Barley stripe mosaic virus γb protein disrupts chloroplast antioxidant defenses to optimize viral replication

The plant antioxidant system plays important roles in response to diverse abiotic and biotic stresses. However, the effects of virus infection on host redox homeostasis and how antioxidant defense pathway is manipulated by viruses remain poorly understood. We previously demonstrated that the Barley stripe mosaic virus (BSMV) γb protein is recruited to the chloroplast by the viral αa replicase to enhance viral replication. Here, we show that BSMV infection induces chloroplast oxidative stress. The versatile γb protein interacts directly with NADPH‐dependent thioredoxin reductase C (NTRC), a core component of chloroplast antioxidant systems. Overexpression of NbNTRC significantly impairs BSMV replication in Nicotiana benthamiana plants, whereas disruption of NbNTRC expression leads to increased viral accumulation and infection severity. To counter NTRC‐mediated defenses, BSMV employs the γb protein to competitively interfere with NbNTRC binding to 2‐Cys Prx. Altogether, this study indicates that beyond acting as a helicase enhancer, γb also subverts NTRC‐mediated chloroplast antioxidant defenses to create an oxidative microenvironment conducive to viral replication.     

Chloroplast Proteome of <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> Infected by Tomato Blistering Mosaic Virus

Tymovirus is a genus of plant pathogenic viruses that infects several dicotyledonous plants worldwide, causing serious diseases in economically important crops. The known cytopathic effect on the host cell organelles involves chloroplast membrane deformation and the induction of vesicles in its periphery. These vesicles are known to be the location where tymoviral genomic RNA replication occurs. Tomato blistering mosaic virus (ToBMV) is a tymovirus recently identified in tomato plants in Brazil, which is able to infect several other plants, including tobacco. In this work, we investigated the chloroplast proteomic profile of ToBMV-infected N. benthamiana using bidimensional electrophoresis (2-DE) and mass spectrometry, aiming to study the virus-host interaction related to the virus replication and infection. A total of approximately 200 spots were resolved, out of which 36 were differentially abundant. Differential spots were identified by mass spectrometry including photosynthesis-related and defense proteins. We identified proteins that may be targets of a direct interaction with viral proteins, such as ATP synthase β subunit, RNA polymerase beta-subunit, 50S ribosomal protein L6 and Trigger factor-like protein. The identification of these candidate proteins gives support for future protein–protein interaction studies to confirm their roles in virus replication and disease development.     

Sequential Recruitment of the Endoplasmic Reticulum and Chloroplasts for Plant Potyvirus Replication

