The Ins and Outs of Nondestructive Cell-to-Cell and Systemic Movement of Plant Viruses

Propagation of viral infection in host plants comprises two distinct and sequential stages: viral transport from the initially infected cell into adjacent neighboring cells, a process termed local or cell-to-cell movement, and a chain of events collectively referred to as systemic movement that consists of entry into the vascular tissue, systemic distribution with the phloem stream, and unloading of the virus into noninfected tissues. To achieve intercellular transport, viruses exploit plasmodesmata, complex cytoplasmic bridges interconnecting plant cells. Viral transport through plasmodesmata is aided by virus-encoded proteins, the movement proteins (MPs), which function by two distinct mechanisms: MPs either bind viral nucleic acids and mediate passage of the resulting movement complexes (M-complexes) between cells, or MPs become a part of pathogenic tubules that penetrate through host cell walls and serve as conduits for transport of viral particles. In the first mechanism, M-complexes pass into neighboring cells without destroying or irreversibly altering plasmodesmata, whereas in the second mechanism plasmodesmata are replaced or significantly modified by the tubules. Here we summarize the current knowledge on both local and systemic movement of viruses that progress from cell to cell as M-complexes in a nondestructive fashion. For local movement, we focus mainly on movement functions of the 30 K superfamily viruses, which encode MPs with structural homology to the 30 kDa MP of Tobacco mosaic virus, one of the most extensively studied plant viruses, whereas systemic movement is primarily described for two well-characterized model systems, Tobacco mosaic virus and Tobacco etch potyvirus. Because local and systemic movement are intimately linked to the molecular infrastructure of the host cell, special emphasis is placed on host factors and cellular structures involved in viral transport.     

Plant virus transport: motions of functional equivalence

Plant virus cell-to-cell movement and subsequent systemic transport are governed by a series of mechanisms involving various virus and plant factors. Specialized virus encoded movement proteins (MPs) control the cell-to-cell transport of viral nucleoprotein complexes through plasmodesmata. MPs of different viruses have diverse properties and each interacts with specific host factors that also have a range of functions. Most viruses are then transported via the phloem as either nucleoprotein complexes or virions, with contributions from host and virus proteins. Some virus proteins contribute to the establishment and maintenance of systemic infection by inhibiting RNA silencing-mediated degradation of viral RNA. In spite of all the different movement strategies and the viral and host components, there are possible functional commonalities in virus-host interactions that govern viral spread through plants.     

Distinct functional domains in the cowpea mosaic virus movement protein.

Cell-to-cell movement of cowpea mosaic virus particles in plants takes place with the help of tubules that penetrate presumably modified plasmodesmata. These tubules, which are built up by the virus-encoded 48-kDa movement protein (MP), are also formed on single protoplast cells. To determine whether the MP contains different functional domains, the effect of mutations in its coding region was studied. Mutations between amino acids 1 and 313 led to complete abolishment of the tubule-forming capacity, while a deletion in the C-terminal region resulted in tubules that could not take up virus particles. From these observations, it is concluded that the MP contains at least two distinct domains, one that is involved in tubule formation and that spans amino acids 1 and 313 and a second that is probably involved in the incorporation of virus particles in the tubule and that is located in the C terminus between amino acids 314 and 331.     

Histone H3 interacts and colocalizes with the nuclear shuttle protein and the movement protein of a geminivirus

