Reticulon-like properties of a plant virus-encoded movement protein.

Plant viruses encode movement proteins (MPs) that ensure the transport of viral genomes through plasmodesmata (PD) and use cell endomembranes, mostly the endoplasmic reticulum (ER), for delivery of viral genomes to PD and formation of PD-anchored virus replication compartments. Here, we demonstrate that the Hibiscus green spot virus BMB2 MP, an integral ER protein, induces constrictions of ER tubules, decreases the mobility of ER luminal content, and exhibits an affinity to highly curved membranes. These properties are similar to those described for reticulons, cellular proteins that induce membrane curvature to shape the ER tubules. Similar to reticulons, BMB2 adopts a W-like topology within the ER membrane. BMB2 targets PD and increases their size exclusion limit, and these BMB2 activities correlate with the ability to induce constrictions of ER tubules. We propose that the induction of ER constrictions contributes to the BMB2-dependent increase in PD permeability and formation of the PD-associated replication compartments, therefore facilitating the virus intercellular spread. Furthermore, we show that the ER tubule constrictions also occur in cells expressing TGB2, one of the three MPs of Potato virus X (PVX), and in PVX-infected cells, suggesting that reticulon-like MPs are employed by diverse RNA viruses.

Similarities in intracellular transport of plant viral movement proteins BMB2 and TGB3.

The cell-to-cell transport of many plant viruses through plasmodesmata requires viral movement proteins (MPs) encoded by a 'triple gene block' (TGB) and termed TGB1, TGB2 and TGB3. TGB3 is a small integral membrane protein that contains subcellular targeting signals and directs both TGB2 and the helicase domain-containing TGB1 protein to plasmodesmata-associated structures. Recently, we described a 'binary movement block' (BMB) coding for two MPs, BMB1 and BMB2. The BMB2 protein associates with endoplasmic reticulum (ER) membranes, accumulates at plasmodesmata-associated membrane bodies and directs the BMB1 helicase to these structures. TGB3 transport to cell peripheral bodies was previously shown to bypass the secretory pathway and involve a non-conventional mechanism. Here, we provide evidence that the intracellular transport of both poa semilatent virus TGB3 and hibiscus green spot virus BMB2 to plasmodesmata-associated sites can occur via lateral translocation along the ER membranes. Agrobacterium-mediated transient co-expression in Nicotiana benthamiana leaves revealed that green fluorescent protein (GFP)-fused actin-binding domains of Arabidopsis fimbrin (ABD2-GFP) and mouse talin (TAL-GFP) inhibited the subcellular targeting of TGB3 and BMB2 to plasmodesmata-associated bodies, which resulted in TGB3 and BMB2 accumulation in the cytoplasm in association with aberrant ER structures. Inhibition of COPII budding complex formation by the expression of a dominant-negative mutant of the small GTPase Sar1 had no detectable effect on BMB2 subcellular targeting, which therefore could occur without exit from the ER in COPII transport vesicles. Collectively, the presented data support the current view that plant viral MPs exploit the ER:actin network for their intracellular transport.

Plant-specific 4/1 polypeptide interacts with an endoplasmic reticulum protein related to human BAP31.

MAIN CONCLUSION: The plant-specific 4/1 protein interacts, both in yeast two-hybrid system and in vitro, and co-localizes in plant cells with plant BAP-like protein, the orthologue of human protein BAP31. In yeast two-hybrid system, we identified a number of Nicotiana benthamiana protein interactors of Nt-4/1, the protein known to affect systemic transport of potato spindle tuber viroid. For one of these interactors, an orthologue of human B-cell receptor-associated protein 31 (BAP31) termed plant BAP-like protein (PBL), the ability to interact with Nt-4/1 was studied in greater detail. Analyses of purified proteins expressed in bacterial cells carried out in vitro with the surface plasmon resonance (SPR) spectroscopy revealed that the N. tabacum PBL (NtPBL) was able to interact with Nt-4/1 with high-affinity, and that their complex can form at physiologically relevant concentrations of both proteins. Subcellular localization studies of 4/1-GFP and NtPBL-mRFP transiently co-expressed in plant cells revealed the co-localization of the two fusion proteins in endoplasmic reticulum-associated bodies, suggesting their interaction in vivo. The N-terminal region of the Nt-4/1 protein was found to be required for the specific subcellular targeting of the protein, presumably due to a predicted amphipathic helix mediating association of the Nt-4/1 protein with cell membranes. Additionally, this region was found to contain a trans-activator domain responsible for the Nt-4/1 ability to activate transcription of a reporter gene in yeast.

