Cucumber Mosaic Virus Genome Research Articles

Mixed viral infection constrains the genome formula of multipartite cucumber mosaic virus

Many plant viruses have a multipartite organization, with multiple genome segments packaged into separate virus particles. The genome formula describes the relative frequencies of all viral genome segments, and previous work suggests rapid changes in these frequencies facilitate virus adaptation. Many studies have reported mixed viral infections in plants, often resulting in strong virus–virus interactions. Here, we tested whether mixed infections with tripartite alfalfa mosaic virus (AMV) and monopartite potato virus Y (PVY) affected the genome formula of the tripartite cucumber mosaic virus (CMV), our experimental model. We found that the CMV titer was reduced in mixed infections with its tripartite Bromoviridae relative AMV and in triple infections with both AMV and PVY, indicating notable virus–virus interactions. The variability of the CMV genome formula was significantly lower in mixed infections (CMV and AMV, CMV and PVY, and CMV and AMV and PVY) than in single infections (CMV only). These observations led to the surprising conclusion that mixed infections with two distinct viruses constrain the CMV genome formula. It remains unclear how common these effects are for different combinations of virus species and strains and what the underlying mechanisms are. We, therefore, extended a simulation model to consider three putative scenarios in which a second virus affected the genome formula. The simulation results also suggested that shifts in the genome formula occur, but may not be widespread due to the required conditions. One scenario modeled—co-infection exclusion through niche differentiation—was congruent with the experimental data, as this scenario led to reductions in genome formula variability and titer of the multipartite virus. Whereas previous studies highlighted host–species effects, our results indicate that the genome formula is also affected by mixed infections, suggesting that there is a broader set of environmental cues that affect the genome formula.

Visual tracking of viral infection dynamics reveals the synergistic interactions between cucumber mosaic virus and broad bean wilt virus 2

Cucumber mosaic virus (CMV) is one of the most prevalent plant viruses in the world, and causes severe damage to various crops. CMV has been studied as a model RNA virus to better understand viral replication, gene functions, evolution, virion structure, and pathogenicity. However, CMV infection and movement dynamics remain unexplored due to the lack of a stable recombinant virus tagged with a reporter gene. In this study, we generated a CMV infectious cDNA construct tagged with a variant of the flavin-binding LOV photoreceptor (iLOV). The iLOV gene was stably maintained in the CMV genome after more than four weeks of three serial passages between plants. Using the iLOV-tagged recombinant CMV, we visualized CMV infection and movement dynamics in living plants in a time course manner. We also examined whether CMV infection dynamics is influenced by co-infection with broad bean wilt virus 2 (BBWV2). Our results revealed that no spatial interference occurred between CMV and BBWV2. Specifically, BBWV2 facilitated the cell-to-cell movement of CMV in the upper young leaves. In addition, the BBWV2 accumulation level increased after co-infection with CMV.

RNA In Situ Hybridization of Detecting Cucumber Mosaic Virus in Shoots of Nicotiana benthamiana Plants.

RNA in situ hybridization, a histological technique derived from Southern blotting and northern blotting, has been an important approach in biology studies for many years. In the studies of virus-plant interactions, RNA in situ hybridization provides a direct visualization of viral RNA in host plants. Here, we provide a detailed protocol for viral RNA in situ hybridization that has been successfully used to detect Cucumber mosaic virus genome (CMV) RNAs in shoots of N. benthamiana plants.

