruses.     

Stability and transmissibility of SARS-CoV-2 in the environment

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing the ongoing global coronavirus disease 2019 (COVID-19) pandemic, is believed to be transmitted primarily through respiratory droplets and aerosols. However, reports are increasing regarding the contamination of environmental surfaces, shared objects, and cold-chain foods with SARS-CoV-2 RNA and the possibility of environmental fomite transmission of the virus raises much concern and debate. This study summarizes the current knowledge regarding potential mechanisms of environmental transmission of SARS-CoV-2, including the prevalence of surface contamination in various settings, the viability and stability of the virus on surfaces or fomites, as well as environmental factors affecting virus viability and survival such as temperature and relative humidity. Instances of fomite transmission, including cold-chain food transmission, and the importance of fomite transmission in epidemics, are discussed. The knowledge gaps regarding fomite transmission of SARS-CoV-2 are also briefly analyzed.     

A fragile lattice: replacing bacteriophage lambda's head stability gene D with the shp gene of phage 21 generates the Mg2+-dependent virus, lambda shp

Phage lambda DNA packaging is accompanied by prohead expansion, due to structural changes in gpE, the major capsid protein. Rearrangement of the gpE lattice creates binding sites for trimers of gpD, the head stabilization protein. lambda-Like phage 21's shp gene is homologous to lambda's D gene. gpD and gpShp share 49% amino acid identity. To ask whether gpShp could stabilize the lambda head shell, we replaced lambda's D gene with shp, creating lambda shp. Unlike lambda or 21, lambda shp was strictly dependent on the presence of 10(-2) M Mg2+, and lambda shp virions were very sensitive to chelating agents. Density gradient studies indicated that the lambda gpE lattice was underpopulated with gpShp. gpD's N-terminus has been proposed to contact gpE, and we found that lambda D/shp, which produces a chimeric protein with the N-terminus of gpD and the C-terminus of gpShp, was Mg2+-independent and more stable than lambda shp.     

The ecology of Cucumber mosaic virus and sustainable agriculture

An account is given of the ecology of Cucumber mosaic virus (CMV) as a pertinent example of how a virus can affect the sustainability of an important crop. It is now generally accepted that the technologies used in modern agriculture should ensure that production systems are operated in such a way that the quality of the produce is maintained year after year without causing degradation of the environment. Recent experiences in countries of the Mediterranean basin demonstrate that the benefits expected from the introduction of new and highly productive plant varieties may be quickly eroded by the concomitant introduction of new virus strains which can greatly change the structure of the resident virus population. Quarantine inspection of plant propagules and genetic engineering are suggested as powerful tools to help achieve sustainability.     

Purification of pea enation mosaic virus from its pea aphid vector, Acyrthosiphon pisum, and aphid-transmission characteristics

Pea enation mosaic virus (PEMV) was purified from pea aphids by a system involving chloroform-butanol emulsification, differential centrifugation, and density gradient centrifugation. The product was highly infectious when transmitted to pea plants by pea aphids, and it was extracted in sufficiently high concentration to be monitored in centrifuged sucrose columns with a UV scanner. Electron micrographs showed that purified preparations contained an abundance of virus particles that measured ca. 27 nm in diameter and were indistinguishable from those isolated from infected plants. No virus particles were detected in preparations of nonviruliferous aphids, and no infectivity was obtained from these preparations in either aphid or mechanical transmission tests.Virus purified from an aphid source was established in pea plants by mechanical transmission and compared as to aphid-transmissibility with a virus line from a plant source; both lines were initiated with the same PEMV strain. The aphid-source virus line was transmitted by pea aphid first-stage nymphs with significantly (P < 0.05) higher efficiency than the plant-source virus line after 1- and 4-hr acquisition-access periods, and it was transmitted with characteristics superior to those previously recorded for PEMV-pea aphid relationships. First-instar pea aphids were 95.8% efficient in transmission of the aphid-source virus after only a 4-hr acquisition-access period on infected pea plants. The virus line when acquired from infected plants had a median latent period (LP₅₀) in the first instar of only 5.7 hr.     

Production of cucumber mosaic virus RNA5 and its role in recombination

Cucumber Mosaic Virus (CMV) is a plant infecting tripartite positive-strand RNA virus. In addition to three genomic and two known subgenomic RNAs, CMV strains of subgroup II (e.g. Q-CMV), but not subgroup I (e.g. Fny-CMV), produce and package a redundant RNA5 encompassing the 3′ 304-307 nucleotides of RNAs 2 and 3. The mechanism regulating RNA5 production and its role in CMV life cycle is unknown. In this study, transient expression of Q2 or Q3 by agroinfiltration into Nicotiana benthamiana plants resulted in efficient accumulation of RNA5 suggesting that its production is independent of CMV replication. Deletion and point mutations engineered into a highly conserved region (Box1) adjacent to the 5′ end of RNA5 identified sequences required for its efficient production. An experimental system, involving a chimera of Q3 (Q3B3) characterized by having a 3′ tRNA-like structure (3′TLS) from Brome mosaic virus (BMV) and RNA5 defective variants of Q1 (Q1Δ), Q2 (Q2Δ) and Q3B3 (Q3ΔB3), was used to evaluate in vivo the contribution of RNA5 in promoting RNA recombination. Generation of precise homologous recombinants was strictly dependent on sequence identity. When both parental RNAs carried the Box1, recombination occurred preferentially within the Box1. In contrast, generation of non-homologous recombinants occurred only when Q1 and Q2 were competent to produce RNA5. A mechanistic model explaining the functional role played by the RNA5 in generating CMV recombinants was presented.     