Geminiviruses are plant-infecting viruses with small circular single-stranded DNA genomes. These viruses utilize nuclear shuttle proteins (NSPs) and movement proteins (MPs) for trafficking of infectious DNA through the nuclear pore complex and plasmodesmata, respectively. Here, a biochemical approach was used to identify host factors interacting with the NSP and MP of the geminivirus Bean dwarf mosaic virus (BDMV). Based on these studies, we identified and characterized a host nucleoprotein, histone H3, which interacts with both the NSP and MP. The specific nature of the interaction of histone H3 with these viral proteins was established by gel overlay and in vitro and in vivo coimmunoprecipitation (co-IP) assays. The NSP and MP interaction domains were mapped to the N-terminal region of histone H3. These experiments also revealed a direct interaction between the BDMV NSP and MP, as well as interactions between histone H3 and the capsid proteins of various geminiviruses. Transient-expression assays revealed the colocalization of histone H3 and NSP in the nucleus and nucleolus and of histone H3 and MP in the cell periphery and plasmodesmata. Finally, using in vivo co-IP assays with a Myc-tagged histone H3, a complex composed of histone H3, NSP, MP, and viral DNA was recovered. Taken together, these findings implicate the host factor histone H3 in the process by which an infectious geminiviral DNA complex forms within the nucleus for export to the cell periphery and cell-to-cell movement through plasmodesmata.     

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.     

Caulimoviridae Tubule-Guided Transport Is Dictated by Movement Protein Properties

Plant viruses move through plasmodesmata (PD) either as nucleoprotein complexes (NPCs) or as tubule-guided encapsidated particles with the help of movement proteins (MPs). To explore how and why MPs specialize in one mechanism or the other, we tested the exchangeability of MPs encoded by DNA and RNA virus genomes by means of an engineered alfalfa mosaic virus (AMV) system. We show that Caulimoviridae (DNA genome virus) MPs are competent for RNA virus particle transport but are unable to mediate NPC movement, and we discuss this restriction in terms of the evolution of DNA virus MPs as a means of mediating DNA viral genome entry into the RNA-trafficking PD pathway.Following virus entry and replication, successful infection of a host requires viral spread to distal parts of the organism through the vascular tissue. In plants, virus movement involves mostly symplastic trafficking of different viral components through the connections of plasmodesmata (PD) (13). With this aim, plant viruses encode one or more movement proteins (MPs), which allow viral genomes to cross the host cell wall by altering the size exclusion limit (SEL) or the structure of PD (6, 11). Plant viruses have evolved distinct mechanisms to move their genomes within the host. These mechanisms can be grouped into two general strategies: one in which the genome is transported in the form of a nucleoprotein complex (NPC) and another in which nucleic acids are encapsidated and move as virus particles. In both cases, besides altering PD SEL, MPs are involved either in NPC assembly or in forming tubules traversing modified PD and helping transport of either NPC or virions to the neighboring cell. Within these two major strategies, there exists a wide range of variability in terms of the number and type of viral and host proteins helping MPs to mediate virus spread within the host (11).In spite of such variability, several different MPs have been classified into a 30K superfamily; these MPs, from 20 genera including both RNA and DNA genome viruses, are structurally related to the 30-kDa MP of Tobacco mosaic virus (TMV), independent of the movement strategy followed (14). Members of this family have a common core of predicted secondary structure elements (α-helices and β-elements) containing a nucleic acid binding domain. Distinct MPs belong to this family, including several tubule-forming MPs, although these are phylogenetically separated from the other members (14). Thus, 30K superfamily MPs are closely related, and some of them are functionally interchangeable in the viral context (2, 20). In particular, MPs from five distinct genera with an RNA genome can successfully replace the corresponding gene of Alfalfa mosaic virus (AMV) (19), indicating that one or more basic and fundamental movement properties might be associated with the common 30K structural core.Among all known plant viruses, only three viral families have evolved a DNA genome: Geminiviridae, Caulimoviridae, and Nanoviridae (6). One possible explanation for this restriction is that endogenous cell-to-cell transport via PD is specialized to use RNA as the communication and signaling molecule (12). To circumvent this restriction, and to allow the efficient exploitation of endogenous transport machineries, DNA genome viruses have evolved appropriate mechanisms involving their MPs. Interestingly, Begomovirus and Caulimovirus MPs also belong to the 30K superfamily discussed above (14). The MP encoded by Cauliflower mosaic virus (CaMV), the type member of Caulimoviridae, forms tubules that guide the movement of encapsidated virus via an indirect MP-virion interaction (16, 21), whereas geminivirus MPs selectively bind their genomes and transport them as NPCs (6, 9, 17). In this study, we investigated the evolutionary convergence of MPs encoded by DNA and RNA viruses by testing their exchangeability in the viral context.     

