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1.  Microscopic Analysis of Severe Structural Rearrangements of the Plant Endoplasmic Reticulum and Golgi Caused by Overexpression of Poa semilatent virus Movement Protein 
The Scientific World Journal  2012;2012:416076.
Cell-to-cell transport of plant viruses is mediated by virus-encoded movement proteins and occurs through plasmodesmata interconnecting neighboring cells in plant tissues. Three movement proteins coded by the “triple gene block” (TGB) and named TGBp1, TGBp2 and TGBp3 have distinct functions in viral transport. TGBp1 binds viral genomic RNAs to form ribonucleoprotein complexes representing the transport form of viral genome, while TGBp2 and TGBp3 are necessary for intracellular delivery of such complexes to plasmodesmata. Recently, it was revealed that overexpression of Potato virus X TGBp3 triggers the unfolded protein response mitigating the endoplasmic reticulum (ER) stress leading to cell death if this protein reaches high levels in the ER. Here we report microscopic studies of the influence of the Poa semilatent hordeivirus TGBp3 overexpressed in Nicotiana benthamiana epidermal cells by particle bombardment on cell endomembranes and demonstrate that the protein C-terminal transmembrane segment contains a determinant responsible for vesiculation and coalescence of the endoplasmic reticulum and Golgi presumably accompanying the ER stress that can be induced upon high-level TGBp3 expression.
PMCID: PMC3259505  PMID: 22272174
2.  Phylogenetic relationship of some “accessory” helicases of plant positive-stranded RNA viruses: toward understanding the evolution of triple gene block 
Recently, we hypothesized that silencing suppression activity gained by a viral replicative helicase led to the emergence of the second helicase possessing activity of the viral silencing suppressor and/or movement protein (MP). Our hypothesis accounted for the evolutionary origin of the specialized ‘triple gene block’ (TGB) in plant virus genomes encoding the MPs TGB1, TGB2, and TGB3 required for viral cell-to-cell transport through plasmodesmata. Here, we used public transcriptome databases to identify previously unrecognized viruses. The analysis of novel viral genomes further supported the previously proposed scenario of TGB origin and evolution, which included the following steps. First, the accessory helicase gene could have been acquired by horizontal gene transfer (HGT) presumably occured independently in different virus groups. Second, the TGB2 gene evolved by HGT or autonomization of the C-terminal transmembrane domain found in at least one TGB1 helicase. Third, the TGB3 gene has most likely emerged in the genomic block consisting of the TGB1 and TGB2 genes.
PMCID: PMC4436898  PMID: 26042118
plant virus; virus genome; RNA helicase; evolution; movement protein; triple gene block
3.  Making sense of nuclear localization: A zinc-finger protein encoded by a cytoplasmically replicating plant RNA virus acts a transcription factor 
Plant Signaling & Behavior  2013;8(8):e25263.
Recent studies have uncovered numerous nucleus-localized proteins encoded by plant RNA viruses. Whereas for some of these viruses nuclear (or, more specifically, nucleolar) passage of the proteins is needed for the virus movement within the plant or suppression of host defense, the nuclear function of these proteins remains largely unknown. Recently, the situation has been clarified for one group of plant RNA viruses, the Carlaviruses. Being positive-stranded RNA viruses, carlaviruses multiply exclusively in the cytoplasm. Chrysanthemum virus B (CVB, a carlavirus) encodes a zinc-finger protein p12 targeted to the nucleus in a nuclear localization signal-dependent manner. In a recent work, we demonstrated that p12 directly interacts with chromatin and plant promoters, thus, acts as a eukaryotic transcription factor (TF) and activates expression of a host TF involved in regulation of cell size and proliferation to favor virus infection. Therefore our studies identified a novel nuclear stage of in CVB infection involving modulation of host gene expression and plant development. Whereas it is well established that any RNA virus actively replicating in the cell causes changes in the transcriptome, our study expanded this view by showing that some positive-stranded RNA viruses can directly manipulate host transcription by encoding eukaryotic TFs.
