A Structural Perspective of Reps from CRESS-DNA Viruses and Their Bacterial Plasmid Homologues.

Rolling circle replication (RCR) is ubiquitously used by cellular and viral systems for genome and plasmid replication. While the molecular mechanism of RCR has been described, the structural mechanism is desperately lacking. Circular-rep encoded single stranded DNA (CRESS-DNA) viruses employ a viral encoded replicase (Rep) to initiate RCR. The recently identified prokaryotic homologues of Reps may also be responsible for initiating RCR. Reps are composed of an endonuclease, oligomerization, and ATPase domain. Recent structural studies have provided structures for all these domains such that an overall mechanism of RCR initiation can begin to be synthesized. However, structures of Rep in complex with its various DNA substrates and/or ligands are lacking. Here we provide a 3D bioinformatic review of the current structural information available for Reps. We combine an excess of 1590 sequences with experimental and predicted structural data from 22 CRESS-DNA groups to identify similarities and differences between Reps that lead to potentially important functional sites. Experimental studies of these sites may shed light on how Reps execute their functions. Furthermore, we identify Rep-substrate or Rep-ligand structures that are urgently needed to better understand the structural mechanism of RCR.

Molecular characterization and In Vitro synthesis of infectious RNA of a Turnip vein-clearing virus isolated from Alliaria petiolata in Hungary.

A tobamovirus was isolated from leaves of Alliaria petiolata plants, showing vein-clearing, interveinal chlorosis, and moderate deformation. Host range experiments revealed a high similarity of isolate ApH both to ribgrass mosaic viruses and turnip vein-clearing viruses. The complete nucleotide sequence of the viral genome was determined. The genomic RNA is composed of 6312 nucleotides and contains four open reading frames (ORF). ORF1 is 3324 nt-long and encodes a polypeptide of about 125.3 kDa. The ORF1 encoded putative replication protein contains an Alphavirus-like methyltransferase domain. ORF2 is 4806 nt-long and encodes a polypeptide of about 182 kDa. The ORF2 encoded putative replication protein contains an RNA-dependent RNA polymerase, catalytic domain. ORF3 encodes the putative cell-to-cell movement protein with a molecular weight of 30.1 kDa. ORF4 overlaps with ORF3 and encodes the coat protein with a size of 17.5 kDa. Sequence comparisons revealed that the ApH isolate has the highest similarity to turnip vein-clearing viruses and should be considered an isolate of Turnip vein-clearing virus (TVCV). This is the first report on the occurrence of TVCV in Hungary. In vitro transcripts prepared from the full-length cDNA clone of TVCV-ApH were highly infectious and induced typical symptoms characteristic to the original isolate of the virus. Since infectious clones of TVCV-ApH and crTMV (another isolate of TVCV) markedly differed in respect to recovery phenotype in Arabidopsis thaliana, it is feasible to carry out gene exchange or mutational studies to determine viral factors responsible for the symptom recovery phenotype.

Viruses infecting cool season crops in the northern Turkey.

Virus diseases of cool season vegetable crops (mainly cabbage, white and red head cabbage, broccoli, kale, radish, rocket salad, garden cress, and turnip) were surveyed in Bafra Plain, Turkey during winter 2017, and 2018. Leaf samples were collected from different species of the Brassicaceae family showing mosaic, mottling, necrotic spots, malformation, and chlorosis symptoms. These samples were tested for the presence of Cauliflower mosaic virus (CaMV), Cucumber mosaic virus (CMV), Beet western yellows virus (BWYV), Radish mosaic virus (RaMV), Turnip mosaic virus (TuMV), Turnip yellow mosaic virus (TYMV), and Turnip yellows virus (TuYV) by biological and serological methods. A total of 455 samples were collected from cole crop fields and tested for the seven viruses by double-antibody sandwich ELISA using specific polyclonal antibodies. According to the results, out of these, 7 % of the samples were infected by at least one of these viruses. TuMV was the most prevalent virus detected in cole crops. TuMV, CaMV, and CMV were detected in 3 %, 2 %, and 2 % of infected samples, respectively, and the infection rate of these three viruses changed significantly among Brassica species.