The replication of positive-strand RNA viruses occurs in cytoplasmic membrane-bound virus replication complexes (VRCs). Depending on the virus, distinct cellular organelles such as the endoplasmic reticulum (ER), chloroplast, mitochondrion, endosome, and peroxisome are recruited for the formation of VRC-associated membranous structures. Previously, the 6,000-molecular-weight protein (6K) of plant potyviruses was shown to be an integral membrane protein that induces the formation of 6K-containing membranous vesicles at endoplasmic reticulum (ER) exit sites for potyvirus genome replication. Here, we present evidence that the 6K-induced vesicles predominantly target chloroplasts, where they amalgamate and induce chloroplast membrane invaginations. The vesicular transport pathway and actomyosin motility system are involved in the trafficking of the 6K vesicles from the ER to chloroplasts. Viral RNA, double-stranded RNA, and viral replicase components are concentrated at the 6K vesicles that associate with chloroplasts in infected cells, suggesting that these chloroplast-bound 6K vesicles are the site for potyvirus replication. Taken together, these results suggest that plant potyviruses sequentially recruit the ER and chloroplasts for their genome replication.The replication of eukaryotic positive-strand RNA viruses in infected cells is closely associated with unique virus-induced intracellular membranous vesicles (22). These membranous vesicles have been proposed to provide a scaffold for anchoring the virus replication complex (VRC), confine the process of RNA replication to a specific safeguarded cytoplasmic location, and prevent the activation of certain host defense mechanisms that can be triggered by double-stranded RNA (dsRNA) intermediates during virus replication (33, 47). Depending on the type of virus, the virus-induced membranous vesicles are derived from various intracellular organelles in the host. Many plant and animal viruses remodel and utilize the endoplasmic reticulum (ER) in VRCs (1, 6, 17, 33, 34, 36, 38, 39, 46). Other cellular organelles such as endosomes, lysomes, chloroplasts, peroxisomes, and mitochondria have also been suggest to be the replication site for togaviruses, tymoviruses, and tombusviruses, respectively (25, 27, 31). Given that the ER appears to be the site where the host cell translation machinery is hijacked for the biosynthesis of the first set of viral proteins, the subcellular location of virus replication (either in the vicinity of the ER or elsewhere) and the mechanism of transport to locations other than the ER are poorly understood.Plant potyviruses, accounting for ∼30% of known plant viruses including many agriculturally important viruses, e.g., Turnip mosaic virus (TuMV), Maize dwarf mosaic virus (MDMV), Tobacco etch virus (TEV), and Potato virus Y (PVY), are related to picornaviruses and picorna-like viruses (20, 21, 43). The potyviral genome is a single-stranded positive-sense RNA of about 10 kb in length and encodes at least 11 mature viral proteins (8, 43). Of these 11 proteins, the 6-kDa protein (designated 6K or 6K2) contains a central hydrophobic domain (35). In seminal work, Carrington and colleagues determined that 6K induces the formation of the ER-derived vesicles for TEV replication (35, 38). More recently, viral proteins required for replication and several host factors, namely, eukaryotic initiation factor (isoform) 4E, poly(A)-binding protein, eukaryotic elongation factor 1A, and heat shock cognate 70-3 protein, have been shown to associate with the TuMV 6K-induced vesicles (9, 41), raising the possibility that the potyviral 6K vesicles represent sites of viral genome replication. Furthermore, we have demonstrated that the biogenesis of the potyviral 6K vesicles occurs at COPII-accumulating ER exit sites (ERES) on the ER membrane (45). In this study, we further studied the trafficking of 6K-induced vesicles and found that the 6K-induced mobile vesicles trafficked predominantly from the ER to the periphery of chloroplasts. We show that these 6K vesicles docked on the outer chloroplast envelope and induced chloroplast invaginations. The chloroplast-associated 6K vesicles contained viral replicase components and dsRNA and were concentrated with viral RNA. We provide evidence that the early secretory pathway and actomyosin motility system were required for the trafficking of 6K vesicles from the ER to chloroplasts. These results suggest that plant potyviruses sequentially recruit the ER and chloroplasts for their genome replication.     

Co-opted Oxysterol-Binding ORP and VAP Proteins Channel Sterols to RNA Virus Replication Sites via Membrane Contact Sites

Viruses recruit cellular membranes and subvert cellular proteins involved in lipid biosynthesis to build viral replicase complexes and replication organelles. Among the lipids, sterols are important components of membranes, affecting the shape and curvature of membranes. In this paper, the tombusvirus replication protein is shown to co-opt cellular Oxysterol-binding protein related proteins (ORPs), whose deletion in yeast model host leads to decreased tombusvirus replication. In addition, tombusviruses also subvert Scs2p VAP protein to facilitate the formation of membrane contact sites (MCSs), where membranes are juxtaposed, likely channeling lipids to the replication sites. In all, these events result in redistribution and enrichment of sterols at the sites of viral replication in yeast and plant cells. Using in vitro viral replication assay with artificial vesicles, we show stimulation of tombusvirus replication by sterols. Thus, co-opting cellular ORP and VAP proteins to form MCSs serves the virus need to generate abundant sterol-rich membrane surfaces for tombusvirus replication.

Authors Summary

Cellular proteins and cellular membranes are usurped by positive-stranded RNA viruses to assemble viral replicase complexes required for their replication. Tombusviruses, which are small RNA viruses of plants, depend on sterol-rich membranes for replication. The authors show that the tombusviral replication protein binds to cellular oxysterol-binding ORP proteins. Moreover, the endoplasmic reticulum resident cellular VAP proteins also co-localize with viral replication proteins. These protein interactions likely facilitate the formation of membrane contact sites that are visible in cells replicating tombusvirus RNA. The authors also show that sterols are recruited and enriched to the sites of viral replication. In vitro replication assay was used to show that sterols indeed stimulate tombusvirus replication. In summary, tombusviruses use subverted cellular proteins to build sterol-rich membrane microdomain to promote the assembly of the viral replicase complex. The paper connects efficient virus replication with cellular lipid transport and membrane structures.     

