Class VIII myosins are required for plasmodesmatal localization of a closterovirus Hsp70 homolog

The Hsp70 homolog (Hsp70h) of Beet yellows virus (BYV) functions in virion assembly and cell-to-cell movement and is autonomously targeted to plasmodesmata in association with the actomyosin motility system (A. I. Prokhnevsky, V. V. Peremyslov, and V. V. Dolja, J. Virol. 79:14421-14428, 2005). Myosins are a diverse category of molecular motors that possess a motor domain and a tail domain involved in cargo binding. Plants have two classes of myosins, VIII and XI, whose specific functions are poorly understood. We used dominant negative inhibition to identify myosins required for Hsp70h localization to plasmodesmata. Six full-length myosin cDNAs from the BYV host plant Nicotiana benthamiana were sequenced and shown to encode apparent orthologs of the Arabidopsis thaliana myosins VIII-1, VIII-2, VIII-B, XI-2, XI-F, and XI-K. We found that the ectopic expression of the tail domains of each of the class VIII, but not the class XI, myosins inhibited the plasmodesmatal localization of Hsp70h. In contrast, the overexpression of the motor domains or the entire molecules of the class VIII myosins did not affect Hsp70h targeting. Further mapping revealed that the minimal cargo-binding part of the myosin VIII tails was both essential and sufficient for the inhibition of the proper Hsp70h localization. Interestingly, plasmodesmatal localization of the Tobacco mosaic virus movement protein and Arabidopsis protein RGP2 was not affected by myosin VIII tail overexpression. Collectively, our data implicate class VIII myosins in protein delivery to plasmodesmata and suggest that more than one mechanism of such delivery exist in plants.     

Covering common ground: F-actin-dependent transport of plant viral protein inclusions reveals a novel mechanism for movement utilized by unrelated viral proteins

Plant viruses are composed of diverse genomes (e.g., RNA or DNA) encoding proteins that vary widely in sequence. It is becoming clear, however, that some apparently unrelated viral proteins have similar functions. The P6 protein encoded by Cauliflower mosaic virus (CaMV) and the 126-kDa protein encoded by Tobacco mosaic virus (TMV) are examples of this convergence in protein function. Although having no apparent sequence similarity, both proteins are pathogenicity determinants during infection, are components of novel intracellular cytoplasmic inclusions and suppress RNA silencing. Here we review our recent results demonstrating an additional novel convergent activity between these proteins: both proteins traffic along the actin cytoskeleton (microfilaments). We also discuss results showing a unique property of the P6 protein: a non-mobile strong association with microtubules. Lastly, we discuss the potential mechanism by which the P6 and 126-kDa proteins traffic along microfilaments. We provide new results suggesting that actin filament polymerization-driven movement does not support 126-kDa protein transport, thus leading to a focus on myosins as the driving force for this movement.Key words: actin polymerization, cytoskeleton, cauliflower mosaic virus, microfilaments, microtubules, myosin, tobacco mosaic virus, virus movement, intracellular transport     

Tobacco mosaic virus movement protein associates with the cytoskeleton in tobacco cells.

Tobacco mosaic virus movement protein P30 complexes with genomic viral RNA for transport through plasmodesmata, the plant intercellular connections. Although most research with P30 focuses on its targeting to and gating of plasmodesmata, the mechanisms of P30 intracellular movement to plasmodesmata have not been defined. To examine P30 intracellular localization, we used tobacco protoplasts, which lack plasmodesmata, for transfection with plasmids carrying P30 coding sequences under a constitutive promoter and for infection with tobacco mosaic virus particles. In both systems, P30 appears as filaments that colocalize primarily with microtubules. To a lesser extent, P30 filaments colocalize with actin filaments, and in vitro experiments suggested that P30 can bind directly to actin and tubulin. This association of P30 with cytoskeletal elements may play a critical role in intracellular transport of the P30-viral RNA complex through the cytoplasm to and possibly through plasmodesmata.     

Interaction between long-distance transport factor and Hsp70-related movement protein of Beet yellows virus

Systemic spread of viruses in plants involves local movement from cell to cell and long-distance transport through the vascular system. The cell-to-cell movement of the Beet yellows virus (BYV) is mediated by a movement protein that is an Hsp70 homolog (Hsp70h). This protein is required for the assembly of movement-competent virions that incorporate Hsp70h. By using the yeast two-hybrid system, in vitro coimmunoprecipitation, and in planta coexpression approaches, we show here that the Hsp70h interacts with a 20-kDa BYV protein (p20). We further demonstrate that p20 is associated with the virions presumably via binding to Hsp70h. Genetic and immunochemical analyses indicate that p20 is dispensable for assembly and cell-to-cell movement of BYV but is required for the long-distance transport of virus through the phloem. These results reveal a novel activity for the Hsp70h that provides a molecular link between the local and systemic spread of a plant virus by docking a long-distance transport factor to virions.     