Intracellular targeting of a hordeiviral membrane-spanning movement protein: sequence requirements and involvement of an unconventional mechanism

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.     

A family of plasmodesmal proteins with receptor-like properties for plant viral movement proteins

Plasmodesmata (PD) are essential but poorly understood structures in plant cell walls that provide symplastic continuity and intercellular communication pathways between adjacent cells and thus play fundamental roles in development and pathogenesis. Viruses encode movement proteins (MPs) that modify these tightly regulated pores to facilitate their spread from cell to cell. The most striking of these modifications is observed for groups of viruses whose MPs form tubules that assemble in PDs and through which virions are transported to neighbouring cells. The nature of the molecular interactions between viral MPs and PD components and their role in viral movement has remained essentially unknown. Here, we show that the family of PD-located proteins (PDLPs) promotes the movement of viruses that use tubule-guided movement by interacting redundantly with tubule-forming MPs within PDs. Genetic disruption of this interaction leads to reduced tubule formation, delayed infection and attenuated symptoms. Our results implicate PDLPs as PD proteins with receptor-like properties involved the assembly of viral MPs into tubules to promote viral movement.     

Role of the alfalfa mosaic virus movement protein and coat protein in virus transport

The movement protein (MP) and coat protein (CP) encoded by Alfalfa mosaic virus (AMV) RNA 3 are both required for virus transport. RNA 3 vectors that expressed nonfused green fluorescent protein (GFP), MP:GPF fusions, or GFP:CP fusions were used to study the functioning of mutant MP and CP in protoplasts and plants. C-terminal deletions of up to 21 amino acids did not interfere with the function of the CP in cell-to-cell movement, although some of these mutations interfered with virion assembly. Deletion of the N-terminal 11 or C-terminal 45 amino acids did not interfere with the ability of MP to assemble into tubular structures on the protoplast surface. Additionally, N- or C-terminal deletions disrupted tubule formation. A GFP:CP fusion was targeted specifically into tubules consisting of a wild-type MP. All MP deletion mutants that showed cell-to-cell and systemic movement in plants were able to form tubular structures on the surface of protoplasts. Brome mosaic virus (BMV) MP did not support AMV transport. When the C-terminal 48 amino acids were replaced by the C-terminal 44 amino acids of the AMV MP, however, the BMV/AMV chimeric protein permitted wild-type levels of AMV transport. Apparently, the C terminus of the AMV MP, although dispensable for cell-to-cell movement, confers specificity to the transport process.     

Plant Virus Cell-to-Cell Movement Is Not Dependent on the Transmembrane Disposition of Its Movement Protein