Translation of the shallot virus X TGB3 gene depends on non-AUG initiation and leaky scanning.

Triple gene block (TGB), a conserved gene module found in the genomes of many filamentous and rod-shaped plant viruses, encodes three proteins, TGB1, TGB2 and TGB3, required for viral cell-to-cell movement through plasmodesmata and systemic transport via the phloem. The genome of Shallot virus X, the type species of the genus Allexivirus, includes TGB1 and TGB2 genes, but contains no canonical ORF for TGB3 protein. However, a TGB3-like protein-encoding sequence lacking an AUG initiator codon has been found in the shallot virus X (ShVX) genome in a position typical for TGB3 genes. This putative TGB3 gene is conserved in all allexiviruses. Here, we carried out sequence analysis to predict possible non-AUG initiator codons in the ShVX TGB3-encoding sequence. We further used an agroinfiltration assay in Nicotiana benthamiana to confirm this prediction. Site-directed mutagenesis was used to demonstrate that the ShVX TGB3 could be translated on a bicistronic mRNA template via a leaky scanning mechanism.

Defense Responses Induced by Viral Movement Protein and Its Nuclear Localization Modulate Virus Cell-to-Cell Transport.

Movement proteins (MPs) encoded by plant viruses are essential for cell-to-cell transport of viral genomes through plasmodesmata. The genome of hibiscus green spot virus contains a module of two MP genes termed 'binary movement block' (BMB), encoding the proteins BMB1 and BMB2. Here, BMB1 is shown to induce a defense response in Nicotiana benthamiana plants that inhibits BMB-dependent virus transport. This response is characterized by the accumulation of reactive oxygen species, callose deposition in the cell wall, and upregulation of 9-LOX expression. However, the BMB1-induced response is inhibited by coexpression with BMB2. Furthermore, BMB1 is found to localize to subnuclear structures, in particular to Cajal bodies, in addition to the cytoplasm. As shown in experiments with a BMB1 mutant, the localization of BMB1 to nuclear substructures enhances BMB-dependent virus transport. Thus, the virus transport mediated by BMB proteins is modulated by (i) a BMB1-induced defense response that inhibits transport, (ii) suppression of the BMB1-induced response by BMB2, and (iii) the nuclear localization of BMB1 that promotes virus transport. Collectively, the data presented demonstrate multiple levels of interactions between viral pathogens and their plant hosts during virus cell-to-cell transport.

Virus Genome-Based Reporter for Analyzing Viral Movement Proteins and Plasmodesmata Permeability.

Plant virus movement proteins (MPs) mediate cell-to-cell movement of the virus genome through plasmodesmata (PD). MPs target PD to increase their size exclusion limit (SEL), and this MP function is essential for virus intercellular trafficking. In this chapter, we describe the use of a Potato virus X genome-derived reporter for agroinfiltration-based identification of virus genome-encoded MPs and analysis of the ability of individual viral MPs or plant proteins to increase the PD SEL.

Interaction between Movement Proteins of Hibiscus green spot virus.