Distribution,phosphorylation, and activities of Hsp25 in heat-stressed H9c2 myoblasts: a functional link to cytoprotection

The behavior of the endogenous heat shock protein 25 (Hsp25) in heat-stressed rat H9c2 myoblasts was studied. After mild or severe heating, this protein became less extractable with Triton X-100 and displayed characteristic immunofluorescence patterns, namely (1) granules in the nucleus, and (2) association with F-actin bundles in the cytoplasm. The intranuclear granulation of Hsp25 and its association with F-actin were sensitive to drugs affecting Hsp25 phosphorylation (cantharidin, sodium orthovanadate, SB203580, SB202190). Isoform analysis of Hsp25 translocated to the nucleus-free cytoskeletal fraction revealed only mono- and biphosphorylated Hsp25 and no unphosphorylated Hsp25. Transfected luciferase with initial localization in the nucleosol became colocalized with the Hsp25-containing granules after a heat shock treatment that denatured the enzyme in the cells. The association of Hsp25 with actin filaments after a mild heat stress conferred protection from subsequent F-actin-damaging treatments with cytochalasins (D and B) or severe heat stress. We hypothesize that (1) the binding of heat-denatured nucleosolic proteins to the Hsp25 contained in specific granular structures may serve for the subsequent chaperoning or degradation of the bound proteins, and (2) the actin cytoskeleton is stabilized by the direct targeting of phosphorylated Hsp25 to microfilament bundles.     

The role of Hsp27 and actin in the regulation of movement in human cancer cells responding to heat shock

Human heat shock 27-kDa protein 1 (HSPB1)/heat shock protein (Hsp) 27 is a small heat shock protein which is thought to have several roles within the cell. One of these roles includes regulating actin filament dynamics in cell movement, since Hsp27 has previously been found to inhibit actin polymerization in vitro. In this study, the role of Hsp27 in regulating actin filament dynamics is further investigated. Hsp27 protein levels were reduced using siRNA in SW480 cells, a human colon cancer cell line. An in vitro wound closure assay showed that cells with knocked down Hsp27 levels were unable to close wounds, indicating that this protein is involved in regulating cell motility. Immunoprecipitation pull down assays were done, to observe if and when Hsp27 and actin are in the same complex within the cell, before and after heat shock. At all time points tested, Hsp27 and actin were present in the same cell lysate fraction. Lastly, indirect immunostaining was done before and after heat shock to evaluate Hsp27 and actin interaction in cells. Hsp27 and actin showed colocalization before heat shock, little association 3 h after heat shock, and increased association 24 h after heat shock. Cytoprotection was observed as early as 3 h after heat shock, yet cells were still able to move. These results show that Hsp27 and actin are in the same complex in cells and that Hsp27 is important for cell motility. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The 5′ Cap of Tobacco Mosaic Virus (TMV) Is Required for Virion Attachment to the Actin/Endoplasmic Reticulum Network During Early Infection

Almost nothing is known of the earliest stages of plant virus infections. To address this, we microinjected Cy3 (UTP)‐labelled tobacco mosaic virus (TMV) into living tobacco trichome cells. The Cy3‐virions were infectious, and the viral genome trafficked from cell to cell. However, neither the fluorescent vRNA pool nor the co‐injected green fluorescent protein (GFP) left the injected trichome, indicating that the synthesis of (unlabelled) progeny viral (v)RNA is required to initiate cell‐to‐cell movement, and that virus movement is not accompanied by passive plasmodesmatal gating. Cy3‐vRNA formed granules that became anchored to the motile cortical actin/endoplasmic reticulum (ER) network within minutes of injection. Granule movement on actin/ER was arrested by actin inhibitors indicating actin‐dependent RNA movement. The 5′ methylguanosine cap was shown to be required for vRNA anchoring to the actin/ER. TMV vRNA lacking the 5′ cap failed to form granules and was degraded in the cytoplasm. Removal of the 3′ untranslated region or replicase both inhibited replication but did not prevent granule formation and movement. Dual‐labelled TMV virions in which the vRNA and the coat protein were highlighted with different fluorophores showed that both fluorescent signals were initially located on the same ER‐bound granules, indicating that TMV virions may become attached to the ER prior to uncoating of the viral genome.     

