Apple latent spherical virus structure with stable capsid frame supports quasi-stable protrusions expediting genome release.

Picorna-like plant viruses are non-enveloped RNA spherical viruses of ~30 nm. Part of the survival of these viruses depends on their capsid being stable enough to harbour the viral genome and yet malleable enough to allow its release. However, molecular mechanisms remain obscure. Here, we report a structure of a picorna-like plant virus, apple latent spherical virus, at 2.87 Å resolution by single-particle cryo-electron microscopy (cryo-EM) with a cold-field emission beam. The cryo-EM map reveals a unique structure composed of three capsid proteins Vp25, Vp20, and Vp24. Strikingly Vp25 has a long N-terminal extension, which substantially stabilises the capsid frame of Vp25 and Vp20 subunits. Cryo-EM images also resolve RNA genome leaking from a pentameric protrusion of Vp24 subunits. The structures and observations suggest that genome release occurs through occasional opening of the Vp24 subunits, possibly suppressed to a low frequency by the rigid frame of the other subunits.

A new virus, classifiable in the family Tombusviridae, found infecting Solanum tuberosum in the UK.

A novel virus was discovered in a freeze-dried collection held at SASA, UK, originating from potato (Solanum tuberosum) cv. Nadine. The complete sequence of the viral RNA was determined to be 3674 nucleotides in length encoding five predicted proteins. Based on the deduced genome organization and phylogenetic analysis, this virus represents a putative new member of the genus Alphanecrovirus, family Tombusviridae, most closely related to isolates of Olive mild mosaic virus. The virus was easily transmitted to indicator plants with symptoms that were slower to develop and less severe than those of related viruses. To distinguish this virus, the clearest symptom differences occurred with Nicotiana debneyi, Chenopodium amaranticolor and Ch. quinoa. The virus was detected with antisera to the related viruses tobacco necrosis virus A and tobacco necrosis virus D. The close association to the tobacco necrosis viruses would suggest this virus is not a new introduction to potato but in the past has been misidentified as one of these viruses. The virus isolate has been named potato necrosis virus.

Production of fluorescent antibody-labeling proteins in plants using a viral vector and the application in the detection of Acidovorax citrulli and Bamboo mosaic virus.

Serological methods are relatively convenient and simple for the detection of pathogens for front-line workers. On-site visualization of the test results plays a pivotal role in the process. However, an efficient, universal labeling agent for antibodies is needed for the development of efficient serological detection tools. In this study, a Bamboo mosaic virus (BaMV)-based viral vector was employed to express recombinant proteins, collectively designated GfED, consisting of Staphylococcus aureus Protein A domain ED (SpaED) fused to either the N- or C-terminal of an improved green florescent protein (GFP) with or without the coat protein (CP) of BaMV, efficiently in Chenopodium quinoa. The GfED in crude leaf extracts could specifically attach to IgG molecules of rabbits and mice, effectively labeling IgG with GFP, emitting green light at 506 nm when excited at 450 nm using simple, handheld equipment. To demonstrate the applicability of GfED in serological assays, we have developed a fluorescent dot blot assay for the rapid detection of Acidovorax citrulli (Ac), a bacterial pathogen of cucurbits, and BaMV, a viral pathogen of bamboos. By using the crude extracts of inoculated C. quinoa leaves expressing GfED as an IgG-labeling agent, the pathogens were easily and quickly detected through uncomplicated operations using simple equipment, with results observable by the naked eye. Examination using fluorescent microscopy and transmission electron microscopy revealed that the GfED subunits may assemble into virus-like particles, which were further involved in the formation of aggregates of GfED-antibody-antigen complexes with the potential for fluorescence signal enhancement. The results suggested that plant-expressed GfED may serve as a promising alternative of IgG-labeling agent for current serological assays.

Characterization of the genome of a phylogenetically distinct tospovirus and its interactions with the local lesion-induced host Chenopodium quinoa by whole-transcriptome analyses.

