Two mutually exclusive evolutionary scenarios for allexiviruses that overcome host RNA silencing and autophagy by regulating viral CRP expression.

The genus Allexivirus currently includes eight virus species that infect allium plants. Previously, we showed that there are two distinct groups of allexiviruses (deletion [D]-type and insertion [I]-type) based on the presence or absence of a 10- to 20-base insert (IS) between the coat protein (CP) and cysteine rich protein (CRP) genes. In the present study of CRPs to analyze their functions, we postulated that evolution of allexiviruses may have been largely directed by CRPs and thus proposed two evolutionary scenarios for allexiviruses based mainly on the presence or absence of IS and determined by how the allexiviruses challenge host resistance mechanisms (RNA silencing and autophagy). We found that both CP and CRP are RNA silencing suppressors (RSS), that they can inhibit each other's RSS activity in the cytoplasm, and that CRP becomes a target of host autophagy in the cytoplasm but not CP. To mitigate CRP interference with CP, and to increase the CP's RSS activity, allexiviruses developed two strategies: confinement of D-type CRP in the nucleus and degradation of I-type CRP by autophagy in the cytoplasm. Here, we demonstrate that viruses of the same genus achieve two completely different evolutionary scenarios by controlling expression and subcellular localization of CRP.

Transmission of Grapevine leafroll-associated virus-1 (Ampelovirus) and Grapevine virus A (Vitivirus) by the Cottony Grape Scale, Pulvinaria vitis (Hemiptera: Coccidae).

The cottony grape scale Pulvinaria vitis is a scale insect colonizing grapevine; however, its capacity as a vector of grapevine viruses is poorly known in comparison to other scale species that are vectors of viral species in the genera Ampelovirus and Vitivirus. The ability of P. vitis to transmit the ampeloviruses Grapevine leafroll-associated viruses [GLRaV]-1, -3, and -4, and the vitivirus Grapevine virus A (GVA), to healthy vine cuttings was assessed. The scale insects used originated from commercial vine plots located in Alsace, Eastern France. When nymphs sampled from leafroll-infected vineyard plants were transferred onto healthy cuttings, only one event of transmission was obtained. However, when laboratory-reared, non-viruliferous nymphs were allowed to acquire viruses under controlled conditions, both first and second instar nymphs derived from two vineyards were able to transmit GLRaV-1 and GVA. This is the first report of GLRaV-1 and GVA transmission from grapevine to grapevine by this species.

Impact of Grapevine Red Blotch Disease on Cabernet Sauvignon and Merlot Wine Composition and Sensory Attributes.

Grapevine red blotch disease (GRBD) is a recently identified viral disease that affects grapevines. GRBD has been shown to impact grapevine physiology and grape composition by altering specific ripening events. However, no studies have been reported on the impact of GRBD on wine composition and its sensory attributes. This study evaluated the impact of GRBD on wine primary and secondary metabolites, in addition to its sensory properties, when making wines from Cabernet Sauvignon and Merlot grapes during two seasons. Wines made with GRBD-impacted fruit were lower in ethanol content when compared to wines made with grapes from healthy grapevines. This was attributed to the lower total soluble sugar (TSS) levels of diseased grapes due to delayed ripening at harvest. GRBD impacted wine phenolic composition by decreasing anthocyanin concentrations and increasing flavonol concentrations in some instances. Additionally, proanthocyanidin concentrations were also consistently higher in GRBD wines compared to wines made from healthy fruit. Descriptive analysis demonstrated that GRBD can impact wine style by altering aroma, flavor, and mouthfeel attributes. However, the extent of GRBD impact on wine composition and sensory properties were site and season dependent.

Revisiting the cysteine-rich proteins encoded in the 3'-proximal open reading frame of the positive-sense single-stranded RNA of some monopartite filamentous plant viruses: functional dissection of p15 from grapevine virus B.