Analysis of the complete genome sequence of cucumber mosaic virus from Vinca minor and Wisteria sinensis in Iran

We present here the first complete sequence of cucumber mosaic virus (CMV) from RNA sequencing of a dwarf periwinkle (Vinca minor) and a Wisteria sinensis in Iran by de novo assembly and annotation of contigs. The tripartite genome of CMV-Ir-VM and Ir-WS consists of a 3391-nucleotide (nt) RNA1, a 3038-nt RNA2, and a 2197-nt RNA3 segment. The sequence comparisons and phylogenetic analyses revealed that all three RNA segments of these two isolates belonged to the CMV subgroup II. RNA1, RNA2, and RNA3 of these two isolates, respectively shared 97.61–99.06%, 97.83–98.88%, and 97.19–98.86% nucleotide identities to those of CMV subgroup II isolates. In genetic analysis, the 2b gene revealed more genetic variability (π = 0.2614) compared with other regions. Our results indicated that purifying selection pressure was the major force acting upon the CMV genome, although some positively selected sites were identified for all encoded proteins.

Detection and Characterization of Tomato spotted wilt virus and Cucumber mosaic virus on Pepper Growing Areas in Antalya

The most efficient method to control the plant virus diseases is breeding resistant cultivars. However, the resistance could be broken down after using resistant cultivars. This study was aimed to determine the prevalence and also serological and molecular characterization of Tomato spotted wilt virus (TSWV) and Cucumber mosaic virus (CMV) that cause infections, especially, in resistant pepper cultivars. For this reason, samples were collected from pepper growing greenhouses and open fields during vegetation period of 2015 in different parts of Antalya province including Kumluca, Demre, Serik and Aksu districts. Out of 148 pepper samples collected, 53 (35.81%) were infected with TSWV and 11 (7.34%) with CMV as a result of Double-Antibody Sandwich Enzyme-Linked Immunosorbent Assay (DASELISA) test. Some regions on S RNA (nucleocapsid protein gene), M RNA (glycoprotein gene) and L RNA (RNAdependent RNA polymerase gene) of TSWV genome; RNA 1 (helicase/methyltransferase gene) and RNA 3 (coat protein gene) of CMV genome of DAS-ELISA positive samples were amplified by RT-PCR with specific primers. Nucleotide similarity rates of nucleocapsid protein gene, glycoprotein gene and RNA-dependent RNA polymerase gene regions of TSWV isolate varied between 92-98% identity with other isolates in GenBank and CMV isolate varied between 89-96%. TSWV isolate showed nucleotide identity varied between 92-97% with Tsw resistance breaking isolates from Capsicum plants in Turkey, Italy and Spain. Genetic determinant for overcoming pepper Tsw resistance is located in S segment but aminoacid substitutions responsible for TSWV breakdown remain contradictory in several reports.

Profiling of small RNAs derived from cucumber mosaic virus in infected Nicotiana benthamiana plants by deep sequencing

In plants, RNA silencing is a conserved mechanism underlying antiviral immunity. To investigate antiviral responses in Nicotiana benthamiana, we analyzed the profiles of the virus-derived small RNAs (vsRNAs) in wild-type N. benthamiana and NbRDR6 mutant plants infected with the cucumber mosaic virus (CMV) 2b-deficient mutant. We observed that NbRDR6 regulates RNA silencing by producing vsRNAs that trigger an effective antiviral response, while NbRDR1 may nonredundantly and synergistically function with NbRDR6 to mediate immune responses. The vsRNAs in N. benthamiana and NbRDR6 mutant plants mainly comprised 21 or 22 nucleotides, and mostly consisted of a 5′-terminal adenine. Additionally, NbAGO2 expression was significantly up-regulated in N. benthamiana and NbRDR6 mutant plants, suggesting that NbAGO2 is closely associated with the antiviral activities of vsRNAs. The distribution of vsRNAs in the CMV genome was biased toward RNA sense strands in both N. benthamiana and NbRDR6 mutant plants. These findings indicate the specific and conserved antiviral immunity in Nicotiana benthamiana.

Characterization of siRNAs derived from cucumber mosaic virus in infected tobacco plants.