A Survey on Plant Viruses in Natural Brassicaceae Communities Using RNA-Seq.

Studies on plant viruses are biased towards crop diseases and little is known about viruses in natural vegetation. We conducted extensive surveys of plant viruses in wild Brassicaceae plants occurring in three local plant communities in central Japan. We applied RNA-Seq with selective depletion of rRNA, which allowed us to detect infections of all genome-reported viruses simultaneously. Infections of Turnip mosaic virus (TuMV), Cucumber mosaic virus (CMV), Brassica yellows virus, Pelargonium zonate spot virus, and Arabidopsis halleri partitivirus 1 were detected from the two perennial species, Arabidopsis halleri subsp. gemmifera and Rorippa indica. De novo assembly further detected partial sequences of a putative novel virus in Arabis fragellosa. Virus species composition and infection rate differed depending on site and plant species. Viruses were most frequently detected from the perennial clonal plant, A. halleri, in which a high clonal transmission rate of viruses across multiple years was confirmed. Phylogenetic analysis of TuMV and CMV showed that virus strains from wild Brassicaceae were included as a major clade of these viruses with other reported strains from crop plants, suggesting that viruses were shared among wild plants and crops. Our studies indicated that distribution of viruses in natural plant populations are determined by the combinations of life histories of viruses and hosts. Revealing viral distribution in the natural plant communities improves our knowledge on the ecology of plant viruses.

Migration of plant viruses: Time correlations with the agriculture history and human immigration.

In this review, I made the phylodynamic comparisons of three plant viruses, Turnip mosaic virus (TuMV), Cauliflower mosaic virus (CaMV) and Cucumber mosaic virus (CMV), using the genomic sequences of a large numbers of isolates collected worldwide. We analyzed these genomic nucleotide sequences, in combination with published sequences, to estimate the timescale and rate of evolution of the individual genes of TuMV, CaMV and CMV. The main hosts of the viruses are Brassicaceae crops. We also compared these estimates from complete sequences with those from which non-synonymous and invariate codons had been removed. Our analyses provided a preliminary definition of the present geographical structure of three plant virus populations in the world, and showed that the time of migration of three plant viruses correlate well with agriculture history and human immigration.

Phylodynamic evidence of the migration of turnip mosaic potyvirus from Europe to Australia and New Zealand.

Turnip mosaic virus (TuMV) is a potyvirus that is transmitted by aphids and infects a wide range of plant species. We investigated the evolution of this pathogen by collecting 32 isolates of TuMV, mostly from Brassicaceae plants, in Australia and New Zealand. We performed a variety of sequence-based phylogenetic and population genetic analyses of the complete genomic sequences and of three non-recombinogenic regions of those sequences. The substitution rates, divergence times and phylogeographical patterns of the virus populations were estimated. Six inter- and seven intralineage recombination-type patterns were found in the genomes of the Australian and New Zealand isolates, and all were novel. Only one recombination-type pattern has been found in both countries. The Australian and New Zealand populations were genetically different, and were different from the European and Asian populations. Our Bayesian coalescent analyses, based on a combination of novel and published sequence data from three non-recombinogenic protein-encoding regions, showed that TuMV probably started to migrate from Europe to Australia and New Zealand more than 80 years ago, and that distinct populations arose as a result of evolutionary drivers such as recombination. The basal-B2 subpopulation in Australia and New Zealand seems to be older than those of the world-B2 and -B3 populations. To our knowledge, our study presents the first population genetic analysis of TuMV in Australia and New Zealand. We have shown that the time of migration of TuMV correlates well with the establishment of agriculture and migration of Europeans to these countries.

Two novel mastreviruses from chickpea (Cicer arietinum) in Australia.