Chenopodium quinoa is a natural local lesion host of numerous plant viruses, including tospoviruses (family Bunyaviridae). Groundnut chlorotic fan-spot tospovirus (GCFSV) has been shown to consistently induce local lesions on the leaves of C. quinoa 4 days post-inoculation (dpi). To reveal the whole genome of GCFSV and its interactions with C. quinoa, RNA-seq was performed to determine the transcriptome profiles of C. quinoa leaves. The high-throughput reads from infected C. quinoa leaves were used to identify the whole genome sequence of GCFSV and its single nucleotide polymorphisms. Our results indicated that GCFSV is a phylogenetically distinct tospovirus. Moreover, 27,170 coding and 29,563 non-coding sequences of C. quinoa were identified through de novo assembly, mixing reads from mock and infected samples. Several key genes involved in the modulation of hypersensitive response (HR) were identified. The expression levels of 4,893 deduced complete genes annotated using the Arabidopsis genome indicated that several HR-related orthologues of pathogenesis-related proteins, transcription factors, mitogen-activated protein kinases, and defense proteins were significantly expressed in leaves that formed local lesions. Here, we also provide new insights into the replication progression of a tospovirus and the molecular regulation of the C. quinoa response to virus infection.

Differentially expressed genes of Chenopodium amaranticolor in response to cymbidium mosaic virus infection.

Cymbidium mosaic virus (CymMV)-induced expressed sequence tag (EST) clones from Chenopodium amaranticolor were identified. CymMV was mechanically inoculated onto C. amaranticolor, and local lesion symptoms were observed. Inoculated leaves were collected on serial days post inoculation (dpi) to identify activated or suppressed genes. mRNA isolation and suppression subtractive hybridization (SSH) were then performed to identify differentially expressed genes related to the local lesion response. Fifty-three ESTs, including genes related to defense and stress responses (e.g., lipoxygenase, jasmonate-induced protein, and heat shock protein), were generated. In addition, a large proportion of the ESTs were found to be involved in photosynthesis, as determined by their functional categories. Expression levels of several EST genes were observed using quantitative real-time reverse transcription-polymerase chain reaction, and the evaluated genes showed varying levels of expression during the experimental period. In this study, differentially expressed sequences via SSH were identified from CymMV-infected C. amaranticolor, and profiling and annotation were carried out to determine the expression pattern of CymMV and its interaction with C. amaranticolor.

Molecular identification and biological characterization of a new potyvirus in lettuce.

A potyvirus causing necrosis and leaf distortion on lettuce was found in the Lazio region of Italy. Host range analysis showed its ability to infect only Chenopodium quinoa and C. amaranticolor in addition to some lettuce cultivars. The virus could be transmitted by aphids of the species Myzus persicae. The complete 9829-nt genome was characterized. BLAST analysis of sequence of the complete encoded polyprotein showed that the most closely related virus is asparagus virus 1, with 52 % amino acid sequence identity. These results suggest that this virus should be considered a member of a new species in the genus Potyvirus.

Resistance to Plum pox virus strain C in Arabidopsis thaliana and Chenopodium foetidum involves genome-linked viral protein and other viral determinants and might depend on compatibility with host translation initiation factors.

Research performed on model herbaceous hosts has been useful to unravel the molecular mechanisms that control viral infections. The most common Plum pox virus (PPV) strains are able to infect Nicotiana species as well as Chenopodium and Arabidopsis species. However, isolates belonging to strain C (PPV-C) that have been adapted to Nicotiana spp. are not infectious either in Chenopodium foetidum or in Arabidopsis thaliana. In order to determine the mechanism underlying this interesting host-specific behavior, we have constructed chimerical clones derived from Nicotiana-adapted PPV isolates from the D and C strains, which differ in their capacity to infect A. thaliana and C. foetidum. With this approach, we have identified the nuclear inclusion a protein (VPg+Pro) as the major pathogenicity determinant that conditions resistance in the presence of additional secondary determinants, different for each host. Genome-linked viral protein (VPg) mutations similar to those involved in the breakdown of eIF4E-mediated resistance to other potyviruses allow some PPV chimeras to infect A. thaliana. These results point to defective interactions between a translation initiation factor and the viral VPg as the most probable cause of host-specific incompatibility, in which other viral factors also participate, and suggest that complex interactions between multiple viral proteins and translation initiation factors not only define resistance to potyviruses in particular varieties of susceptible hosts but also contribute to establish nonhost resistance.

Presence of poly(A) and poly(A)-rich tails in a positive-strand RNA virus known to lack 3׳ poly(A) tails.