This study characterized the viral small interfering RNAs (vsiRNAs) from Nicotiana tabacum cv. Samsun infected with a cucumber mosaic virus (CMV) 2b-deficient mutant. Most vsiRNAs were 21 -22 nucleotides in length and the 5'-terminal ends were dominated by A and U, respectively. The observed vsiRNAs were heterogeneously distributed throughout the CMV genome; however, most of the vsiRNAs were derived from sense strands, as opposed to antisense strands. These results demonstrate the conserved and specific function of Dicer-like (DCL), Argonaute (AGO) and RNA-dependent RNA polymerase (RDR) proteins in tobacco. Finally, it was revealed that vsiRNAs target abundant host genes, indicating complex roles for CMV vsiRNAs during the development of symptoms.

Genetic structure and molecular variability of Cucumber mosaic virus isolates in the United States.

Cucumber mosaic virus (CMV) has a worldwide distribution and the widest host range of any known plant virus. From 2000 to 2012, epidemics of CMV severely affected the production of snap bean (Phaseulos vulgaris L.) in the Midwest and Northeastern United States. Virus diversity leading to emergence of new strains is often considered a significant factor in virus epidemics. In addition to epidemics, new disease phenotypes arising from genetic exchanges or mutation can compromise effectiveness of plant disease management strategies. Here, we captured a snapshot of genetic variation of 32 CMV isolates collected from different regions of the U.S including new field as well as historic isolates. Nucleotide diversity (π) was low for U.S. CMV isolates. Sequence and phylogenetic analyses revealed that CMV subgroup I is predominant in the US and further showed that the CMV population is a mixture of subgroups IA and IB. Furthermore, phylogenetic analysis suggests likely reassortment between subgroups IA and IB within five CMV isolates. Based on phylogenetic and computational analysis, recombination between subgroups I and II as well as IA and IB in RNA 3 was detected. This is the first report of recombination between CMV subgroups I and II. Neutrality tests illustrated that negative selection was the major force operating upon the CMV genome, although some positively selected sites were detected for all encoded proteins. Together, these data suggest that different regions of the CMV genome are under different evolutionary constraints. These results also delineate composition of the CMV population in the US, and further suggest that recombination and reassortment among strain subgroups does occur but at a low frequency, and point towards CMV genomic regions that differ in types of selection pressure.

Suppression of Antiviral Silencing by Cucumber Mosaic Virus 2b Protein in Arabidopsis Is Associated with Drastically Reduced Accumulation of Three Classes of Viral Small Interfering RNAs

We investigated the genetic pathway in Arabidopsis thaliana targeted during infection by cucumber mosaic virus (CMV) 2b protein, known to suppress non-cell-autonomous transgene silencing and salicylic acid (SA)-mediated virus resistance. We show that 2b expressed from the CMV genome drastically reduced the accumulation of 21-, 22-, and 24-nucleotide classes of viral small interfering RNAs (siRNAs) produced by Dicer-like4 (DCL4), DCL2, and DCL3, respectively. The defect of a CMV 2b-deletion mutant (CMV-Delta2b) in plant infection was efficiently rescued in Arabidopsis mutants producing neither 21- nor 22-nucleotide viral siRNAs. Since genetic analysis further identifies a unique antiviral role for DCL3 upstream of DCL4, our data indicate that inhibition of the accumulation of distinct viral siRNAs plays a key role in 2b suppression of antiviral silencing. Strikingly, disease symptoms caused by CMV-Delta2b in Arabidopsis mutants defective in antiviral silencing were as severe as those caused by CMV, demonstrating an indirect role for the silencing suppressor activity in virus virulence. We found that production of CMV siRNAs without 2b interference depended largely on RNA-dependent RNA polymerase 1 (RDR1) inducible by SA. Given the known role of RDR6-dependent transgene siRNAs in non-cell-autonomous silencing, our results suggest a model in which 2b inhibits the production of RDR1-dependent viral siRNAs that confer SA-dependent virus resistance by directing non-cell-autonomous antiviral silencing.