Two novel mastreviruses (genus Mastrevirus; family Geminiviridae), with proposed names chickpea chlorosis virus (CpCV) and chickpea redleaf virus, are described from chickpea (Cicer arietinum) from eastern Australia. The viruses have genomes of 2,582 and 2,605 nucleotides, respectively, and share similar features and organisation with typical dicot-infecting mastreviruses. Two distinct strains of CpCV were suggested by phylogenetic analysis. Additionally, a partial mastrevirus Rep sequence from turnip weed (Rapistrum rugosum) indicated the presence of a distinct strain of Tobacco yellow dwarf virus (TYDV). In phylogenetic analyses, isolates of Bean yellow dwarf virus, Chickpea chlorotic dwarf Pakistan virus and Chickpea chlorotic dwarf Sudan virus from southern and northern Africa and south-central and western Asia clustered separately from these three viruses from Australia. An Australian, eastern Asian, or south-eastern Asian origin for the novel mastreviruses and TYDV is discussed.

Identification of plant viruses using one-dimensional gel electrophoresis and peptide mass fingerprints.

A generic assay to detect and partially characterize unknown viruses from plants was developed. Proteins extracted from virus-infected and uninfected plants were separated in one dimension by SDS polyacrylamide gel electrophoresis. Differentially expressed protein bands were eluted after trypsin digestion and resulting peptide fragments separated according to their mass by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Resulting peptide mass fingerprints (PMF) were compared with those in protein databases. The assay was used to identify three known viruses: the potyviruses Zucchini yellow mosaic virus and Turnip mosaic virus, and an alfamovirus Alfalfa mosaic virus. It was also used to identify a virus that manifested symptoms in wild Cakile maritima plants, tentatively identified as Pelargonium zonate spot virus (PZSV) (genus Anulavirus) by its PMF, and then confirmed by nucleotide sequencing. The detection of PZSV constitutes a first record of this virus in Australia and in this host. It is proposed that this rapid and simple assay is a useful approach for analysis of plant samples known to harbor viruses that could not be identified using antisera or nucleic acid-based assays.

Diverse poly(A) binding proteins mediate internal translational initiation by a plant viral IRES.

During 5'-cap-dependent translation, methylated 5'-cap and 3'-poly(A) tail work synergistically in a poly(A) binding protein (PABP)-dependent manner to facilitate translation via promoting the formation of a closed mRNA loop. On the other hand, during internal translation initiation, the requirement for and the roles of 3'-poly(A) tail and PABP vary depending on specific characteristics of each internal ribosomal entry site (IRES). In this study, we analyzed the effect of 3'-poly(A) tail and phylogenetically divergent PABPs on a polypurine tract-containing IRES element derived from the coat protein gene of crucifer-infecting tobamovirus (CrTMV IRES(CP)). We find that mutations in the internal polypurine tract decrease IRES activity in a heterologous (mammalian) system in vivo. Moreover, these mutations decrease the high-affinity binding of all phylogenetically divergent PABPs derived from Arabidopsis and yeast in electro mobility gel shift assays in vitro. Partial PABP depletion and reconstitution assays using Arabidopsis-derived PABP2, 3, 5, 8 and yeast Pab1p provide further evidence that CrTMV IRES(CP) requires PABP for maximal activity. Furthermore, stronger enhancement in the presence of 3'-poly(A) and the absence of 5'-methylated cap suggests a potential joint interaction between PABP, the CrTMV IRES(CP) and the 3'-poly(A).

Identification of new isolates of Turnip mosaic virus that cluster with less common viral strains.

