Actin cytoskeleton is involved in targeting of a viral Hsp70 homolog to the cell periphery

The cell-to-cell movement of plant viruses involves translocation of virus particles or nucleoproteins to and through the plasmodesmata (PDs). As we have shown previously, the movement of the Beet yellows virus requires the concerted action of five viral proteins including a homolog of cellular approximately 70-kDa heat shock proteins (Hsp70h). Hsp70h is an integral component of the virus particles and is also found in PDs of the infected cells. Here we investigate subcellular distribution of Hsp70h using transient expression of Hsp70h fused to three spectrally distinct fluorescent proteins. We found that fluorophore-tagged Hsp70h forms motile granules that are associated with actin microfilaments, but not with microtubules. In addition, immobile granules were observed at the cell periphery. A pairwise appearance of these granules at the opposite sides of cell walls and their colocalization with the movement protein of Tobacco mosaic virus indicated an association of Hsp70h with PDs. Treatment with various cytoskeleton-specific drugs revealed that the intact actomyosin motility system is required for trafficking of Hsp70h in cytosol and its targeting to PDs. In contrast, none of the drugs interfered with the PD localization of Tobacco mosaic virus movement protein. Collectively, these findings suggest that Hsp70h is translocated and anchored to PDs in association with the actin cytoskeleton.     

The Cauliflower Mosaic Virus Protein P6 Forms Motile Inclusions That Traffic along Actin Microfilaments and Stabilize Microtubules