A reexamination of proteins with conserved cysteines and basic amino acids encoded by the 3'-proximal gene of the positive-sense single-stranded RNA of some monopartite filamentous plant viruses has been carried out. The cysteines are involved in a putative Zn-finger domain, which, together with the basic amino acids, form part of the nuclear or nucleolar localization signals. An in-depth study of one of these proteins, p15 from grapevine B virus (GVB), has shown: (i) a three-dimensional structure with four α-helices predicted by two independent in silico approaches, (ii) the nucleolus as the main accumulation site by applying confocal laser microscopy to a fusion between p15 and the green fluorescent protein, (iii) the involvement of the basic amino acids and the putative Zn-finger domain, mapping at the N-terminal region of p15, in the nucleolar localization signal, as revealed by the effect of six alanine substitution mutations, (iv) the p15 suppressor function of sense-mediated RNA silencing as revealed by agroinfiltration in a transgenic line of Nicotiana benthamiana, and (v) the enhancer activity of p15 on viral pathogenicity in N. benthamiana when expressed from a potato virus X vector. In addition, we elaborate on an evolutionary scenario for these filamentous viruses, invoking takeover by a common ancestor(s) of viral or host genes coding for those cysteine-rich proteins, followed by divergence, which would also explain why they are encoded in the 3'-proximal gene of the genomic single-stranded viral RNA.

Functional analysis of apple stem pitting virus coat protein variants.

BACKGROUND: Although the canonical function of viral coat protein (CP) is to encapsidate the viral genome, they have come to be recognized as multifunctional proteins, involved in almost every stage of the viral infection cycle. However, CP functions of Apple stem pitting virus (ASPV) has not been comprehensively documented. This study aimed to characterize the functions of ASPV CP and any functional diversification caused by sequence diversity of six ASPV CP variants and studied their biological, serological, pathogenic and viral suppressor of RNA silencing (VSR) functions. METHODS: Six ASPV CP variants that have previously been shown to belong to different subgroups were selected here to study their diversity functions. Agrobacterium mediated infiltration (Agroinfiltration) was used to express YFP-ASPV-CPs in Nicotiana. benthamiana and infect Nicotiana. occidental with PVX-ASPV-CPs in. Confocal microscopy was used to detect YFP-ASPV-CPs florescence. CPs expressed in Escherichia coli BL21 (DE3) were induced by IPTG. RESULTS: In this study, we showed that recombinant CPs expressed in Escherichia coli BL21 (DE3) had different levels of serological reactivity to three anti-ASPV antibodies used to detect ASPV. Furthermore, fusion CPs with YFP (YFP-CPs) expressed in N. benthamiana cells differed in their ability to form aggregates. We also showed that ASPV isolates that harbour these CPs induced different biological symptoms on its herbaceous host N. occidentalis. At the same time, we found that all six CPs when expressed in PVX vector showed similar VSR activity and produced similar symptoms in N. occidentalis, despite their differences in amino acids. CONCLUSIONS: Different ASPV isolates induced different symptoms in N. occidentalis, however, ASPV CP variants expressed in PVX vector showed the same symptoms in N. occidentalis plants. Also, we showed that ASPV CP variants has the same level of VSR activity, but they have different abilities to aggregate in N. benthamiana.

Screening of Potential Inhibitor against Coat Protein of Apple Chlorotic Leaf Spot Virus.

In this study, we analyzed Coat protein (CP) of Apple chlorotic leaf spot virus (ACLSV), an important latent virus on Apple. Incidence of the virus is upto 60% in various apple cultivars, affecting yield losses of the order of 10-40% (depending upon the cultivar). CP plays an important role as the sole building block of the viral capsid. Homology approach was used to model 193 amino acid sequence of the coat protein. We used various servers such as ConSurf, TargetS, OSML, COACH, COFACTOR for the prediction of active site residues in coat protein. Virtual screening strategy was employed to search potential inhibitors for CP. Top twenty screened molecules considered for drugability, and toxicity analysis and one potential molecule was further analyzed by docking analysis. Here, we reported a potent molecule which could inhibit the formation of viron assembly by targeting the CP protein of virus.

Yeast two-hybrid and pull-down assays propose an interaction between P50 of apple chlorotic leaf spot virus and PR-10 of Malus sylvestris cv. R12740-7A.

Apple chlorotic leaf spot virus (ACLSV) movement protein (P50) is involved in cell-to-cell transport and influences the long-distance spread of silencing activity. Previously, we obtained 69 P50-interacting proteins from Malus sylvestris cv. R12740-7A and using bioinformatics analyzed their biological functions. In this study, we used the GAL4-based two-hybrid yeast system and His pull-down assays to confirm an interaction between PR-10 of M. sylvestris cv. R12740-7A and ACLSV P50. Our results provide a theoretical basis for further research on the biological function of PR-10 in ACLSV infection and the interacting mechanism between host and virus.