Turnip mosaic virus (TuMV) was found infecting cultivated brassicas and wild and cultivated ornamental Brassicaceae plants in different regions of Spain. Five new TuMV isolates, originating from different host plant species (Brassica cretica, Brassica juncea, Brassica napus, Eruca vesicaria subsp. sativa and Sisymbrium orientale), have been identified. The nucleotide sequences of the coat protein (CP) genes of the five isolates were determined. Phylogenetic analysis of the CP sequences showed that the five isolates grouped into two different clusters. The three isolates from the central region of Spain clustered with a previously reported Pisum sativum isolate from southeastern Spain, whereas the other two isolates from the eastern region clustered with two Italian and two Greek isolates. Both clusters were genetically distinct and belonged to the multi-lineage group OBR. The OBR group contains mainly TuMV isolates from hosts other than Brassica spp. and Raphanus sativus and mostly originating from Mediterranean countries. These new sequences provide further phylogenetic resolution of the OBR group. Although new TuMV isolates have been found in Spain, they were not associated with any serious disease outbreaks.

An internal ribosome entry site located upstream of the crucifer-infecting tobamovirus coat protein (CP) gene can be used for CP synthesis in vivo.

It was previously shown that, unlike the type member of the genus Tobamovirus (TMV U1), a crucifer-infecting tobamovirus (crTMV) contains a 148 nt internal ribosome entry site (IRES)(CP,148)(CR) upstream of the coat protein (CP) gene. Here, viral vectors with substitutions in the stem-loop (SL) region of CP subgenomic promoters (TMV U1-CP-GFP/SL-mut and crTMV-CP-GFP/SL-mut) were constructed and the levels of CP synthesis in agroinoculation experiments were compared. No CP-GFP (green fluorescent protein) synthesis was detected in Nicotiana benthamiana leaves inoculated with TMV U1-CP-GFP/SL-mut, whereas a small amount of CP-GFP synthesis was obtained in crTMV-CP-GFP/SL-mut-injected leaves. Northern blots proved that both promoters were inactive. It could be hypothesized that IRES-mediated early production of the CP by crTMV is needed for realization of its crucifer-infecting capacity.

Aphid transmission of a potyvirus depends on suitability of the helper component and the N terminus of the coat protein.

The present study investigates the specificity of potyviruses for aphid species. Two potyviruses differing in their host range were used: Zucchini yellow mosaic virus (ZYMV) mainly infecting cucurbits and Turnip mosaic virus (TuMV) mainly infecting crucifers. Two sets of aphids species were used as vectors, one polyphagous (Myzus persicae and Aphis gossypii) and the other from crucifers (Brevicoryne brassicae and Lipaphis erysimi). Evidence is provided that the specificity between a vector and a potyvirus depends either on the affinity between the aphid species and the helper component (HC) protein used or on the affinity between the HC and the virions. The difference between the two potyviruses cannot be attributed to the DAG domain which is unaltered in both N termini of the CP. Therefore, a ZYMV full length clone served to exchange a fragment encoding for the N terminus of the ZYMV CP by that of TuMV. This partial exchange in the ZYMV CP, allowed the TuMV HC to transmit the chimeric virus but not the wild type ZYMV. The significance of the N terminus context of the CP in the specificity for the HC is discussed.

Molecular evolution of Turnip mosaic virus: evidence of host adaptation, genetic recombination and geographical spread.

Turnip mosaic virus (TuMV), a species of the genus Potyvirus, occurs worldwide. Seventy-six isolates of TuMV were collected from around the world, mostly from Brassica and Raphanus crops, but also from several non-brassica species. Host tests grouped the isolates into one or other of two pathotypes; Brassica (B) and Brassica-Raphanus (BR). The nucleotide sequences of the first protein (P1) and coat protein (CP) genes of the isolates were determined. One-tenth of the isolates were found to have anomalous and variable phylogenetic relationships as a result of recombination. The 5'-terminal 300 nt of the P1 gene of many isolates was also variable and phylogenetically anomalous, whereas the 380 nt 3' terminus of the CP gene was mostly conserved. Trees calculated from the remaining informative parts of the two genes of the non-recombinant sequences by neighbour-joining, maximum-likelihood and maximum-parsimony methods were closely similar, and so these parts of the sequences were concatenated and trees calculated from the resulting 1150 nt. The isolates fell into four consistent groups; only the relationships of these groups with one another and with the outgroup differed. The "basal-B" cluster of eight B-pathotype isolates was most variable, was not monophyletic, and came from both brassicas and non-brassicas from southwest and central Eurasia. Closest to it, and forming a monophyletic subgroup of it in most trees, and similarly variable, was the "basal-BR" group of eight BR pathotype Eurasian isolates. The third and least variable group, the "Asian-BR" group, was of 22 BR-pathotype isolates, all from brassicas, mostly Raphanus, and all from east Asia mostly Japan. The fourth group of 36 isolates, the "world-B" group, was from all continents, most were isolated from brassicas and most were of the B-pathotype. The simplest of several possible interpretations of the trees is that TuMV originated, like its brassica hosts, in Europe and spread to the other parts of the world, and that the BR pathotype has recently evolved in east Asia.