The gene VI product (P6) of Cauliflower mosaic virus (CaMV) is a multifunctional protein known to be a major component of cytoplasmic inclusion bodies formed during CaMV infection. Although these inclusions are known to contain virions and are thought to be sites of translation from the CaMV 35S polycistronic RNA intermediate, the precise role of these bodies in the CaMV infection cycle remains unclear. Here, we examine the functionality and intracellular location of a fusion between P6 and GFP (P6-GFP). We initially show that the ability of P6-GFP to transactivate translation is comparable to unmodified P6. Consequently, our work has direct application for the large body of literature in which P6 has been expressed ectopically and its functions characterized. We subsequently found that P6-GFP forms highly motile cytoplasmic inclusion bodies and revealed through fluorescence colocalization studies that these P6-GFP bodies associate with the actin/endoplasmic reticulum network as well as microtubules. We demonstrate that while P6-GFP inclusions traffic along microfilaments, those associated with microtubules appear stationary. Additionally, inhibitor studies reveal that the intracellular movement of P6-GFP inclusions is sensitive to the actin inhibitor, latrunculin B, which also inhibits the formation of local lesions by CaMV in Nicotiana edwardsonii leaves. The motility of P6 along microfilaments represents an entirely new property for this protein, and these results imply a role for P6 in intracellular and cell-to-cell movement of CaMV.Cauliflower mosaic virus (CaMV), the type member of the genus Caulimovirus, has a circular double-stranded DNA genome known to encode six open reading frames (ORFs). The gene product of ORF VI (P6) is a multifunctional protein whose ascribed functions have increased in number since its initial characterization over 20 years ago. P6 was originally described as the most abundant CaMV protein in infected plants (Odell and Howell, 1980) and was later shown to be the major constituent of amorphous, electron-dense inclusion bodies that are thought to be the sites of virion assembly (Fujisawa et al., 1967; Rubio-Huertos et al., 1968; Himmelbach et al., 1996; Cecchini et al., 1997). Indeed, despite the detection of other viral proteins in CaMV inclusions, the P6 protein on its own is capable of forming inclusion bodies (Cecchini et al., 1997; Li and Leisner, 2002; Haas et al., 2005).P6 is the major pathogenicity determinant for CaMV (Daubert et al., 1984; Baughman et al., 1988; Stratford and Covey, 1989; Zijlstra and Hohn, 1992) and was recently shown to be a suppressor of RNA silencing (Love et al., 2007). In addition, P6 also functions as an avirulence determinant, as it has been shown to be responsible for eliciting a hypersensitive response in Nicotiana edwardsonii and Datura stramonium, as well as nonnecrotic resistance in Nicotiana bigelovii and Arabidopsis (Arabidopsis thaliana) ectotype Tsu-O (Daubert et al., 1984; Schoelz et al., 1986; Wintermantel et al., 1993; Agama et al., 2002). The portion of the P6 protein recognized by plants is localized to the N-terminal third of the protein (Wintermantel et al., 1993; Palanichelvam et al., 2000; Agama et al., 2002). P6 also has a significant effect on plant metabolism, as it is responsible for down-regulating or inducing expression of several plant genes (Geri et al., 1999), including genes involved in ethylene signaling (Geri et al., 2004).Replication of CaMV involves the production of a polycistronic RNA intermediate, the 35S RNA, and P6 acts as a translational transactivator (TAV) by modifying the host translational machinery to allow for reinitiation of translation on this RNA (Ryabova et al., 2002). To carry out this function, the P6 protein physically interacts with the initiation factor eIF3 (Park et al., 2001), as well as ribosomal proteins L13 (Bureau et al., 2004), L18 (Leh et al., 2000), and L24 (Park et al., 2001). Finally, P6 is also a nucleocytoplasmic shuttle protein whose nuclear export is dependent upon a Leu-rich sequence near its N terminus, a region that is also involved in inclusion body formation (Li and Leisner, 2002; Haas et al., 2005). Although the precise role of the P6 protein''s nucleocytoplasmic shuttle function during infection remains to be elucidated, P6 does have the capacity to bind RNA (De Tapia et al., 1993; Cerritelli et al., 1998) and as such may act to control export of the 35S RNA from the nucleus to the cytoplasm, drawing the 35S RNA into the nascent P6 inclusion bodies where viral proteins are translated.Despite the recognized intracellular movement of P6 from cytoplasm to nucleus and the disparate cytoplasmic functions of this protein, factors controlling intracellular transport of P6 remain unknown. The cytoskeleton has been implicated in the intracellular trafficking of a number of plant viral proteins. For example, proteins encoded by several viruses have been found to colocalize with actin microfilaments, including the TGBp2 movement protein from Potato virus X (PVX), TGBp2 and TGBp3 from Potato mop-top virus, the Hsp70 homolog from Beet yellows virus, as well as both the movement (MP) and 126-kD proteins from Tobacco mosaic virus (TMV; McLean et al., 1995; Haupt et al., 2005; Ju et al., 2005; Liu et al., 2005; Prokhnevsky et al., 2005) In addition, inhibitor studies recently demonstrated that the intracellular trafficking of potato leafroll virus MP to the plasmodesmata (PD) is dependent upon an intact actin cytoskeleton (Vogel et al., 2007). Together, these studies suggest that the trafficking of viral proteins along actin filaments is a mechanism utilized by highly divergent RNA viruses.The only documented example of a plant viral protein found to colocalize with both microfilaments and microtubules in cells is the TMV MP (McLean et al., 1995; reviewed in Beachy and Heinlein, 2000; Lucas, 2006), which has been shown to associate with and stabilize microtubules and contains a motif thought to mimic the region of tubulin responsible for lateral junctions between microtubules (Boyko et al., 2000; Ashby et al., 2006). Interestingly, the CaMV gene II product (P2), an aphid transmission factor, was previously shown by immunoelectron microscopy to associate with microtubules in both insect and plant cells, although the significance of this interaction remains unclear (Blanc et al., 1996). In addition to these two viral proteins found to colocalize with microtubules in planta, the Hsp70 homolog from Beet yellows virus and the coat protein from PVX have both been shown to interact with microtubules in vitro (Karasev et al., 1992; Serazev et al., 2003). Evidence that the intracellular localization of grapevine fanleaf virus MP is disturbed by oryzalin, as well as the finding that the geminivirus replication protein AL1 interacts with a kinesin by yeast two-hybrid assay, may also indicate a potential association of these proteins with microtubules (Kong and Hanley-Bowdoin, 2002; Laporte et al., 2003).In this study, we utilize a fusion between the C terminus of P6 and GFP to visualize P6 inclusions in live cells. We demonstrate that the fusion of P6 with GFP does not interfere with its ability to act as a TAV. We further demonstrate that P6-GFP inclusion bodies move intracellularly and are associated with microtubules, actin microfilaments, and the endoplasmic reticulum (ER). Although P6-GFP inclusion bodies associated with microtubules appear stationary, we show that P6-GFP bodies can traffic along microfilaments and that this movement is severely reduced by treatment with the actin inhibitor latrunculin B (LatB). LatB treatment of N. edwardsonii leaves inhibits the formation of local lesions by CaMV, indicating the potential that P6 trafficking on microfilaments is necessary for CaMV cell-to-cell movement. Additionally, the association of P6-GFP inclusion bodies with microtubules prevents the disruption of microtubules by oryzalin, denoting a tight association between these two proteins. We discuss the potential role of P6 movement and cytoskeletal association in CaMV infection.     