Here we show that Tobacco mosaic virus (TMV), a positive-strand RNA virus known to end with 3׳ tRNA-like structures, does possess a small fraction of gRNA bearing polyadenylate tails. Particularly, many tails are at sites corresponding to the 3׳ end of near full length gRNA, and are composed of poly(A)-rich sequences containing the other nucleotides in addition to adenosine, resembling the degradation-stimulating poly(A) tails observed in all biological kingdoms. Further investigations demonstrate that these polyadenylated RNA species are not enriched in chloroplasts. Silencing of cpPNPase, a chloroplast-localized polynucleotide polymerase known to not only polymerize the poly(A)-rich tails but act as a 3׳ to 5׳ exoribonuclease, does not change the profile of polyadenylate tails associated with TMV RNA. Nevertheless, because similar tails were also detected in other phylogenetically distinct positive-strand RNA viruses lacking poly(A) tails, such kind of polyadenylation may reflect a common but as-yet-unknown interface between hosts and viruses.

De novo foliar transcriptome of Chenopodium amaranticolor and analysis of its gene expression during virus-induced hypersensitive response.

BACKGROUND: The hypersensitive response (HR) system of Chenopodium spp. confers broad-spectrum virus resistance. However, little knowledge exists at the genomic level for Chenopodium, thus impeding the advanced molecular research of this attractive feature. Hence, we took advantage of RNA-seq to survey the foliar transcriptome of C. amaranticolor, a Chenopodium species widely used as laboratory indicator for pathogenic viruses, in order to facilitate the characterization of the HR-type of virus resistance. METHODOLOGY AND PRINCIPAL FINDINGS: Using Illumina HiSeq™ 2000 platform, we obtained 39,868,984 reads with 3,588,208,560 bp, which were assembled into 112,452 unigenes (3,847 clusters and 108,605 singletons). BlastX search against the NCBI NR database identified 61,698 sequences with a cut-off E-value above 10(-5). Assembled sequences were annotated with gene descriptions, GO, COG and KEGG terms, respectively. A total number of 738 resistance gene analogs (RGAs) and homology sequences of 6 key signaling proteins within the R proteins-directed signaling pathway were identified. Based on this transcriptome data, we investigated the gene expression profiles over the stage of HR induced by Tobacco mosaic virus and Cucumber mosaic virus by using digital gene expression analysis. Numerous candidate genes specifically or commonly regulated by these two distinct viruses at early and late stages of the HR were identified, and the dynamic changes of the differently expressed genes enriched in the pathway of plant-pathogen interaction were particularly emphasized. CONCLUSIONS: To our knowledge, this study is the first description of the genetic makeup of C. amaranticolor, providing deep insight into the comprehensive gene expression information at transcriptional level in this species. The 738 RGAs as well as the differentially regulated genes, particularly the common genes regulated by both TMV and CMV, are suitable candidates which merit further functional characterization to dissect the molecular mechanisms and regulatory pathways of the HR-type of virus resistance in Chenopodium.

Two distinct sites are essential for virulent infection and support of variant satellite RNA replication in spontaneous beet black scorch virus variants.

Spontaneous point mutations of virus genomes are important in RNA virus evolution and often result in modifications of their biological properties. Spontaneous variants of beet black scorch virus (BBSV) and its satellite (sat) RNA were generated from cDNA clones by serial propagation in Chenopodium amaranticolor and Nicotiana benthamiana. Inoculation with recombinant RNAs synthesized in vitro revealed BBSV variants with divergent infectious phenotypes that affected either symptom expression or replication of satRNA variants. Sequence alignments showed a correlation between the phenotypes and distinct BBSV genomic loci in the 3'UTR or in the domain encoding the viral replicase. Comparative analysis between a virulent variant, BBSV-m294, and the wild-type (wt) BBSV by site-directed mutagenesis indicated that a single-nucleotide substitution of a uridine to a guanine at nt 3477 in the 3'UTR was responsible for significant increases in viral pathogenicity. Gain-of-function analyses demonstrated that the ability of the BBSV variants to support replication of variant satRNAs was mainly determined by aa 516 in the P82 replicase. In this case, an arginine substitution for a glutamine residue was essential for high levels of replication, and alterations of other residues surrounding position 516 in the wtBBSV isolate led to only minor phenotypic effects. These results provide evidence that divergence of virus functions affecting pathogenicity and supporting parasitic replication can be determined by a single genetic site, either a nucleotide or an amino acid. The results suggest that complex interactions occur between virus and associated satRNAs during virus evolution.