The cell-to-cell transport of plant viruses depends on one or more virus-encoded movement proteins (MPs). Some MPs are integral membrane proteins that interact with the membrane of the endoplasmic reticulum, but a detailed understanding of the interaction between MPs and biological membranes has been lacking. The cell-to-cell movement of the Prunus necrotic ringspot virus (PNRSV) is facilitated by a single MP of the 30K superfamily. Here, using a myriad of biochemical and biophysical approaches, we show that the PNRSV MP contains only one hydrophobic region (HR) that interacts with the membrane interface, as opposed to being a transmembrane protein. We also show that a proline residue located in the middle of the HR constrains the structural conformation of this region at the membrane interface, and its replacement precludes virus movement.Plant viruses encode movement proteins (MPs) that mediate the intra- and intercellular spread of the viral genome via plasmodesmata, membranous channels that traverse the walls of plant cells and enable intercellular transport and communication. There is a range of diversity in the number and type of viral proteins required for viral movement (21). Research on tobacco mosaic virus (TMV) has played a leading role in understanding MP activity (2). The genome of TMV encodes a single 30-kDa multidomain protein, the namesake of the 30K superfamily (7). Viral RNA is associated with the membrane of the endoplasmic reticulum (ER) and microtubules in the presence of this MP (23, 30).A large number of plant viruses have 30K MPs, which share common abilities, including binding nucleic acids, localizing and increasing the size exclusion limit of plasmodesmata, and interacting with the ER membrane. A topological model has been proposed in which the TMV MP has two putative transmembrane (TM) helices, both the N and C termini oriented toward the cytoplasm, and a short loop exposed in the ER lumen (4). There is less experimental information for other 30K MPs, but they are likely to have some membrane interaction.Direct experimental evidence of the integration of MPs into the membrane has been obtained only for small hydrophobic MPs that do not belong to the 30K superfamily. There are two TM segments in the p9 protein of carnation mottle virus (41), whereas the p6 protein of beet yellow virus (29) and the p7B protein of melon necrotic spot virus (22) have a single TM segment. In viruses with genomes that include three partially overlapping open reading frames, termed the triple-gene block (TGB), all three TGB proteins are required for movement where the two smaller proteins, TGBp2 and TGBp3, are also TM proteins (24). Furthermore, cross-linking experiments with carnation mottle virus p9 protein demonstrated that its membrane insertion occurs cotranslationally in a signal recognition particle-dependent manner and throughout the cellular membrane integration components, the translocon (33, 34).Prunus necrotic ringspot virus (PNRSV) is a tripartite, positive-strand RNA virus in the genus Ilarvirus of the family Bromoviridae. RNAs 1 and 2 encode the polymerase proteins P1 and P2, respectively. RNA 3 is translated into a single 30K-type MP. The coat protein is translated from a subgenomic RNA 4 produced during virus replication.The present study tackled the association of the PNRSV MP with biological membranes. The in vitro translation of model integral membrane protein constructs in the presence of microsomal membranes demonstrated that the hydrophobic region (HR) of the PNRSV MP did not span the membranes. Different biochemical and biophysical experiments suggested that the protein is tightly associated with, but does not traverse, the membrane, leaving both its N- and C-terminal hydrophilic regions facing the cytosol. Finally, a mutational analysis of the HR revealed that both the helicity and hydrophobicity of the region are essential for viral cell-to-cell movement.     

The P30 movement protein of tobacco mosaic virus is a single-strand nucleic acid binding protein

The P30 protein of tobacco mosaic virus (TMV) is required for cell to cell movement of viral RNA, which presumably occurs through plant intercellular connections, the plasmodesmata. The mechanism by which P30 mediates transfer of TMV RNA molecules through plasmodesmata channels is unknown. We have identified P30 as an RNA and single-stranded (ss) DNA binding protein. Binding of purified P30 to ss nucleic acids is strong, highly cooperative, and sequence nonspecific with a minimal binding site of 4-7 nucleotides per P30 monomer. In-frame deletions across P30 were used to localize the ss nucleic acid binding domain to within amino acid residues 65-86 of the protein. We propose that binding of P30 to TMV RNA creates an unfolded protein-RNA complex that functions as an intermediate in virus cell to cell movement through plasmodesmata.     

葡萄扇叶病毒移动蛋白在寄主体内的动态检测和免疫金标记

利用葡萄扇叶病毒法国分离物F13(Grapenive fanleaf virus, GFLVF13)移动蛋白抗体对杭州分离物(GFLVH)移动蛋白进行Western blot,分析表明移动蛋白在接种GFLVH 3d后的苋色藜(Chenopodium amaranticolor)系统叶中就可检测到,随着时间推移,其积累量逐渐升高,接种16d后达到最高值。接种32d后的病叶已经枯黄,但移动蛋白积累量并没有减少。超薄切片电镜观察发现,在感染GFLVH的昆诺藜(C.quinoa)和苋色藜的叶肉组织薄壁细胞中,病毒粒子呈纵列整齐地排列在小管状结构中,在胞间连丝中也发现有管状结构。免疫金标记显示胶体金能定位在细胞质、细胞壁和胞间连丝上,在管状结构也发现有少量的金粒子。这些结果进一步说明了GFLV是通过管状结构实现细胞间移动的。     