PMCID: PMC3999073  PMID: 23759549
Carlavirus; CVB; transcription factor; cell proliferation; zinc-finger protein
4.  Possible role of the Nt-4/1 protein in macromolecular transport in vascular tissue 
Plant Signaling & Behavior  2013;8(10):e25784.
The Arabidopsis thaliana 4/1 (At-4/1) protein has a highly α-helical structure with potential to interact both with itself and other protein ligands, including the movement proteins of some plant viruses; the Nicotiana tabacum ortholog (Nt-4/1) has similar structure. Here we describe localization of GUS expression in transgenic N. tabacum seedlings under control of the Nt-4/1 promoter, which indicates that transcription is associated with the veins at certain developmental stages, and especially in the hypocotyl. Viroid accumulation and movement was altered in plants in which 4/1 expression was reduced by virus-induced gene silencing. These localization studies support a role of 4/1 in signaling in the vasculature, including mobility of pathogen-related and cellular RNAs.
PMCID: PMC4091084  PMID: 23887490
5.  Plant 4/1 protein: potential player in intracellular, cell-to-cell and long-distance signaling 
Originally isolated as a result of its ability to interact with the movement protein of Tomato spotted wilt virus in a yeast two-hybrid system, the 4/1 protein is proving to be an excellent tool for studying intracellular protein trafficking and intercellular communication. Expression of 4/1 in vivo is tightly regulated, first appearing in the veins of the cotyledon and later in the vasculature of the leaf and stem in association with the xylem parenchyma and phloem parenchyma. Structural studies indicate that 4/1 proteins contain as many as five coiled–coil (CC) domains; indeed, the highest level of sequence identity among 4/1 proteins involves their C-terminal CC domains, suggesting that protein–protein interaction is important for biological function. Recent data predict that the tertiary structure of this C-terminal CC domain is strikingly similar to that of yeast protein She2p; furthermore, like She2p, 4/1 protein exhibits RNA-binding activity, and mutational analysis has shown that the C-terminal CC domain is responsible for RNA binding. The 4/1 protein contains a nuclear export signal. Additional microscopy studies involving leptomycin and computer prediction suggest the presence of a nuclear localization signal as well.
PMCID: PMC3933784  PMID: 24611067
intracellular transport; cell-to-cell transport; long-distance signaling; phloem transport; RNA binding protein
6.  Peculiar Evolutionary History of miR390-Guided TAS3-Like Genes in Land Plants 
The Scientific World Journal  2013;2013:924153.
PCR-based approach was used as a phylogenetic profiling tool to probe genomic DNA samples from representatives of evolutionary distant moss taxa, namely, classes Bryopsida, Tetraphidopsida, Polytrichopsida, Andreaeopsida, and Sphagnopsida. We found relatives of all Physcomitrella patens miR390 and TAS3-like loci in these plant taxa excluding Sphagnopsida. Importantly, cloning and sequencing of Marchantia polymorpha genomic DNA showed miR390 and TAS3-like sequences which were also found among genomic reads of M. polymorpha at NCBI database. Our data suggest that the ancient plant miR390-dependent TAS molecular machinery firstly evolved to target AP2-like mRNAs in Marchantiophyta and only then both ARF- and AP2-specific mRNAs in mosses. The presented analysis shows that moss TAS3 families may undergone losses of tasiAP2 sites during evolution toward ferns and seed plants. These data confirm that miR390-guided genes coding for ARF- and AP2-specific ta-siRNAs have been gradually changed during land plant evolution.
PMCID: PMC3835848  PMID: 24302881
7.  Structural Lability of Barley Stripe Mosaic Virus Virions 
PLoS ONE  2013;8(4):e60942.