Quantitative Determination of <italic>Cucumber Mosaic Virus</italic> Genome RNAs in Virions by Real-Time Reverse Transcription-Polymerase Chain Reaction

A real-time RT-PCR procedure using the green fluorescent dye SYBR Green I was developed for determining the absolute and relative copies of cucumber mosaic virus (CMV) genomic RNAs contained in purified virions. Primers specific to each CMV ORF were designed and selected. Sequences were then amplified with length varying from 61 to 153 bp. Using dilution series of CMV genome RNAs prepared by in vitro transcription as the standard samples, a good linear correlation was observed between their threshold cycle (Ct) values and the logarithms of the initial template amounts. The copies of genomic RNA 1, RNA 2, RNA 3 and the subgenomic RNA 4 in CMV virions were quantified by this method, and the ratios were about 1.00:1.17:3.58:5.81. These results were confirmed by Lab-on-a-chip and northern blot hybridization assays. Our work is the first report concerning the relative amounts of different RNA fragments in CMV virions as a virus with tripartite genome.

Differential selection of genes of cucumber mosaic virus subgroups.

Cucumber mosaic virus (CMV) has an extremely broad plant-host range, a large number of vector species, and a wide geographical distribution. CMV is, therefore, a model by which to understand plant virus adaptation. The selective constraints exerted on the five proteins expressed from the CMV genome were evaluated by application of newly developed maximum-likelihood algorithms to analyze sequences available in data banks. The ratio between nonsynonymous and synonymous substitution rates (omega) was used to detect positive selection on particular codon sites. Amino acid sequences were conserved with omega ranging from 0.07 to 0.60 in different proteins. However, a small proportion of amino acids in proteins 1a, 2a, and 3b, the coat protein (CP), were positively selected (omega > 1). Moreover, the evolution of the CP in the three subgroups of CMV strains revealed different selection profiles along the sequence and significantly different speed of evolution at many positions. Constraints exerted by aphid transmission, rather than plant adaptation, seemed to be responsible for these patterns of evolution in the CP.

Cucumoviruses

Research on the molecular biology of cucumoviruses and their plant-virus interactions has been very extensive in the last decade. Cucumovirus genome structures have been analyzed, giving new insights into their genetic variability, evolution, and taxonomy. A new viral gene has been discovered, and its role in promoting virus infection has been delineated. The localization and various functions of each viral-encoded gene product have been established. The particle structures of Cucumber mosaic virus (CMV) and Tomato aspermy virus have been determined. Pathogenicity domains have been mapped, and barriers to virus infection have been localized. The movement pathways of the viruses in some hosts have been discerned, and viral mutants affecting the movement processes have been identified. Host responses to viral infection have been characterized, both temporally and spatially. Progress has been made in determining the mechanisms of replication, gene expression, and transmission of CMV. The pathogenicity determinants of various satellite RNAs have been characterized, and the importance of secondary structure in satellite RNA-mediated interactions has been recognized. Novel plant genes specifying resistance to infection by CMV have been identified. In some cases, these genes have been mapped, and one resistance gene to CMV has been isolated and characterized. Pathogen-derived resistance has been demonstrated against CMV using various segments of the CMV genome, and the mechanisms of some of these forms of resistances have been analyzed. Finally, the nature of synergistic interactions between CMV and other viruses has been characterized. This review highlights these various achievements in the context of the previous work on the biology of cucumoviruses and their interactions with plants.

Reverse transcription-polymerase chain reaction (RT-PCR) for direct detection of cucumber mosaic virus (CMV) in gladiolus.

A diagnostic method based on Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was performed for detection of CMV genome directly in crude extracts of both healthy and infected gladiolus tissue using primers from the conserved sequences of CMV RNA-3. RT-PCR resulted in the amplification of 540 bp long fragment of CMV coat protein gene (CMV-CP), as expected in most of the plant parts of infected gladiolus samples. Positive signals in Southern hybridization of amplified fragments with cloned CMV-CP cDNA probe also confirmed the presence of CMV genome in gladiolus.