The R-rich motif of Beet black scorch virus P7a movement protein is important for the nuclear localization, nucleolar targeting and viral infectivity.

Beet black scorch virus (BBSV) encodes three movement proteins (P7a, P7b and P5') that facilitate its cell-to-cell movement. An arginine-rich motif of P7a N-terminus was found to determine nuclear and nucleolar localization. Amino acids substitution or deletion of the R-rich motif interfered with P7a nuclear and nucleolar localization. Bimolecular fluorescence complementation (BiFC) assays revealed that P7a protein interacted with Nicotiana benthamiana nuclear import factor importin α, suggesting that P7a is translocated into the nucleus by the classical importin α/ß-dependent pathway. Moreover, P7a also interacted with the nucleolar protein fibrillarin. Mutations in the R-rich motif of P7a diminished P7a interactions with importin α and fibrillarin, influenced viral replication in Nicotiana benthamiana protoplasts and altered the symptom phenotype and viral RNA accumulation in Chenopodium amaranticolor plants. These results demonstrate that the R-rich motif of P7a is correlated with nuclear and nucleolar localization, viral replication and virus infection.

Antiphytoviral activity of essential oil from endemic species Teucrium arduini.

The essential oil of Teucrium arduini L. was characterized by a high concentration of sesquiterpene hydrocarbons (43.8%) of which beta-caryophyllene (19.9%) being the major compound, followed by oxygenated sesquiterpenes (19.6%) of which caryophyllene-oxide (14.6%) was dominant. When applied to plants of Chenopodium amaranticolor and Ch. quinoa for two successive days prior inoculation, the oil was effective in reducing lesion numbers on plants infected with Tobacco mosaic virus (25.7%) and Cucumber mosaic virus (21.9%). The main components of oil, beta-caryophyllene and caryophyllene oxide showed potent antiviral activity against CMV, but weak activity against TMV infection.

Genetic diversity of the ordinary strain of Potato virus Y (PVY) and origin of recombinant PVY strains.

The ordinary strain of Potato virus Y (PVY), PVY(O), causes mild mosaic in tobacco and induces necrosis and severe stunting in potato cultivars carrying the Ny gene. A novel substrain of PVY(O) was recently reported, PVY(O)-O5, which is spreading in the United States and is distinguished from other PVY(O) isolates serologically (i.e., reacting to the otherwise PVY(N)-specific monoclonal antibody 1F5). To characterize this new PVY(O)-O5 subgroup and address possible reasons for its continued spread, we conducted a molecular study of PVY(O) and PVY(O)-O5 isolates from a North American collection of PVY through whole-genome sequencing and phylogenetic analysis. In all, 44 PVY(O) isolates were sequenced, including 31 from the previously defined PVY(O)-O5 group, and subjected to whole-genome analysis. PVY(O)-O5 isolates formed a separate lineage within the PVY(O) genome cluster in the whole-genome phylogenetic tree and represented a novel evolutionary lineage of PVY from potato. On the other hand, the PVY(O) sequences separated into at least two distinct lineages on the whole-genome phylogenetic tree. To shed light on the origin of the three most common PVY recombinants, a more detailed phylogenetic analysis of a sequence fragment, nucleotides 2,406 to 5,821, that is present in all recombinant and nonrecombinant PVY(O) genomes was conducted. The analysis revealed that PVY(N:O) and PVY(N-Wi) recombinants acquired their PVY(O) segments from two separate PVY(O) lineages, whereas the PVY(NTN) recombinant acquired its PVY(O) segment from the same lineage as PVY(N:O). These data suggest that PVY(N:O) and PVY(N-Wi) recombinants originated from two separate recombination events involving two different PVY(O) parental genomes, whereas the PVY(NTN) recombinants likely originated from the PVY(N:O) genome via additional recombination events.

Genetic variability in the coat protein gene of Potato virus S isolates and distinguishing its biologically distinct strains.