Targeting of the turnip yellow mosaic virus 66K replication protein to the chloroplast envelope is mediated by the 140K protein

Turnip yellow mosaic virus (TYMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two replication proteins, 140K and 66K, both being required for its RNA genome replication. The 140K protein contains domains indicative of methyltransferase, proteinase, and NTPase/helicase, and the 66K protein encompasses the RNA-dependent RNA polymerase domain. During viral infection, the 66K protein localizes to virus-induced chloroplastic membrane vesicles, which are closely associated with TYMV RNA replication. To investigate the determinants of its subcellular localization, the 66K protein was expressed in plant protoplasts from separate plasmids. Green fluorescent protein (GFP) fusion and immunofluorescence experiments demonstrated that the 66K protein displayed a cytoplasmic distribution when expressed individually but that it was relocated to the chloroplast periphery under conditions in which viral replication occurred. The 66K protein produced from an expression vector was functional in viral replication since it could transcomplement a defective replication template. Targeting of the 66K protein to the chloroplast envelope in the course of the viral infection appeared to be solely dependent on the expression of the 140K protein. Analysis of the subcellular localization of the 140K protein fused to GFP demonstrated that it is targeted to the chloroplast envelope in the absence of other viral factors and that it induces the clumping of the chloroplasts, one of the typical cytological effects of TYMV infection. These results suggests that the 140K protein is a key organizer of the assembly of the TYMV replication complexes and a major determinant for their chloroplastic localization and retention.     

Effect of salt stress on genes encoding translation-associated proteins in Arabidopsis thaliana

Salinity negatively affects plant growth and disturbs chloroplast integrity. Here, we aimed at identifying salt-responsive translation-related genes in Arabidopsis thaliana with an emphasis on those encoding plastid-located proteins. We used quantitative real-time PCR to test the expression of 170 genes after short-term salt stress (up to 24 h) and identified several genes affected by the stress including: PRPL11, encoding plastid ribosomal protein L11, ATAB2, encoding a chloroplast-located RNA-binding protein presumably functioning as an activator of translation, and PDF1B, encoding a peptide deformylase involved in N-formyl group removal from nascent proteins synthesized in chloroplasts. These genes were previously shown to have important functions in chloroplast biology and may therefore represent new targets for biotechnological optimization of salinity tolerance.     

NbEXPA1, an α‐expansin,is plasmodesmata‐specific and a novel host factor for potyviral infection

Plasmodesmata (PD), unique to the plant kingdom, are structurally complex microchannels that cross the cell wall to establish symplastic communication between neighbouring cells. Viral intercellular movement occurs through PD. To better understand the involvement of PD in viral infection, we conducted a quantitative proteomic study on the PD‐enriched fraction from Nicotiana benthamiana leaves in response to infection by Turnip mosaic virus (TuMV). We report the identification of a total of 1070 PD protein candidates, of which 100 (≥2‐fold increase) and 48 (≥2‐fold reduction) are significantly differentially accumulated in the PD‐enriched fraction, when compared with protein levels in the corresponding healthy control. Among the differentially accumulated PD protein candidates, we show that an α‐expansin designated NbEXPA1, a cell wall loosening protein, is PD‐specific. TuMV infection downregulates NbEXPA1 mRNA expression and protein accumulation. We further demonstrate that NbEXPA1 is recruited to the viral replication complex via the interaction with NIb, the only RNA‐dependent RNA polymerase of TuMV. Silencing of NbEXPA1 inhibits plant growth and TuMV infection, whereas overexpression of NbEXPA1 promotes viral replication and intercellular movement. These data suggest that NbEXPA1 is a host factor for potyviral infection. This study not only generates a PD‐proteome dataset that is useful in future studies to expound PD biology and PD‐mediated virus–host interactions but also characterizes NbEXPA1 as the first PD‐specific cell wall loosening protein and its essential role in potyviral infection.     

The Crucial Role of Chloroplast-Related Proteins in Viral Genome Replication and Host Defense against Positive-Sense Single-Stranded RNA Viruses

Plant viruses are responsible for worldwide production losses of numerous economically important crops. The most common plant RNA viruses are positive-sense single-stranded RNA viruses [(+)ss RNA viruses]. These viruses have small genomes that encode a limited number of proteins. The viruses depend on their host’s machinery for the replication of their RNA genome, assembly, movement, and attraction to the vectors for dispersal. Recently researchers have reported that chloroplast proteins are crucial for replicating (+)ss plant RNA viruses. Some chloroplast proteins, including translation initiation factor [eIF(iso)4E] and 75 DEAD-box RNA helicase RH8, help viruses fulfill their infection cycle in plants. In contrast, other chloroplast proteins such as PAP2.1, PSaC, and ATPsyn-α play active roles in plant defense against viruses. This is also consistent with the idea that reactive oxygen species, salicylic acid, jasmonic acid, and abscisic acid are produced in chloroplast. However, knowledge of molecular mechanisms and functions underlying these chloroplast host factors during the virus infection is still scarce and remains largely unknown. Our review briefly summarizes the latest knowledge regarding the possible role of chloroplast in plant virus replication, emphasizing chloroplast-related proteins. We have highlighted current advances regarding chloroplast-related proteins’ role in replicating plant (+)ss RNA viruses.     