The fate of pea enation mosaic virus in its pea aphid vector, Acyrthosiphon pisum (Harris)

The fate of pea enation mosaic virus (PEMV) in its primary vector the pea aphid, Acyrthosiphon pisum (Harris), was studied by ultrathin sectioning and conventional transmission electron microscopy. PEMV virions were highly concentrated in the lumen of the midgut and hindgut, and also invaded a variety of tissues in the vector. Virions were detected in viroplasmlike areas, in defined structures in the cytoplasm, and in the nuclei of midgut epithelial cells. Virions also were seen in electron-dense areas in the cytoplasm of midgut muscle cells and hemocytes. Electron-dense structures containing viruslike particles occurred in the cytoplasm of the fat body. Organs and tissues in which PEMV could not be detected include the epidermis, mycetome, ovaries, tracheal system, foregut and hindgut epithelium and musculature, skeletal musculature, eye tissues, nervous system, and salivary system.     

Molecular epidemiology of Cucumber mosaic virus and its satellite RNA

Molecular analysis of viral isolates can yield information that facilitates an understanding of virus epidemiology and has been termed molecular epidemiology. This approach has only recently been applied to plant viruses. Results on the molecular epidemiology of Cucumber mosaic virus (CMV) and its satellite RNA (satRNA) in Spain, where CMV is endemic in vegetable crops are presented here. To characterise the genetic structure of CMV populations, c. 300 isolates, representing 17 outbreaks (i.e. sub-populations) in different crops, regions and years, were compared. Genetic analyses of CMV isolates were done by ribonuclease protection assay of cRNA probes representing RNA1, RNA2 and the two open reading frames in RNA3. All isolates belonged to one of three genetic types: Sub-group II and two types of Sub-group I. The genetic structure of the 17 sub-populations varied randomly, without correlation with location, year, or host plant species. Thus, CMV in Spain shows a metapopulation structure with local extinction and random recolonisation from local or distant virus reservoirs. The frequency of mixed infections and of new genetic types generated by reassortment of genomic segments or by recombination was also estimated. Results indicate that heterologous genetic combinations are not favoured. About 30% of CMV isolates were supporting a satRNA. The frequency of CMV isolates with a satRNA differed for each sub-population, being c. 1 in eastern Spain in 1990 and decreasing to c. 0 in distant regions and in subsequent years. Molecular analyses of CMV-satRNA isolates show high genetic diversity, due both to the accumulation of point mutations and to recombination. The CMV-satRNA population is a single, unstructured one. Thus, the CMV-satRNA population has a genetic structure and dynamics different from those of its helper virus. This indicates that CMV-satRNA has spread epidemically on the extant virus population from an original reservoir in eastern Spain. The relevance of these results for the control of CMV infections is discussed.     

Long-distance movement and viral assembly of tobacco mosaic virus mutants

Spreading of tobacco mosaic virus in infected plants is of two modes: cell-to-cell movement (to adjacent cells) and long-distance movement (to distant parts of the plant). Viral coat protein has been suggested to be involved in long-distance movement. To analyze the function of coat protein in the movement, we used mutants with modifications in the coat protein gene or in the assembly origin on the genomic RNA. A mutant which has the coding region for the C-terminal 5 amino acids of the protein deleted and mutants with 1 amino acid inserted after residue 101 or 152 of the protein retained both the abilities of long-distance movement and assembly into virus particles. Other mutants in the coat protein gene eliminated the two abilities. A mutant with modifications in the assembly origin displayed greatly reduced abilities of both the movement and assembly. These results suggest that both the coat protein with its ability to assemble into virus particles and the assembly origin are involved in long-distance movement, and that virus particles may play a pivotal role in the movement.     

Development of a new cucumber mosaic virus-based plant expression vector with truncated 3a movement protein

We have developed a Cucumber mosaic virus (CMV)-based expression vector for the production of heterologous proteins in plants. Cell-to-cell movement of CMV is dependent on the presence of coat protein (CP). Previous studies have shown that deletion of 33 amino acids (aa) from the carboxy-terminus of the 3a movement protein facilitates cell-to-cell movement that is independent of CP. The CMV-based expression vector that we have designed utilizes this truncated 3a protein, allowing the expression of target genes from the strong CP subgenomic promoter and without the need for providing CP in trans for cell-to-cell spread. Using this vector we achieved expression levels of ~ 450 mg/kg leaf tissue of green fluorescent protein (GFP) when the vector was delivered into Nicotiana benthamiana plants by agroinfiltration. Human growth hormone (hGH), on the other hand, accumulated to ~ 170 mg/kg of leaf tissue when the same approach was used to deliver the vector.     

Replication, stability, and gene expression of tobacco mosaic virus mutants with a second 30K ORF

A series of tobacco mosaic virus (TMV)-hybrids containing a second 30K open reading frame (ORF) inserted into different positions of the genome 3' region were constructed. These insertional mutants were used to evaluate the effects of a modified viral genome organization on replication and gene expression. They were evaluated for stability upon systemic infection and subsequent host passage using RNase protection assays. A mutant with the second 30K ORF fused in frame to two-thirds of the coat protein reading frame replicated as a free-RNA virus and produced increased amounts of the hybrid protein compared to the wild-type 30K protein, but substantially reduced amounts compared to the wild-type coat protein. A mutant with the second 30K ORF inserted between the native 30K and coat protein ORFs produced reduced amounts of 30K protein but replicated efficiently and was maintained for weeks of systemic infection before the population gradually shifted to progeny wild-type TMV. Mutants with the second 30K ORF fused behind different lengths of the coat protein subgenomic RNA promoter/leader region and inserted between the coat protein gene and the 3' nontranslated sequences replicated poorly and the mutations were not maintained during continued replication in plants.