The complete coat protein (CP) nucleotide sequences of 13 Potato virus S (PVS) isolates from Australia and three from Europe were compared to those of 37 others. On phylogenetic analysis, the Australian sequences were in PVS(O) sub-clades III and IV, and the European isolates were in sub-clades I and VII. The European isolates invaded Chenopodium spp. systemically, but eight Australian isolates did not. Amino acid sequence differences at the N-terminal ends of the CPs were unrelated to the ability to invade Chenopodium spp. systemically. The acronym PVS(O-CS) is suggested for isolates that invade Chenopodium spp. systemically but are not within clade PVS(A).

Development of Tobacco necrosis virus A as a vector for efficient and stable expression of FMDV VP1 peptides.

Plant virus-based expression systems provide attractive alternatives for production of animal virus-originated antigenic peptides. In the present study, an infectious cDNA clone of Tobacco necrosis virus A Chinese isolate (TNV-A(C)) was used for expression of different peptides derived from Foot and mouth disease virus (FMDV) serotype O VP1 fused downstream of the coat protein (CP) open reading frame (ORF). Chenopodium amaranticolor inoculated with in vitro transcripts of the chimaeras developed symptoms similar to those caused by wild-type TNV-A(C). Western blot and RT-PCR detection of the infected leaves demonstrated that the chimaeras were infective, and a large number of self-assembled virions could be purified and observed under electron microscopy. Immunogold labelling revealed that highly expressed FMDV VP1 peptides could be displayed on the surfaces of virus particles. Additional immunoblotting and DNA sequence analyses showed that most of the chimaeras contained unmodified foreign peptides even after six successive passages in C. amaranticolor and three passages in Nicotiana benthamiana. Our results also suggest that the amino acid sequence and peptide length have a substantial influence on viral morphogenesis and systemic infections. Finally, animal experiments showed that purified chimaeric virus particles (CVPs) could induce a strong immune response against FMDV structural protein VP1 via an intramuscular route. And when inoculated nasally, CVPs could induce systemic and mucosal immune responses in mice.

Analysis of cell-to-cell and long-distance movement of apple latent spherical virus in infected plants using green, cyan, and yellow fluorescent proteins.

Apple latent spherical virus (ALSV) expressing green, cyan, and yellow fluorescent proteins (GFP, CFP, and YFP) was constructed and used to analyze the local and systemic movement of the virus in infected plants. In Chenopodium quinoa plants inoculated with GFP-ALSV, the infection foci first appeared as small fluorescent spots 2-3 days post inoculation (dpi). The GFP spots expanded as rings from 5 dpi, then fused to each other, and most fluorescence faded out at 10-12 dpi. In upper uninoculated leaves, GFP fluorescence was first observed 6-7 dpi on the basal area of mature leaves and on the entire area of young developing leaves. The appearance of fluorescent flecks on young leaves was first found on and near the class III and IV veins. ALSV labeled with two different fluorescent proteins (CFP-ALSV and YFP-ALSV) were used to investigate the distribution of identical, but differently labeled viruses in mixed infection. Fluorescence from CFP and YFP was in each case observed in separate areas in both inoculated and upper uninoculated leaves, indicating that populations of identical, but differently labeled viruses were replicated and distributed in discrete areas of infected leaves.

Electrophoretic mobility of the capsid protein of the Plum pox virus strain PPV-Rec indicates its partial phosphorylation.

A double-band SDS-PAGE profile was found reproducible for capsid protein (CP) of Plum pox virus (PPV) isolates belonging to the strain PPV-Rec. The double-band was also present in the virus population multiplied in various plants. A single-lesion passage in a hypersensitive host Chenopodium foetidum showed that its presence was not a result of a mixed infection. We found that the two electrophoretic forms of CP shared identical N-terminus. Therefore, they did not originate from an alternative proteolytic processing, but were different in their posttranslational modification. The slower band of CP could be converted to the faster one by the phosphatase treatment. We assumed that CP protein was present in both phosphorylated and dephosphorylated forms in the infected plants.

Analysis of the subgenomic RNAs and the small open reading frames of Beet black scorch virus.