Assembly of turnip yellow mosaic virus replication complexes: interaction between the proteinase and polymerase domains of the replication proteins

Turnip yellow mosaic virus (TYMV), a positive-strand RNA virus in the alphavirus-like supergroup, encodes two nonstructural replication proteins (140K and 66K), both of which are required for its RNA genome replication. The 140K protein contains domains indicative of methyltransferase, proteinase, and NTPase/helicase activities, while the 66K protein encompasses the RNA-dependent RNA polymerase domain. Recruitment of the 66K protein to the sites of viral replication, located at the periphery of chloroplasts, is dependent upon the expression of the 140K protein. Using antibodies raised against the 140K and 66K proteins and confocal microscopy, we report the colocalization of the TYMV replication proteins at the periphery of chloroplasts in transfected or infected cells. The replication proteins cofractionated in functional replication complexes or with purified chloroplast envelope membranes prepared from infected plants. Using a two-hybrid system and coimmunoprecipitation experiments, we also provide evidence for a physical interaction of the TYMV replication proteins. In contrast to what has been found for other members of the alphavirus-like supergroup, the interaction domains were mapped to the proteinase domain of the 140K protein and to a large region encompassing the core polymerase domain within the 66K protein. Coexpression and colocalization experiments confirmed that the helicase domain of the 140K protein is unnecessary for the proper recruitment of the 66K protein to the chloroplast envelope, while the proteinase domain appears to be essential for that process. These results support a novel model for the interaction of TYMV replication proteins and suggest that viruses in the alphavirus-like supergroup may have selected different pathways to assemble their replication complexes.     

The Ubiquitin-Proteasome System Regulates the Accumulation of Turnip yellow mosaic virus RNA-Dependent RNA Polymerase during Viral Infection

Replication of positive-strand RNA viruses, the largest group of plant viruses, is initiated by viral RNA-dependent RNA polymerase (RdRp). Given its essential function in viral replication, understanding the regulation of RdRp is of great importance. Here, we show that Turnip yellow mosaic virus (TYMV) RdRp (termed 66K) is degraded by the proteasome at late time points during viral infection and that the accumulation level of 66K affects viral RNA replication in infected Arabidopsis thaliana cells. We mapped the cis-determinants responsible for 66K degradation within its N-terminal noncatalytic domain, but we conclude that 66K is not a natural N-end rule substrate. Instead, we show that a proposed PEST sequence within 66K functions as a transferable degradation motif. In addition, several Lys residues that constitute target sites for ubiquitylation were mapped; mutation of these Lys residues leads to stabilization of 66K. Altogether, these results demonstrate that TYMV RdRp is a target of the ubiquitin-proteasome system in plant cells and support the idea that proteasomal degradation may constitute yet another fundamental level of regulation of viral replication.     

The unfolded protein response plays dual roles in rice stripe virus infection through fine-tuning the movement protein accumulation

The movement of plant viruses is a complex process that requires support by the virus-encoded movement protein and multiple host factors. The unfolded protein response (UPR) plays important roles in plant virus infection, while how UPR regulates viral infection remains to be elucidated. Here, we show that rice stripe virus (RSV) elicits the UPR in Nicotiana benthamiana. The RSV-induced UPR activates the host autophagy pathway by which the RSV-encoded movement protein, NSvc4, is targeted for autophagic degradation. As a counteract, we revealed that NSvc4 hijacks UPR-activated type-I J-domain proteins, NbMIP1s, to protect itself from autophagic degradation. Unexpectedly, we found NbMIP1 stabilizes NSvc4 in a non-canonical HSP70-independent manner. Silencing NbMIP1 family genes in N. benthamiana, delays RSV infection, while over-expressing NbMIP1.4b promotes viral cell-to-cell movement. Moreover, OsDjA5, the homologue of NbMIP1 family in rice, behaves in a similar manner toward facilitating RSV infection. This study exemplifies an arms race between RSV and the host plant, and reveals the dual roles of the UPR in RSV infection though fine-tuning the accumulation of viral movement protein.     