Plant viruses spread by diffusion on ER-associated movement-protein-rafts through plasmodesmata gated by viral induced host β-1,3-glucanases

Plant viruses spread cell-to-cell by exploiting and modifying plasmodesmata, coaxial membranous channels that cross cell walls and interlink the cytoplasm, endoplasmic reticulum and plasma-membranes of contiguous cells. To facilitate viral spread, viruses encode for one or more movement proteins that interact with ER and ER derived membranes, bind vRNA and target to Pd. Mounting evidence suggests that RNA viruses that do not spread as virions employ the same basic mechanism to facilitate cell-to-cell spread. In light of the research reviewed here, we propose a general functional model for the cell-to-cell spread of these viruses. This model posits that MPs have multiple functions: one function involves directing virus induced β-1,3-glucanases which accumulate in ER derived vesicles to the cell wall to hydrolyze Pd associated callose in order to gate open the Pd; independently, the MPs form ER-associated protein rafts which transport bound vRNA by diffusion along ER to adjacent cells via the ER component of the plasmodesmata. The driving force for spread is the diffusion gradient between infected and non-infected adjacent cells.     

How do plant virus nucleic acids move through intercellular connections?

In addition to their function in transport of water, ions, small metabolites, and growth factors in normal plant tissue, the plasmodesmata presumably serve as routes for cell-to-cell movement of plant viruses in infected tissue. Virus cell-to-cell spread through plasmodesmata is an active process mediated by specialized virus encoded movement proteins; however, the mechanism by which these proteins operate is not clear. We incorporate recent information on the biochemical properties of plant virus movement proteins and their interaction with plasmodesmata in a model for transport of nucleic acids through plasmodesmatal channels. We propose that only single stranded (ss) nucleic acids can be transported efficiently through plasmodesmata, and that movement proteins function as molecular chaperones for ss nucleic acids to form unfolded movement protein-ss nucleic acid complexes. These complexes are targeted to plasmodesmata. Plasmodesmatal permeability is then increased following interaction with movement protein and the entire movement complex or its nucleic acid component is translocated across the plasmodesmatal channel.     

Analysis of deletion mutants indicates that the 2A polypeptide of hepatitis A virus participates in virion morphogenesis

Unlike all other picornaviruses, the primary cleavage of the hepatitis A virus (HAV) polyprotein occurs at the 2A/2B junction and is carried out by the only proteinase encoded by the virus, 3C(pro). The resulting P1-2A capsid protein precursor is subsequently cleaved by 3C(pro) to generate VP0, VP3, and VP1-2A, which associate as pentamers. An unidentified cellular proteinase acting at the VP1/2A junction releases the mature capsid protein VP1 from VP1-2A later in the morphogenesis process. Although these aspects of polyprotein processing are well characterized, the function of 2A is unknown. To study its role in the viral life cycle, we assessed the infectivity of synthetic, genome-length RNAs containing 11 different in-frame deletions in the 2A region. Deletions in the N-terminal 40% of 2A abolished infectivity, whereas deletions in the C-terminal 60% resulted in viruses with a small-focus replication phenotype. C-terminal deletions in 2A had no effect on RNA replication kinetics under one-step growth conditions, nor did they have an effect on capsid protein synthesis and 3C(pro)-mediated processing. However, C-terminal deletions in 2A altered the VP1/2A cleavage, resulting in accumulation of uncleaved VP1-2A precursor in virions and possibly accounting for a delay in the appearance of infectious particles with these mutants, as well as a fourfold decrease in specific infectivity of the virus particles. When the capsid proteins were expressed from recombinant vaccinia viruses, the N-terminal part of 2A was required for efficient cleavage of the P1-2A precursor by 3C(pro) and assembly of structural precursors into pentamers. These data indicate that the N-terminal domain of 2A must be present as a C-terminal extension of P1 for folding of the capsid protein precursor to allow efficient 3C(pro)-mediated cleavages and to promote pentamer assembly, after which cleavage at the VP1/2A junction releases the mature VP1 protein, a process that appears to be necessary to produce highly infectious particles.     