The tobacco mosaic virus 126-kilodalton protein, a constituent of the virus replication complex, alone or within the complex aligns with and traffics along microfilaments

Virus-induced cytoplasmic inclusion bodies (referred to as virus replication complexes [VRCs]) consisting of virus and host components are observed in plant cells infected with tobacco mosaic virus, but the components that modulate their form and function are not fully understood. Here, we show that the tobacco mosaic virus 126-kD protein fused with green fluorescent protein formed cytoplasmic bodies (126-bodies) in the absence of other viral components. Using mutant 126-kD:green fluorescent fusion proteins and viral constructs expressing the corresponding mutant 126-kD proteins, it was determined that the size of the 126-bodies and the corresponding VRCs changed in synchrony for each 126-kD protein mutation tested. Through colabeling experiments, we observed the coalignment and intracellular trafficking of 126-bodies and, regardless of size, VRCs, along microfilaments (MFs). Disruption of MFs with MF-depolymerizing agents or through virus-induced gene silencing compromised the intracellular trafficking of the 126-bodies and VRCs and virus cell-to-cell movement, but did not decrease virus accumulation to levels that would affect virus movement or prevent VRC formation. Our results indicate that (1) the 126-kD protein modulates VRC size and traffics along MFs in cells; (2) VRCs traffic along MFs in cells, possibly through an interaction with the 126-kD protein, and the negative effect of MF antagonists on 126-body and VRC intracellular movement and virus cell-to-cell movement correlates with the disruption of this association; and (3) virus movement was not correlated with VRC size.     

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.     

Inhibition of Tobacco Mosaic Virus Movement by Expression of an Actin-Binding Protein

The tobacco mosaic virus (TMV) movement protein (MP) required for the cell-to-cell spread of viral RNA interacts with the endoplasmic reticulum (ER) as well as with the cytoskeleton during infection. Whereas associations of MP with ER and microtubules have been intensely investigated, research on the role of actin has been rather scarce. We demonstrate that Nicotiana benthamiana plants transgenic for the actin-binding domain 2 of Arabidopsis (Arabidopsis thaliana) fimbrin (AtFIM1) fused to green fluorescent protein (ABD2:GFP) exhibit a dynamic ABD2:GFP-labeled actin cytoskeleton and myosin-dependent Golgi trafficking. These plants also support the movement of TMV. In contrast, both myosin-dependent Golgi trafficking and TMV movement are dominantly inhibited when ABD2:GFP is expressed transiently. Inhibition is mediated through binding of ABD2:GFP to actin filaments, since TMV movement is restored upon disruption of the ABD2:GFP-labeled actin network with latrunculin B. Latrunculin B shows no significant effect on the spread of TMV infection in either wild-type plants or ABD2:GFP transgenic plants under our treatment conditions. We did not observe any binding of MP along the length of actin filaments. Collectively, these observations demonstrate that TMV movement does not require an intact actomyosin system. Nevertheless, actin-binding proteins appear to have the potential to exert control over TMV movement through the inhibition of myosin-associated protein trafficking along the ER membrane.     