Movement protein of Apple chlorotic leaf spot virus is genetically unstable and negatively regulated by Ribonuclease E in E. coli.

Movement protein (MP) of Apple chlorotic leaf spot virus (ACLSV) belongs to "30 K" superfamily of proteins and members of this family are known to show a wide array of functions. In the present study this gene was found to be genetically unstable in E. coli when transformed DH5α cells were grown at 28 °C and 37 °C. However, genetic instability was not encountered at 20 °C. Heterologous over expression failed despite the use of different transcriptional promoters and translational fusion constructs. Total cell lysate when subjected to western blotting using anti-ACLSV MP antibodies, showed degradation/cleavage of the expressed full-length protein. This degradation pointed at severe proteolysis or instability of the corresponding mRNA. Predicted secondary structure analysis of the transcript revealed a potential cleavage site for an endoribonuclease (RNase E) of E. coli. The negating effect of RNase E on transcript stability and expression was confirmed by northern blotting and quantitative RT-PCR of the RNA extracted from RNase E temperature sensitive mutant (strain N3431). The five fold accumulation of transcripts at non-permissive temperature (43 °C) suggests the direct role of RNase E in regulating the expression of ACLSV MP in E. coli.

[Identification of Host Factors Interacting with the Movement Protein of Apple Chlorotic Leaf Spot Virus by Yeast Two-Hybrid System].

In order to identify host factors which interact with the movement protein (MP) of Apple chlorotic leaf spot virus (ACLSV), ACLSV MP was cloned into the bait vector pGBKT7 and used to screen a cDNA library of Malus sylvestris cv. R12740-7A, which had previously been constructed by yeast two-hybrid sequencing transformation. The protein functions of the identified host factors were determined according to their gene annotations in GenBank. The result showed that the bait plasmid pGBKT7-MP showed no virulence or self-activating effect on yeast strain Y2H Gold. Sixty-nine interactor proteins were identified, which were divided into the following 10 classes according to their described functions: hydrolases; pathogenesis-related proteins; DNA binding proteins; phosphatases; ligases; proteins with catalytic activity; phenylalanine ammonialyases; peroxidases; NAD binding proteins; and proteins of unknown function. Bioinformatic analysis of gene homology suggested that phosphatases, pathogenesis-related proteins and glyceraldehyde-3-phosphate dehydrogenase A may play an important role in the interaction between virus and host. This study may provide a theoretical basis for the further study of viral pathogenesis and virus-host interaction mechanisms.

Subcellular localization and membrane association of the replicase protein of grapevine rupestris stem pitting-associated virus, family Betaflexiviridae.

As a member of the newly established Betaflexiviridae family, grapevine rupestris stem pitting-associated virus (GRSPaV) has an RNA genome containing five ORFs. ORF1 encodes a putative replicase polyprotein typical of the alphavirus superfamily of positive-strand ssRNA viruses. Several viruses of this superfamily have been demonstrated to replicate in structures designated viral replication complexes associated with intracellular membranes. However, structure and cellular localization of the replicase complex have not been studied for members of Betaflexiviridae, a family of mostly woody plant viruses. As a first step towards the elucidation of the replication complex of GRSPaV, we investigated the subcellular localization of full-length and truncated versions of its replicase polyprotein via fluorescent tagging, followed by fluorescence microscopy. We found that the replicase polyprotein formed distinctive punctate bodies in both Nicotiana benthamiana leaf cells and tobacco protoplasts. We further mapped a region of 76 amino acids in the methyl-transferase domain responsible for the formation of these punctate structures. The punctate structures are distributed in close proximity to the endoplasmic reticulum network. Membrane flotation and biochemical analyses demonstrate that the N-terminal region responsible for punctate structure formation associated with cellular membrane is likely through an amphipathic α helix serving as an in-plane anchor. The identity of this membrane is yet to be determined. This is, to our knowledge, the first report on the localization and membrane association of the replicase proteins of a member of the family Betaflexiviridae.