RIBOSOMES BOUND TO CHLOROPLAST MEMBRANES IN CHLAMYDOMONAS REINHARDTII

The amount of chloroplast ribosomal RNAs of Chlamydomonas reinhardtii which sediment at 15,000 g is increased when cells are treated with chloramphenicol. Preparations of chloroplast membranes from chloramphenicol-treated cells contain more chloroplast ribosomal RNAs than preparations from untreated cells. The membranes from treated cells also contain more ribosome-like particles, some of which appear in polysome-like arrangements. About 50% of chloroplast ribosomes are released from membranes in vitro as subunits by 1 mM puromycin in 500 mM KCl. A portion of chloroplast ribosomal subunits is released by 500 mM KCl alone, a portion by 1 mM puromycin alone, and a portion by 1 mM puromycin in 500 mM KCl. Ribosomes are not released from isolated membranes by treatment with ribonuclease. Membranes in chloroplasts of chloramphenicol-treated cells show many ribosomes associated with membranes, some of which are present in polysome-like arrangements. This type of organization is less frequent in chloroplasts of untreated cells. Streptogramin, an inhibitor of initiation, prevents chloramphenicol from acting to permit isolation of membrane-bound ribosomes. Membrane-bound chloroplast ribosomes are probably a normal component of actively growing cells. The ability to isolate membrane-bound ribosomes from chloramphenicol-treated cells is probably due to chloramphenicol-prevented completion of nascent chains during harvesting of cells. Since chloroplasts synthesize some of their membrane proteins, and a portion of chloroplast ribosomes is bound to chloroplast membranes through nascent protein chains, it is suggested that the membrane-bound ribosomes are synthesizing membrane protein.     

The CD225 Domain of IFITM3 Is Required for both IFITM Protein Association and Inhibition of Influenza A Virus and Dengue Virus Replication

The interferon-induced transmembrane protein 3 (IFITM3) gene is an interferon-stimulated gene that inhibits the replication of multiple pathogenic viruses in vitro and in vivo. IFITM3 is a member of a large protein superfamily, whose members share a functionally undefined area of high amino acid conservation, the CD225 domain. We performed mutational analyses of IFITM3 and identified multiple residues within the CD225 domain, consisting of the first intramembrane domain (intramembrane domain 1 [IM1]) and a conserved intracellular loop (CIL), that are required for restriction of both influenza A virus (IAV) and dengue virus (DENV) infection in vitro. Two phenylalanines within IM1 (F75 and F78) also mediate a physical association between IFITM proteins, and the loss of this interaction decreases IFITM3-mediated restriction. By extension, similar IM1-mediated associations may contribute to the functions of additional members of the CD225 domain family. IFITM3''s distal N-terminal domain is also needed for full antiviral activity, including a tyrosine (Y20), whose alteration results in mislocalization of a portion of IFITM3 to the cell periphery and surface. Comparative analyses demonstrate that similar molecular determinants are needed for IFITM3''s restriction of both IAV and DENV. However, a portion of the CIL including Y99 and R87 is preferentially needed for inhibition of the orthomyxovirus. Several IFITM3 proteins engineered with rare single-nucleotide polymorphisms demonstrated reduced expression or mislocalization, and these events were associated with enhanced viral replication in vitro, suggesting that possessing such alleles may impact an individual''s risk for viral infection. On the basis of this and other data, we propose a model for IFITM3-mediated restriction.     

Efficient Herpes Simplex Virus 1 Replication Requires Cellular ATR Pathway Proteins

Herpes simplex virus 1 (HSV-1) is a double-stranded DNA virus that replicates in the nucleus of the host cell and is known to interact with several components of the cellular DNA-damage-signaling machinery. We have previously reported that the DNA damage response kinase, ATR, is specifically inactivated in HSV-1-infected cells. On the other hand, we have also shown that ATR and its scaffolding protein, ATRIP, are recruited to viral replication compartments, where they play beneficial roles during HSV-1 replication. In order to better understand this apparent discrepancy, we tested the hypothesis that some of the components of the ATR pathway may exert an antiviral effect on infection. In fact, we learned that all 10 of the canonical ATR pathway proteins are stable in HSV-infected cells and are recruited to viral replication compartments; furthermore, short hairpin RNA (shRNA) knockdown shows that several, including ATRIP, RPA70, TopBP1, Claspin, and CINP, are required for efficient HSV-1 replication. We also determined that activation of the ATR kinase prior to infection did not affect virus yield but did result in reduced levels of recombination between coinfecting viruses. Together, these data suggest that ATR pathway proteins are not antiviral per se but that activation of ATR signaling may have negative consequences during viral replication, such as inhibiting recombination.     