New Overlapping Gene Encoded by the Cucumber Mosaic Virus Genome

Cucumber mosaic virus (CMV) contains three genomic RNAs (RNAs 1,2, and 3) and a subgenomic RNA (RNA 4), a shared feature of the Bromoviridae family which includes cucumoviruses, bromoviruses, alfalfa mosaic virus, and ilarviruses. We describe in this paper the molecular characterization of a novel subgenomic RNA of the Q strain of CMV (Q-CMV), RNA 4A, which was first reported in 1973 (K. W. C. Peden and R. H. Symons, Virology 53, 487-492, 1973). RNA 4A is 682 nucleotides and is identical in sequence to the 3′-terminal 682 nucleotides of RNA 2. RNA 4A encodes a small open reading frame (ORF) of 100 codons, which, in RNA 2, overlaps the C-terminal portion of the major 2a gene; thus it is likely that RNA 4A functions as the mRNA for the in vivo expression of the ORF, called ORF 2b. Polyclonal antibodies raised against a 2b fusion protein expressed in Escherichia coli specifically detected the 2b gene product in Q-CMV-infected cucumber plants by Western immunoblotting. Examination of published viral RNA sequences revealed the conservation of ORF 2b in all of the four other cucumoviruses sequenced to date; however, it is absent from the rest of the Bromoviridae. We suggest that the proposed ORF 2b may be expressed in other cucumoviruses, most likely via 4A-like subgenomic RNAs, and that the predicted gene product may have a unique functional role in the infection process of cucumoviruses.

Cucumber mosaic virus genome is encapsidated in alfalfa mosaic virus coat protein expressed in transgenic tobacco plants

The expression of viral coat protein (CP) in transgenic plants has been shown to be very effective in virus plant protection. However, the introduction of CP genes into plants presents the potential risk of the encapsidation of a superinfecting viral genome in the transgenic protein, an event which could change the epidemiology of the disease. To detect the potential heterologous encapsidation of the cucumber mosaic virus (CMV) genome by alfalfa mosaic virus (AIMV) CP expressed in transgenic tobacco plants, a system of immunocapture (IC) and amplification by polymerase chain reaction (PCR) was optimized. This provided high sensitivity and reliable selection of the heterologously encapsidated CMV genome in the presence of natural CMV particles. As little as 2 pg of virus could be detected by immunocapture/polymerase chain reaction (IC/PCR) technique. Evidence for heterologous encapsidation of the CMV genome was found in 11 of the 33 transgenic plants tested two weeks after CMV inoculation. This demonstrates a significant rate of heterologous encapsidation events between two unrelated viruses in transgenic plants. Since CP is involved in the interactions of the virus particle with its vector, the release in the field of such transgenic plants could alter the transmission properties of some important viruses.

Nucleotide sequence of cucumber mosaic virus RNA. 1. Presence of a sequence complementary to part of the viral satellite RNA and homologies with other viral RNAs.

The nucleotide sequence of the 3389 residues of RNA 1 (Mr 1.15 X 10(6) of the Q strain of cucumber mosaic virus (CMV) was determined, completing the primary structure of the CMV genome (8617 nucleotides). CMV RNA 1 was sequenced by the dideoxy-chain-termination method using M13 clones carrying RNA 1 sequences as well as synthetic oligonucleotide primers on RNA 1 as a template. At the 5' end of the RNA there are 97 noncoding residues between the cap structure and the first AUG (98-100), which is the start of a single long open-reading frame. This reading frame encodes a translation product of 991 amino acid residues (Mr 110791) and stops 319 nucleotide residues from the 3' end of RNA 1. In addition to the conserved 3' region present in all CMV RNAs (307 residues in RNA 1), RNAs 1 and 2 have highly homologous 5' leader sequences, a 12-nucleotide segment of which is also conserved in the corresponding RNAs of brome mosaic virus (BMV). CMV satellite RNA can form stable base pairs with a region of CMV RNAs 1 and 2 including this 12-nucleotide sequence, implying a regulatory function. This conserved sequence is part of a hairpin structure in RNAs 1 and 2 of CMV and BMV and in CMV satellite RNA. The entire translation products of RNA 1 of CMV and BMV could be aligned with significant homology. Less prominent homologies were found with alfalfa mosaic virus RNA 1 translation product and with tobacco mosaic virus Mr-126000 protein.