A full-length cDNA of the genome of Beet black scorch virus (BBSV), isolate Ningxia, was constructed and modified by site-directed mutagenesis to permit in vitro transcription of mutant viral RNAs. Two subgenomic (sg) RNAs (sgRNA1 and sgRNA2) appeared during BBSV replication. Mutagenesis revealed that sgRNA1 transcription was initiated at G2209 within the P82 polymerase subunit open reading frame (ORF) and that transcription of sgRNA2 began at G2526 within the nested p7b/p5' ORF. Initiation-codon shifting or premature termination of translation of the three ORFs (P7a, P7b and P5') encoded by sgRNA1 indicated that each of the genes was required for localized movement, accumulation of viral RNAs and formation of local lesions on the leaves of Chenopodium amaranticolor. Microscopic observations of the distribution of green fluorescent protein fused to the N-terminal portion of the capsid protein provided additional evidence that the P7a, P7b and P5' proteins are each required for cell-to-cell movement. In contrast, elimination of sgRNA2 showed that the BBSV coat protein was not required for viral RNA accumulation or the appearance of local lesions on C. amaranticolor. In addition, disruption of the small P5 ORF previously predicted by computer analysis to originate at the C terminus of the P82 ORF had no effect on disease phenotype, suggesting that this ORF may represent a cryptic, non-essential gene. These results show that BBSV has a novel cell-to-cell movement protein organization that differs in size and sequence from that of other viruses.

[Translational regulation of potato virus X RNA-coat protein complexes: the key role of a coat protein N-terminal peptide].

The efficiency of in vitro translation of potato virus X (PVX) RNA within vRNP complexes assembled from genomic RNA and viral CP was examined. The vRNP particles contain the 5'-proximal RNA segments encapsidated by helically arranged CP head-like portions heterogeneous in length and the CP-free RNA tail. Translation of RNA is completely repressed upon incubation with PVX CP and is accompanied by vRNP particles production. By contrast, translation is activated in vRNPs in vitro assembled using two CP forms, differing in the principals of their N-terminal peptides modification. The N-terminal peptide of PVX CP represents the major phosphorylation site(s) for Thr/Ser-specific protein kinases. It was shown that: (i) CP phosphorylation results in a translational activation of vRNP; (ii) removal of N-terminal peptide from CP abolished activation and CP retains the translation repressing ability. It was suggested that substitution of Ser/Thr residues by non-phosphorylated Ala/Gly in N-terminal peptide of the mutant CP will led to a complete inhibition of vRNP translation. However, opposite results were obtained in our experiments: (i) RNA of such mutant virus (PVX-ST) was efficiently translated within the virions; (ii) RNA of a wild-type (wt) PVX also efficiently translated in mixedly assembled vRNP "wt PVX RNA + PVX-ST CP"; (iii) opposite result (repression of translation) was obtained with "mixed" vRNP (PVX-ST RNA + wtPVX CP). Therefore, the N-terminal peptide located at the surface of the particle and of the particles plays a key role in translation activation of the RNA encapsidated in vRNP and native virions.

Sensitivity of brome mosaic virus RNA1 replication to mutations in the 3' tRNA-like structure implies a requirement for sustained synthesis of replicase protein 1a.

The replication competence of a series of brome mosaic virus (BMV) RNA1 variants with defined mutations in the 3' tRNA-like structure, previously characterized in vitro to be defective in minus-strand synthesis and several tRNA-associated functions, was analyzed in barley protoplasts. Inocula containing wild type RNAs2 and 3 and RNA1 bearing either Deltaknob or 5'Psk mutation failed to replicate. Two additional RNA1 variants, each bearing either M4 or 5'AGA mutation, resulted in detectable accumulation of progeny but are inhibitory to overall viral replication when supplied in high concentrations. Another aminoacylation-defective mutation Delta5' supported viral replication but did not interfere with viral replication even at higher concentrations. Coinoculation of replication-incompetent variants of RNAl with wt RNAs2 and 3 to Chenopodium hybridum plants resulted in the delayed development of local necrotic lesions characteristic of a wt infection. Sequence analysis of progeny RNA recovered from these lesions indicated that, in each case, a functional 3' noncoding sequence was restored due to homologous recombination with a corresponding sequence from wt RNA3. Taken together the results suggest that, unlike protein 2a which is required in catalytic amounts, the intrinsic involvement of protein 1a at various stages of virus infection cycle demands its sustained synthesis.