The triple gene block movement proteins of a grape virus in the genus Foveavirus confer limited cell-to-cell spread of a mutant Potato virus X.

Grapevine rupestris stem pitting-associated virus (GRSPaV) is a member of the genus Foveavirus in the family Betaflexiviridae. The genome of GRSPaV encodes five proteins, among which are three movement proteins designated the triple gene block (TGB) proteins. The TGB proteins of GRSPaV are highly similar to their counterparts in Potato virus X (PVX), as reflected in size, modular structure, conservation of critical amino acid sequence motifs, as well as similar cellular localization. Based on these similarities, we predicted that the TGB proteins of these two viruses would be interchangeable. To test this hypothesis, we replaced the entire or partial sequence of PVX TGB with the corresponding regions from GRSPaV, creating chimeric viruses that contain the PVX backbone and different sequences from GRSPaV TGB. These chimeric constructs were delivered into plants of Nicotiana benthamiana through agro-infiltration to test whether they were capable of cell-to-cell and systemic movement. To our surprise, viruses derived from pPVX.GFP(CH3) bearing GRSPaV TGB in place of PVX TGB lost the ability to move either cell-to-cell or systemically. Interestingly, another chimeric virus resulting from pPVX.GFP(HY2) containing four TGB genes (TGB1 from PVX and TGB1-3 from GRSPaV), exhibited limited cell-to-cell, but not systemic, movement. Our data question the notion that analogous movement proteins encoded by even distantly related viruses are functionally interchangeable and can be replaced by each other. These data suggest that other factors, besides the TGB proteins, may be required for successful intercellular and/or systemic movement of progeny viruses. This is the first experimental demonstration that the GRSPaV TGB function as movement proteins in the context of a chimeric virus and that four TGB genes were required to support the intercellular movement of the chimeric virus.

Characterization of plant virus-encoded gene silencing suppressors.

Agroinfiltration assay using green fluorescent protein (GFP)-expressing Nicotiana benthamiana line 16c is a powerful method for screening of putative plant virus-encoded gene silencing suppressors. This method allows the investigator to know whether the putative viral suppressor inhibits silencing in a cell (local silencing) and/or spreading of silencing throughout a plant (systemic silencing). Additionally, grafting experiments using transgenic plants expressing the suppressor and the GFP will indicate whether the suppressor blocks systemic silencing steps, which include the production of a silencing signal in a silenced cell, and the cell-to-cell and long-distance movement of a silencing signal throughout a plant. Here, we describe methods and techniques of an agroinfiltration assay and grafting experiments, which were used for the characterization of Apple chlorotic leaf spot virus 50 kDa movement protein as a gene silencing suppressor. This protocol should allow the investigator to characterize putative plant virus-encoded gene silencing suppressors.

Subcellular localization of the triple gene block proteins encoded by a Foveavirus infecting grapevines.

Grapevine rupestris stem pitting-associated virus (GRSPaV; Foveavirus; Flexiviridae) contains a positive-sense, ssRNA genome. GRSPaV occurs worldwide in grapes and is involved in the Rugose Wood disease complex. The GRSPaV genome contains the triple gene block (TGB), a genetic module present in several genera of plant RNA viruses. TGB encodes three proteins (TGBp1, TGBp2 and TGBp3) that are believed to work together to achieve intra- and inter-cellular transport of virions in infected plants. To reveal the subcellular localization of each TGB protein and to examine the impact that different fusion positions may have on the behavior of the native protein, we made a series of expression constructs and expressed the corresponding protein fusions in Nicotiana tabacum BY-2 cells and protoplasts. We demonstrated that TGBp1 had both a cytosolic and nuclear distribution. Two TGBp1 fusions (GFP fused at the N- or C-terminus) differ in subcellular distribution. Through the use of truncation mutants, we mapped TGBp1 regions responsible for the formation of two distinct types of aggregates. Sequence analyses predicted two and one transmembrane domains in TGBp2 and TGBp3, respectively. GFP fusions at either terminus of TGBp2 revealed identical localization to the ER network and ER-derived structures. In contrast, the two TGBp3 fusions to mRFP differed in localization. This is the first report on the subcellular localization of the viral proteins of a member of the Foveavirus genus.