Virions of Barley stripe mosaic virus (BSMV) were neglected for more than thirty years after their basic properties were determined. In this paper, the physicochemical characteristics of BSMV virions and virion-derived viral capsid protein (CP) were analyzed, namely, the absorption and intrinsic fluorescence spectra, circular dichroism spectra, differential scanning calorimetry curves, and size distributions by dynamic laser light scattering. The structural properties of BSMV virions proved to be intermediate between those of Tobacco mosaic virus (TMV), a well-characterized virus with rigid rod-shaped virions, and flexuous filamentous plant viruses. The BSMV virions were found to be considerably more labile than expected from their rod-like morphology and a distant sequence relation of the BSMV and TMV CPs. The circular dichroism spectra of BSMV CP subunits incorporated into the virions, but not subunits of free CP, demonstrated a significant proportion of beta-structure elements, which were proposed to be localized mostly in the protein regions exposed on the virion outer surface. These beta-structure elements likely formed during virion assembly can comprise the N- and C-terminal protein regions unstructured in the non-virion CP and can mediate inter-subunit interactions. Based on computer-assisted structure modeling, a model for BSMV CP subunit structural fold compliant with the available experimental data was proposed.
PMCID: PMC3629216  PMID: 23613760
8.  Beet yellows virus replicase and replicative compartments: parallels with other RNA viruses 
In eukaryotic virus systems, infection leads to induction of membranous compartments in which replication occurs. Virus-encoded subunits of the replication complex mediate its interaction with membranes. As replication platforms, RNA viruses use the cytoplasmic surfaces of different membrane compartments, e.g., endoplasmic reticulum (ER), Golgi, endo/lysosomes, mitochondria, chloroplasts, and peroxisomes. Closterovirus infections are accompanied by formation of multivesicular complexes from cell membranes of ER or mitochondrial origin. So far the mechanisms for vesicles formation have been obscure. In the replication-associated 1a polyprotein of Beet yellows virus (BYV) and other closteroviruses, the region between the methyltransferase and helicase domains (1a central region (CR), 1a CR) is marginally conserved. Computer-assisted analysis predicts several putative membrane-binding domains in the BYV 1a CR. Transient expression of a hydrophobic segment (referred to here as CR-2) of the BYV 1a in Nicotiana benthamiana led to reorganization of the ER and formation of ~1-μm mobile globules. We propose that the CR-2 may be involved in the formation of multivesicular complexes in BYV-infected cells. This provides analogy with membrane-associated proteins mediating the build-up of “virus factories” in cells infected with diverse positive-strand RNA viruses (alpha-like viruses, picorna-like viruses, flaviviruses, and nidoviruses) and negative-strand RNA viruses (bunyaviruses).
PMCID: PMC3589766  PMID: 23508802
RNA virus replication; membrane vesicles; virus replication factory; endoplasmic reticulum modification; intracellular traffic
10.  Recent Advances in Research of Plant Virus Movement Mediated by Triple Gene Block 
The aim of this short review was to summarize recent advances in the field of viral cell-to-cell movement mediated by the triple gene block (TGB). The growing body of new research has uncovered links between virus cell-to-cell trafficking and replication, silencing suppression, virus spread over the plant, as well as suggested the roles of nucleus/nucleolus in plant virus transport and revealed protein-membrane associations occurring during subcellular targeting and cell-to-cell movement. In this context, our review briefly summarized current views on several potentially important functions of TGB proteins and on the development of new experimental systems that improved understanding of the molecular events during TGB-mediated virus movement.
PMCID: PMC3520053  PMID: 23248633
plant virus; virus movement; movement protein; triple gene block; TGB
11.  The Role of Microtubule Association in Plasmodesmal Targeting of Potato mop-top virus Movement Protein TGBp1 
Cell-to-cell movement of Potato mop-top virus (PMTV) is mediated by three virus-encoded ‘triple gene block’ (TGB) proteins termed TGBp1, TGBp2 and TGBp3. TGBp1 binds virus RNAs to form viral ribonucleoprotein complexes (vRNPs), the transport form of viral genome. TGBp2 and TGBp3 are necessary for intracellular delivery of TGBp1-containing vRNPs to plasmodesmata. To analyze subcellular localization and transport of TGBp1 we used a single binary vector for agrobacterium-mediated co-expression of PMTV TGBp1 fused to green fluorescent protein and TGBp2/TGBp3. At two days post infiltration (dpi) TGBp1 was found in the nucleus and in association with microtubules (MTs). Similar localization pattern was revealed in cells expressing GFP-TGBp1 alone after particle bombardment. At 3 dpi, in addition to the nucleus and MTs, TGBp1 was detected in numerous granular bodies located both along the MTs and at the cell wall. The latter structures co-localized with plasmodesmata-associated callose depositions. At 4 dpi, GFP-TGBp1 was detected in cell wall-associated bodies and also in residual MTs, the nucleoplasm and large perinuclear inclusions resembling aggresomes. Therefore GFP-TGBp1 association with MTs preceded to its localization to plasmodesmata. Disassembly of cell MTs by colchicine prevented GFP-TGBp1 targeting to plasmodesmata and the MT-dependent aggresome formation. Deletion analysis also revealed a correlation between TGBp1 microtubule association and plasmodesmata targeting. We propose that TGBp1 interaction with MTs may be important for the formation of vRNP bodies destined for the transport to plasmodesmata as well as degradation of the excessive TGBp1.