Movement proteins (MPs) of plant viruses enable the translocation of viral genomes from infected to healthy cells through plasmodesmata (PD). The MPs functions involve the increase of the PD permeability and routing of viral genome both to the PD entrance and through the modified PD. Hibiscus green spot virus encodes two MPs, termed BMB1 and BMB2, which act in concert to accomplish virus cell-to-cell transport. BMB1, representing an NTPase/helicase domain-containing RNA-binding protein, localizes to the cytoplasm and the nucleoplasm. BMB2 is a small hydrophobic protein that interacts with the endoplasmic reticulum (ER) membranes and induces local constrictions of the ER tubules. In plant cells, BMB2 localizes to PD-associated membrane bodies (PAMBs) consisting of modified ER tubules and directs BMB1 to PAMBs. Here, we demonstrate that BMB1 and BMB2 interact in vitro and in vivo, and that their specific interaction is essential for BMB2-directed targeting of BMB1 to PAMBs. Using mutagenesis, we show that the interaction involves the C-terminal BMB1 region and the N-terminal region of BMB2.

RNA phloem transport mediated by pre-miRNA and viral tRNA-like structures.

Phloem-mobile mRNAs are assumed to contain sequence elements directing RNA to the phloem translocation pathway. One of such elements is represented by tRNA sequences embedded in untranslated regions of many mRNAs, including those proved to be mobile. Genomic RNAs of a number of plant viruses possess a 3'-terminal tRNA-like structures (TLSs) only distantly related to genuine tRNAs, but nevertheless aminoacylated and capable of interaction with some tRNA-binding proteins. Here, we elaborated an experimental system for analysis of RNA phloem transport based on an engineered RNA of Potato virus X capable of replication, but not encapsidation and movement in plants. The TLSs of Brome mosaic virus, Tobacco mosaic virus and Turnip yellow mosaic virus were demonstrated to enable the phloem transport of foreign RNA. A miRNA precursor, pre-miR390b, was also found to render RNA competent for the phloem transport. In line with this, sequences of miRNA precursors were identified in a Cucurbita maxima phloem transcriptome, supporting the hypothesis that, at least in some cases, miRNA phloem signaling can involve miRNA precursors. Collectively, the data presented here suggest that RNA molecules can be directed into the phloem translocation pathway by structured RNA elements such as those of viral TLSs and miRNA precursors.

Mechanical stress-induced subcellular re-localization of N-terminally truncated tobacco Nt-4/1 protein.

The Nicotiana tabacum 4/1 protein (Nt-4/1) of unknown function expressed in plant vasculature has been shown to localize to cytoplasmic bodies associated with endoplasmic reticulum. Here, we analyzed molecular interactions of an Nt-4/1 mutant with a deletion of 90 N-terminal amino acid residues (Nt-4/1d90) having a diffuse GFP-like localization. Upon transient co-expression with VAP27, a membrane protein known to localize to the ER, ER-plasma membrane contact sites and plasmodesmata, Nt-4/1d90 was concentrated around the cortical ER tubules, forming a network matching the shape of the cortical ER. Additionally, in response to mechanical stress, Nt-4/1d90 was re-localized to small spherical bodies, whereas the subcellular localization of VAP27 remained essentially unaffected. The Nt-4/1d90-containing bodies associated with microtubules, which underwent noticeable bundling under the conditions of mechanical stress. The Nt-4/1d90 re-localization to spherical bodies could also be induced by incubation at an elevated temperature, although under heat shock conditions the re-localization was less efficient and incomplete. An Nt-4/1d90 mutant, which had phosphorylation-mimicking mutations in a predicted cluster of four potentially phosphorylated residues, was found to both inefficiently re-localize to spherical bodies and tend to revert back to the initial diffuse localization. The presented data show that Nt-4/1 has a potential for response to stresses that is manifested by its deletion mutant Nt-4/1d90, and this response can be mediated by protein dephosphorylation.

RNA helicase domains of viral origin in proteins of insect retrotransposons: possible source for evolutionary advantages.