Cucumber mosaic virus RNA 5 Partial characterization and evidence for no large sequence homologies with genomic RNAs

Cucumber mosaic virus (CMV) contains four major RNA species (RNAs 1,2,3 and 4) the largest three of which are necessary for infectivity [ 1,2] . RNA 4 serves as a messenger for synthesis of coat protein in a wheat embryo cell-free system [3] . The nucleotide sequence of RNA 4 must also be present in RNA 3 since it is known that RNA 3 carries the information for coat protein synthesis in vivo [4,5] . In addition to the genome RNAs and RNA 4, a fifth RNA component (RNA 5) of low molecular weight (100 000) has been detected in CMV preparations [ 1,6] . Interest in RNA 5 was enhanced by the finding that, even though the small RNA is apparently dependent upon the CMV genome RNAs for replication [7] , the relative proportions of RNA 5 and RNA l-4 is tied to the nature of the plant host of viral propagation and, furthermore, the presence of RNA 5 can dramatically alter the response of certain hosts to CMV infection [8,9]. The above findings make it unlikely that RNA 5 is a host RNA normally present in the plant which is entrapped during virus assembly, but they do not in themselves permit us to distinguish between two further possibilities: (1) RNA 5 originates from the CMV RNA but is nonessential for infectivity, i.e., a deletion fragment or partial transcription product of one of the genome RNAs, or (2) RNA 5 is a ‘satellite’ RNA, that is, a parasite RNA with little or no sequence homology with the helper genome. As a prelude to a more detailed sequence analysis, we have determined some of the properties of RNA 5. In particular, the 5’ and 3’ extremities of the RNA

Translation of the four major RNA species of cucumber mosaic virus in plant and animal cell-free systems and in toad oocytes

Cucumber mosaic virus (CMV) contains four major RNA species, designated RNAs 14 in order of decreasing molecular weight. As a preliminary means of analysing gene content and distribution between these RNA species, each of the RNAs was purified and used to direct protein synthesis in cell-free systems from wheat embryos, wheat germ, and rabbit reticulocytes, and in intact oocytes of the toad, Bufo marinus. In vitro translation products of the CMV RNAs (Q strain) were analysed by electrophoresis on discontinuous slab polyacrylamide gels in the presence of sodium dodecyl sulphate. RNA 1 and RNA 2 proved untranslatable in oocytes but both directed synthesis of a large number of products in the wheat and rabbit reticulocyte cell-free systems. The highest molecular weight products obtained, about 105,000 for RNA 1 and 120,000 for RNA 2, represent most of the coding potential of these RNAs. Thus, RNA 1 and RNA 2 may be monocistronic messengers for the 105,000 and 120,000 MW products, respectively. Translation of RNA 3 in the wheat cell-free systems yielded three major products of molecular weights 34,000–39,000. In contrast, RNA 3 directed synthesis of essentially one product of molecular weight about 34,000 in both the rabbit reticulocyte system and oocytes. RNA 3 did not direct synthesis of coat protein in any of the systems, contrary to our earlier results and the known presence of the coat protein gene on RNA 3. RNA 4 directed synthesis of coat protein and higher- and lower molecular weight minor products in the cell-free systems and in oocytes. A product corresponding to the entire coding potential of RNA 4 was observed in the rabbit reticulocyte system, but not in the wheat systems or toad oocytes. The conflicting results obtained for translation of RNA 3 and RNA 4 in the different systems illustrate the danger in analysing genetic material by in vitro translation. The discussion considers which of the in vitro products are most likely to be synthesized in vivo and a tentative model of the CMV genome is proposed.