PMCID: PMC3109696  PMID: 21660184
Movement protein; virus movement; plasmodesmata; microtubules; subcellular localization.
12.  Intracellular Targeting of a Hordeiviral Membrane-Spanning Movement Protein: Sequence Requirements and Involvement of an Unconventional Mechanism▿  
Journal of Virology  2007;82(3):1284-1293.
The membrane-spanning protein TGBp3 is one of the three movement proteins (MPs) of Poa semilatent virus. TGBp3 is thought to direct other viral MPs and genomic RNA to peripheral bodies located in close proximity to plasmodesmata. We used the ectopic expression of green fluorescent protein-fused TGBp3 in epidermal cells of Nicotiana benthamiana leaves to study the TGBp3 intracellular trafficking pathway. Treatment with inhibitors was used to reveal that the targeting of TGBp3 to plasmodesmata does not require a functional cytoskeleton or secretory system. In addition, the suppression of endoplasmic reticulum-derived vesicle formation by a dominant negative mutant of small GTPase Sar1 had no detectable effect on TGBp3 trafficking to peripheral bodies. Collectively, these results suggested the involvement of an unconventional pathway in the intracellular transport of TGBp3. The determinants of targeting to plasmodesmata were localized to the C-terminal region of TGBp3, including the conserved hydrophilic and terminal membrane-spanning domains.
PMCID: PMC2224415  PMID: 18032484
13.  Long-Distance Movement, Virulence, and RNA Silencing Suppression Controlled by a Single Protein in Hordei- and Potyviruses: Complementary Functions between Virus Families 
Journal of Virology  2002;76(24):12981-12991.
RNA silencing is a natural defense mechanism against genetic stress factors, including viruses. A mutant hordeivirus (Barley stripe mosaic virus [BSMV]) lacking the γb gene was confined to inoculated leaves in Nicotiana benthamiana, but systemic infection was observed in transgenic N. benthamiana expressing the potyviral silencing suppressor protein HCpro, suggesting that the γb protein may be a long-distance movement factor and have antisilencing activity. This was shown for γb proteins of both BSMV and Poa semilatent virus (PSLV), a related hordeivirus. Besides the functions in RNA silencing suppression, γb and HCpro had analogous effects on symptoms induced by the hordeiviruses. Severe BSMV-induced symptoms were correlated with high HCpro concentrations in the HCpro-transgenic plants, and substitution of the γb cistron of BSMV with that of PSLV led to greatly increased symptom severity and an altered pattern of viral gene expression. The efficient systemic infection with the chimera was followed by the development of dark green islands (localized recovery from infection) in leaves and exemption of new developing leaves from infection. Recovery and the accumulation of short RNAs diagnostic of RNA silencing in the recovered tissues in wild-type N. benthamiana were suppressed in HCpro-transgenic plants. These results provide evidence that potyviral HCpro and hordeivirus γb proteins contribute to systemic viral infection, symptom severity, and RNA silencing suppression. HCpro's ability to suppress the recovery of plants from viral infection emphasizes recovery as a manifestation of RNA silencing.
PMCID: PMC136670  PMID: 12438624

Results 1-13 (13)