Recently, a novel phenomenon of horizontal gene transfer of helicase-encoding sequence from positive-stranded RNA viruses to LINE transposons in insect genomes was described. TRAS family transposons encoding an ORF2 protein, which comprised all typical functional domains and an additional helicase domain, were found to be preserved in many families during the evolution of the order Lepidoptera. In the present paper, in species of orders Hemiptera and Orthoptera, we found helicase domain-encoding sequences integrated into ORF1 of retrotransposons of the Jockey family. RNA helicases encoded by transposons of TRAS and Jockey families represented separate brunches in a phylogenetic tree of helicase domains and thus could be considered as independently originated in the evolution of insect transposons. Transcriptome database analyses revealed that both TRAS and Jockey transposons encoding the helicase domain represented transcribed genome sequences. Moreover, the transposon-encoded helicases were found to contain the full set of conserved motifs essential for their enzymatic activities. Taking into account the previously reported ability of RNA helicase encoded by TRAS ORF2 to suppress post-transcriptional RNA silencing, we propose possible scenarios of evolutionary fixation of actively expressed functional helicases of viral origin in insect retrotransposons as genetic elements advantageous for both transposons and their insect hosts.

A novel block of plant virus movement genes.

Hibiscus green spot virus (HGSV) is a recently discovered and so far poorly characterized bacilliform plant virus with a positive-stranded RNA genome consisting of three RNA species. Here, we demonstrate that the proteins encoded by the ORF2 and ORF3 in HGSV RNA2 are necessary and sufficient to mediate cell-to-cell movement of transport-deficient Potato virus X in Nicotiana benthamiana. These two genes represent a specialized transport module called a 'binary movement block' (BMB), and ORF2 and ORF3 are termed BMB1 and BMB2 genes. In agroinfiltrated epidermal cells of N. benthamiana, green fluorescent protein (GFP)-BMB1 fusion protein was distributed diffusely in the cytoplasm and the nucleus. However, in the presence of BMB2, GFP-BMB1 was directed to cell wall-adjacent elongated bodies at the cell periphery, to cell wall-embedded punctate structures co-localizing with callose deposits at plasmodesmata, and to cells adjacent to the initially transformed cell. Thus, BMB2 can mediate the transport of BMB1 to and through plasmodesmata. In general, our observations support the idea that cell-to-cell trafficking of movement proteins involves an initial delivery to membrane compartments adjacent to plasmodesmata, subsequent entry of the plasmodesmata cavity and, finally, transport to adjacent cells. This process, as an alternative to tubule-based transport, has most likely evolved independently in triple gene block (TGB), double gene block (DGB), BMB and the single gene-coded transport system.

Phylogenetic and functional analyses of a plant protein related to human B-cell receptor-associated proteins.

Human B-cell receptor-associated protein BAP31 (HsBAP31) is the endoplasmic reticulum-resident protein involved in protein sorting and transport as well as pro-apoptotic signaling. Plant orthologs of HsBAP31 termed 'plant BAP-like proteins' (PBL proteins) have thus far remained unstudied. Recently, the PBL protein from Nicotiana tabacum (NtPBL) was identified as an interactor of Nt-4/1, a plant protein known to interact with plant virus movement proteins and affect the long-distance transport of potato spindle tuber viroid (PSTVd) via the phloem. Here, we have compared the sequences of PBL proteins and studied the biochemical properties of NtPBL. Analysis of a number of fully sequenced plant genomes revealed that PBL-encoding genes represent a small multigene family with up to six members per genome. Two conserved motifs were identified in the C-terminal region of PBL proteins. The NtPBL C-terminal hydrophilic region (NtPBL-C) was expressed in bacterial cells, purified, and used for analysis of its RNA binding properties in vitro. In gel shift experiments, NtPBL-C was found to bind several tested RNAs, showing the most efficient binding to microRNA precursors (pre-miRNA) and less efficient interaction with PSTVd. Mutational analysis suggested that NtPBL-C has a composite RNA-binding site, with two conserved lysine residues in the most C-terminal protein region being involved in binding of pre-miRNA but not PSTVd RNA. Virus-mediated transient expression of NtPBL-C in plants resulted in stunting and leaf malformation, developmental abnormalities similar to those described previously for blockage of miRNA biogenesis/function. We hypothesize that the NtPBL protein represents a previously undiscovered component of the miRNA pathway.