Insertion and topology of a plant viral movement protein in the endoplasmic reticulum membrane

Virus-encoded movement proteins (MPs) mediate cell-to-cell spread of viral RNA through plant membranous intercellular connections, the plasmodesmata. The molecular pathway by which MPs interact with viral genomes and target plasmodesmata channels is largely unknown. The 9-kDa MP from carnation mottle carmovirus (CarMV) contains two potential transmembrane domains. To explore the possibility that this protein is in fact an intrinsic membrane protein, we have investigated its insertion into the endoplasmic reticulum membrane. By using in vitro translation in the presence of dog pancreas microsomes, we demonstrate that CarMV p9 inserts into the endoplasmic reticulum without the aid of any additional viral or plant host components. We further show that the membrane topology of CarMV p9 is N(cyt)-C(cyt) (N and C termini of the protein facing the cytoplasm) by in vitro translation of a series of truncated and full-length constructs with engineered glycosylation sites. Based on these results, we propose a topological model in which CarMV p9 is anchored in the membrane with its N- and C-terminal tail segments interacting with its soluble, RNA-bound partner CarMV p7, to accomplish the viral cell-to-cell movement function.     

Domains of the TMV movement protein involved in subcellular localization

To identify and map functionally important regions of the tobacco mosaic virus movement protein, deletions of three amino acids were introduced at intervals of 10 amino acids throughout the protein. Mutations located between amino acids 1 and 160 abolished the capacity of the protein to transport virus from cell to cell, while some of the mutations in the C-terminal third of the protein permitted function. Despite extensive tests, no examples were found of intermolecular complementation between mutants, suggesting that function requires each movement protein molecule to be fully competent. Many of the mutants were fused to green fluorescent protein, and their subcellular localizations were determined by fluorescence microscopy in infected plants and protoplasts. Most mutants lost the ability to accumulate in one or more of the multiple subcellular sites targeted by wild-type movement protein, suggesting that specific functional domains were disrupted. The order in which accumulation at subcellular sites occurs during infection does not represent a targeting pathway. Association of the movement protein with microtubules or with plasmodesmata can occur in the absence of other associations. The region of the protein around amino acids 9–11 may be involved in targeting the protein to cortical bodies (probably associated with the endoplasmic reticulum) and to plasmodesmata. The region around residues 49–51 may be involved in co-alignment of the protein with microtubules. The region around residues 88–101 appears to play a role in targeting to both the cortical bodies and microtubules. Thus, the movement protein contains independently functional domains.     

Phosphorylation down-regulates the RNA binding function of the coat protein of potato virus A

Plant viruses encode movement proteins (MPs) to facilitate transport of their genomes from infected into neighboring healthy cells through plasmodesmata. Growing evidence suggests that specific phosphorylation events can regulate MP functions. The coat protein (CP) of potato virus A (PVA; genus Potyvirus) is a multifunctional protein involved both in virion assembly and virus movement. Labeling of PVA-infected tobacco leaves with [(33)P]orthophosphate demonstrated that PVA CP is phosphorylated in vivo. Competition assays established that PVA CP and the well characterized 30-kDa MP of tobacco mosaic virus (genus Tobamovirus) are phosphorylated in vitro by the same Ser/Thr kinase activity from tobacco leaves. This activity exhibits a strong preference for Mn(2+) over Mg(2+), can be inhibited by micromolar concentrations of Zn(2+) and Cd(2+), and is not Ca(2+)-dependent. Tryptic phosphopeptide mapping revealed that PVA CP was phosphorylated by this protein kinase activity on multiple sites. In contrast, PVA CP was not phosphorylated when packaged into virions, suggesting that the phosphorylation sites are located within the RNA binding domain and not exposed on the surface of the virion. Furthermore, two independent experimental approaches demonstrated that the RNA binding function of PVA CP is strongly inhibited by phosphorylation. From these findings, we suggest that protein phosphorylation represents a possible mechanism regulating formation and/or stability of viral ribonucleoproteins in planta.