Influence of cytoplasmic heat shock protein 70 on viral infection of Nicotiana benthamiana

The accumulation of heat shock protein 70 (Hsp70) generally occurs in plants infected with viruses. However, the effect of Hsp70 accumulation on plant viral infection and pathogenesis remains elusive. In this study, the expression of six Hsp70 genes was found to be induced by the four diverse RNA viruses, Tobacco mosaic virus, Potato virus X (PVX), Cucumber mosaic virus and Watermelon mosaic virus, in Nicotiana benthamiana. Heat treatment enhanced the accumulation and systemic infection of these viruses. Similar results were obtained for viral infection in plants heterologously expressing an Arabidopsis cytoplasmic Hsp70 through either a PVX vector or Agrobacterium infiltration. In contrast, viral infection was compromised in cytoplasmic NbHsp70c‐1 gene‐silenced plants. These data demonstrate that the cytoplasmic Hsp70s can enhance the infection of N. benthamiana by diverse viruses.     

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.     

Validation of microtubule-associated Tobacco mosaic virus RNA movement and involvement of microtubule-aligned particle trafficking

Functional studies of Tobacco mosaic virus (TMV) infection using virus derivatives expressing functional, dysfunctional, and temperature-sensitive movement protein (MP) mutants indicated that the cell-to-cell transport of TMV RNA is functionally correlated with the association of MP with microtubules. However, the role of microtubules in the movement process during early infection remains unclear, since MP accumulates on microtubules rather late in infection and treatment of plants with microtubule-disrupting agents fails to strongly interfere with cell-to-cell movement of TMV RNA. To further test the role of microtubules in TMV cell-to-cell movement, we investigated TMV strain Ni2519, which is temperature-sensitive for movement. We demonstrate that the temperature-sensitive defect in movement is correlated with temperature-sensitive changes in the localization of MP to microtubules. Furthermore, we show that during early phases of recovery from non-permissive conditions, the MP localizes to microtubule-associated particles. Similar particles are found in cells at the leading front of spreading TMV infection sites. Initially mobile, the particles become immobile when MP starts to accumulate along the length of the particle-associated microtubules. Our observations confirm a role for microtubules in the spread of TMV infection and associate this role with microtubule-associated trafficking of MP-containing particles in cells engaged in the cell-to-cell movement of the TMV genome.     

Salicylic acid has cell-specific effects on tobacco mosaic virus replication and cell-to-cell movement

Tobacco mosaic virus (TMV) and Cucumber mosaic virus expressing green fluorescent protein (GFP) were used to probe the effects of salicylic acid (SA) on the cell biology of viral infection. Treatment of tobacco with SA restricted TMV.GFP to single-epidermal cell infection sites for at least 6 d post inoculation but did not affect infection sites of Cucumber mosaic virus expressing GFP. Microinjection experiments, using size-specific dextrans, showed that SA cannot inhibit TMV movement by decreasing the plasmodesmatal size exclusion limit. In SA-treated transgenic plants expressing TMV movement protein, TMV.GFP infection sites were larger, but they still consisted overwhelmingly of epidermal cells. TMV replication was strongly inhibited in mesophyll protoplasts isolated from SA-treated nontransgenic tobacco plants. Therefore, it appears that SA has distinct cell type-specific effects on virus replication and movement in the mesophyll and epidermal cell layers, respectively. Thus, SA can have fundamentally different effects on the same pathogen in different cell types.     

The actin cytoskeleton is involved in the regulation of the plasmodesmal size exclusion limit

Plasmodesmata (PD) are the communication channels which allow the trafficking of macromolecules between neighboring cells. Such cell-to-cell movement of macromolecules is regulated during plant growth and development; however, little is known about the regulation mechanism of PD size exclusion limit (SEL). Plant viral movement proteins (MPs) enhance the invasion of viruses from cell to cell by increasing the SEL of the PD and are therefore a powerful means for the study of the plasmodesmal regulation mechanisms. In a recent study, we reported that the actin cytoskeleton is involved in the increase of the PD SEL induced by MPs. Microinjection experiments demonstrated that actin depolymerization was required for the Cucumber mosaic virus (CMV) MP-induced increase in the PD SEL. In vitro experiments showed that CMV MP severs actin filaments (F-actin). Furthermore, through the analyses of two CMV MP mutants, we demonstrated that the F-actin severing ability of CMV MP was required to increase the PD SEL. These results are similar to what has been found in Tobacco mosaic virus MP. Thus, our data suggest that actin dynamics may participate in the regulations of the PD SEL.Key words: plasmodesmata, size exclusion limit, movement protein, actin filaments, F-actin severing     