Relocation of the NIb Gene in the Tobacco Etch Potyvirus Genome

Potyviruses express most of their proteins from a long open reading frame that is translated into a large polyprotein processed by three viral proteases. To understand the constraints on potyvirus genome organization, we relocated the viral RNA-dependent RNA polymerase (NIb) cistron to all possible intercistronic positions of the Tobacco etch virus (TEV) polyprotein. Only viruses with NIb at the amino terminus of the polyprotein or in between P1 and HC-Pro were viable in tobacco plants.     

Optimized expression, solubilization and purification of nuclear inclusion protein b of cardamom mosaic virus

All RNA viruses encode an RNA-dependent RNA polymerase (RdRP) that is required for replication of the viral genome. Nuclear inclusion b (NIb) gene codes for the RdRp in Potyviridae viruses. In this study, expression, solubilization and purification of NIb protein of Cardamom mosaic virus (CdMV) is reported. The objective of the present study was to express and purify the NIb protein of CdMV on a large scale for structural characterization, as the structure of the RdRp from a plant virus is yet to be determined. However, the expression of NIb protein with hexa-histidine tag in Escherichia coli led to insoluble aggregates. Out of all the approaches [making truncated versions to reduce the size of protein; replacing an amino acid residue likely to be involved in hydrophobic intermolecular interactions with a hydrophilic one; expressing the protein along with chaperones; expression in Origami cells for proper disulphide bond formation, in E. coli as a fusion with maltose-binding protein (MBP) and in Nicotiana tabacum] to obtain the RdRp in a soluble form, only expression in E. coli as a fusion with MBP and its expression in N. tabacum were successful. The NIb expressed in plant or as a fusion with MBP in E. coli can be scaled up for further work.     

Formation of Potato Virus A-Induced RNA Granules and Viral Translation Are Interrelated Processes Required for Optimal Virus Accumulation

RNA granules are cellular structures, which play an important role in mRNA translation, storage, and degradation. Animal (+)RNA viruses often co-opt RNA granule proteins for viral reproduction. However, the role of RNA granules in plant viral infections is poorly understood. Here we use Potato virus A (PVA) as a model potyvirus and demonstrate that the helper component-proteinase (HCpro), the potyviral suppressor of RNA silencing, induces the formation of RNA granules. We used confocal microscopy to demonstrate the presence of host RNA binding proteins including acidic ribosomal protein P0, argonaute 1 (AGO1), oligouridylate-binding protein 1 (UBP1), varicose (VCS) and eukaryotic initiation factor iso4E (eIF(iso)4E) in these potyvirus-induced RNA granules. We show that the number of potyviral RNA granules is down-regulated by the genome-linked viral protein (VPg). We demonstrated previously that VPg is a virus-specific translational regulator that co-operates with potyviral RNA granule components P0 and eIF(iso)4E in PVA translation. In this study we show that HCpro and varicose, components of potyviral RNA granules, stimulate VPg-promoted translation of the PVA, whereas UBP1 inhibits this process. Hence, we propose that PVA translation operates via a pathway that is interrelated with potyviral RNA granules in PVA infection. The importance of these granules is evident from the strong reduction in viral RNA and coat protein amounts that follows knock down of potyviral RNA granule components. HCpro suppresses antiviral RNA silencing during infection, and our results allow us to propose that this is also the functional context of the potyviral RNA granules we describe in this study.     

Replication and trafficking of a plant virus are coupled at the entrances of plasmodesmata

Plant viruses use movement proteins (MPs) to modify intercellular pores called plasmodesmata (PD) to cross the plant cell wall. Many viruses encode a conserved set of three MPs, known as the triple gene block (TGB), typified by Potato virus X (PVX). In this paper, using live-cell imaging of viral RNA (vRNA) and virus-encoded proteins, we show that the TGB proteins have distinct functions during movement. TGB2 and TGB3 established endoplasmic reticulum–derived membranous caps at PD orifices. These caps harbored the PVX replicase and nonencapsidated vRNA and represented PD-anchored viral replication sites. TGB1 mediated insertion of the viral coat protein into PD, probably by its interaction with the 5′ end of nascent virions, and was recruited to PD by the TGB2/3 complex. We propose a new model of plant virus movement, which we term coreplicational insertion, in which MPs function to compartmentalize replication complexes at PD for localized RNA synthesis and directional trafficking of the virus between cells.