Comparative analysis of TMV-Cg and TMV-U1 detection methods in infected Arabidopsis thaliana.

The common strain of the tobacco mosaic virus (TMV-U1), and the crucifer-infecting tobacco mosaic virus (TMV-Cg), both members of Tobamovirus genus, infect efficiently the solanaceous plants such as tomato and tobacco. The crucifer-infecting tobacco mosaic virus (TMV-Cg) also infects Arabidopsis thaliana plant, spreading systemically without causing severe symptoms. In contrast, Arabidopsis is a poor host for TMV-U1 infection. Within the past 10 years, Arabidopsis has developed into a powerful model system for studying plant-pathogen interaction. However, a detailed analysis comparing the accuracy of various viral detection methods has not been reported previously. Four detection methods were evaluated in A. thaliana (ecotype Po-1), infected with TMV-U1 or TMV-Cg. Western blots, enzyme-linked immunosorbent assay (ELISA), reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ RNA hybridization methods were used to determine viral spread at various days post inoculation (dpi) in inoculated and apical non-inoculated leaves. The detection of viral spread of TMV-U1 and TMV-Cg in Arabidopsis, using these four detection methods, supports previous studies, which demonstrate that the systemic spreads of these two viruses differ in Arabidopsis. Western blotting and ELISA detected TMV-Cg at 5dpi, and TMV-U1 at 12 dpi in systemic tissues. Viral spread was detected earlier when using RNA detection methods. Reverse transcriptase-polymerase chain reaction (RT-PCR) was very sensitive for detecting TMV-Cg in A. thaliana, but less sensitive for TMV-U1 detection. In situ RNA hybridization showed differential distribution of TMV-Cg and TMV-U1 in the inoculated leaf and systemic tissues.

Nucleotide sequence analysis of the 3' terminal region of a wasabi strain of crucifer tobamovirus genomic RNA: subgrouping of crucifer tobamoviruses.

The 3' terminal 2378 nucleotides of a wasabi strain of crucifer tobamovirus (CTMV-W) infectious to crucifer plants was determined. This includes the 3' non-coding region of 235 nucleotides, coat protein (CP) gene (468 nucleotides), movement protein (MP) gene (798 nucleotides) and C-terminal partial readthrough portion of 180 K protein gene (940 nucleotides). Comparison of the sequence with homologous regions of thirteen other tobamovirus genomes showed that it had much higher identity to those of four other crucifer tobamoviruses, 85.2% to cr-TMV and turnip vein-clearing virus (TVCV), 87.4% to oilseed rape mosaic virus (ORMV) and 87.1% to TMV-Cg, than to those of other tobamoviruses. Thus CTMV-W was most similar to ORMV and TMV-Cg in sequence, but only marginally so, whereas the location and size of its MP gene was the same as cr-TMV amd TVCV. These results, together with other analyses, show that CTMV-W is a new crucifer tobamovirus, that the five crucifer tobamoviruses can be classified into two subgroups based on MP gene organization, and that the rate of sequence change is not the same in all lineages.