Tobamovirus replicase coding region is involved in cell-to-cell movement

Tobacco mosaic virus (TMV) encodes a 30-kDa movement protein (MP) which enables viral movement from cell to cell. It is, however, unclear whether the 126- and 183-kDa replicase proteins are involved in the cell-to-cell movement of TMV. In the course of our studies into TMV-R, a strain with a host range different from that of TMV-U1, we have obtained an interesting chimeric virus, UR-hel. The amino acid sequence differences between UR-hel and TMV-U1 are located only in the helicase-like domain of the replicase. Interestingly, UR-hel has a defect in its cell-to-cell movement. The replication of UR-hel showed a level of replication of the genome, synthesis, and accumulation of MP similar to that observed in TMV-U1-inoculated protoplasts. Such observations support the hypothesis that the replicase coding region may in some fashion be involved in cell-to-cell movement of TMV.     

Hsp70 negatively controls rotavirus protein bioavailability in caco-2 cells infected by the rotavirus RF strain

Previous studies demonstrated that the induction of the heat shock protein Hsp70 in response to viral infection is highly specific and differs from one cell to another and for a given virus type. However, no clear consensus exists so far to explain the likely reasons for Hsp70 induction within host cells during viral infection. We show here that upon rotavirus infection of intestinal cells, Hsp70 is indeed rapidly, specifically, and transiently induced. Using small interfering RNA-Hsp70-transfected Caco-2 cells, we observed that Hsp70 silencing was associated with an increased virus protein level and enhanced progeny virus production. Upon Hsp70 silencing, we observed that the ubiquitination of the main rotavirus structural proteins was strongly reduced. In addition, the use of proteasome inhibitors in infected Caco-2 cells was shown to induce an accumulation of structural viral proteins. Together, these results are consistent with a role of Hsp70 in the control of the bioavailability of viral proteins within cells for virus morphogenesis.     

Chaperone-mediated in vitro disassembly of polyoma- and papillomaviruses

Hsp70 chaperones play a role in polyoma- and papillomavirus assembly, as evidenced by their interaction in vivo with polyomavirus capsid proteins at late times after virus infection and by their ability to assemble viral capsomeres into capsids in vitro. We studied whether Hsp70 chaperones might also participate in the uncoating reaction. In vivo, Hsp70 co-immunoprecipitated with polyomavirus virion VP1 at 3 h after infection of mouse cells. In vitro, prokaryotic and eukaryotic Hsp70 chaperones efficiently disassembled polyoma- and papillomavirus-like particles and virions in energy-dependent reactions. These observations support a role for cell chaperones in the disassembly of these viruses.     

Interaction of the host protein NbDnaJ with Potato virus X minus-strand stem-loop 1 RNA and capsid protein affects viral replication and movement

Plant viruses must interact with host cellular components to replicate and move from cell to cell. In the case of Potato virus X (PVX), it carries stem-loop 1 (SL1) RNA essential for viral replication and movement. Using two-dimensional electrophoresis northwestern blot analysis, we previously identified several host proteins that bind to SL1 RNA. Of those, we further characterized a DnaJ-like protein from Nicotiana benthamiana named NbDnaJ. An electrophoretic mobility shift assay confirmed that NbDnaJ binds only to SL1 minus-strand RNA, and bimolecular fluorescence complementation (BiFC) indicated that NbDnaJ interacts with PVX capsid protein (CP). Using a series of deletion mutants, the C-terminal region of NbDnaJ was found to be essential for the interaction with PVX CP. The expression of NbDnaJ significantly changed upon infection with different plant viruses such as PVX, Tobacco mosaic virus, and Cucumber mosaic virus, but varied depending on the viral species. In transient experiments, both PVX replication and movement were inhibited in plants that over-expressed NbDnaJ but accelerated in plants in which NbDnaJ was silenced. In summary, we suggest that the newly identified NbDnaJ plays a role in PVX replication and movement by interacting with SL1(-) RNA and PVX CP.