Subcellular localization and detection of <Emphasis Type="Italic">Tobacco mosaic virus</Emphasis> ORF6 protein by immunoelectron microscopy

Members of the genus Tobamovirus represent one of the best-characterized groups of plant positive, single stranded RNA viruses. Previous studies have shown that genomes of some tobamoviruses contain not only genes coding for coat protein, movement protein, and the cistron coding for different domains of RNA-polymerase, but also a gene, named ORF6, coding for a poorly conserved small protein. The amino acid sequences of ORF6 proteins encoded by different tobamoviruses are highly divergent. The potential role of ORF6 proteins in replication of tobamoviruses still needs to be elucidated. In this study, using biochemical and immunological methods, we have shown that ORF6 peptide is accumulated after infection in case of two isolates of Tobacco mosaic virus strain U1 (TMV-U1 common and TMV-U1 isolate A15). Unlike virus particles accumulating in the cytoplasm, the product of the ORF6 gene is found mainly in nuclei, which correlates with previously published data about transient expression of ORF6 isolated from TMV-U1. Moreover, we present new data showing the presence of ORF6 genes in genomes of several tobamoviruses. For example, in the genomes of other members of the tobamovirus subgroup 1, including Rehmannia mosaic virus, Paprika mild mottle virus, Tobacco mild green mosaic virus, Tomato mosaic virus, Tomato mottle mosaic virus, and Nigerian tobacco latent virus, sequence comparisons revealed the existence of a similar open reading frame like ORF6 of TMV.     

Patellins 3 and 6, two members of the Plant Patellin family,interact with the movement protein of Alfalfa mosaic virus and interfere with viral movement

Movement proteins (MPs) encoded by plant viruses interact with host proteins to facilitate or interfere with intra‐ and/or intercellular viral movement. Using yeast two‐hybrid and bimolecular fluorescence complementation assays, we herein present in vivo evidence for the interaction between Alfalfa mosaic virus (AMV) MP and Arabidopsis Patellin 3 (atPATL3) and Patellin 6 (atPATL6), two proteins containing a Sec14 domain. Proteins with Sec14 domains are implicated in membrane trafficking, cytoskeleton dynamics, lipid metabolism and lipid‐mediated regulatory functions. Interestingly, the overexpression of atPATL3 and/or atPATL6 interfered with the plasmodesmata targeting of AMV MP and correlated with reduced infection foci size. Consistently, the viral RNA levels increased in the single and double Arabidopsis knockout mutants for atPATL3 and atPATL6. Our results indicate that, in general, MP–PATL interactions interfere with the correct subcellular targeting of MP, thus rendering the intracellular transport of viral MP‐containing complexes less efficient and diminishing cell‐to‐cell movement.     

Homology between the proteins encoded by tobacco mosaic virus and two tricornaviruses

Summary A comparison was made of the amino acid sequences of the proteins encoded by RNAs 1 and 2 of alfalfa mosaic virus (A1MV) and brome mosaic virus (BMV), and the 126K and 183K proteins encoded by tobacco mosaic virus (TMV). Three blocks of extensive homology of about 200 to 350 amino acids each were observed. Two of these blocks are located in the A1MV and BMV RNA 1 encoded proteins and the TMV encoded 126K protein; they are situated at the N-terminus and C-terminus, respectively. The third block is located in the A1MV and BMV RNA 2 encoded proteins and the C-terminal part of the TMV encoded 183K protein. These homologies are discussed with respect to the functional equivalence of these putative replicase proteins and a possible evolutionary connection between A1MV, BMV and TMV.     

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     

Cell-to-cell movement of three genera (+) ss RNA plant viruses

The current investigations of three genera plant virus cell-to-cell movement were presented. Viruses reveal different local transport strategies, but all of them are the results of virus factors–host components interactions. The Tobacco mosaic virus (TMV) does not require capsid protein for translocation through plasmodesmata but 30 K movement protein participates in this process. It was found direct or indirect TMV movement proteins host partners in Tobamovirus movement like: pectin methylesterase, movement protein binding 2C, chaperones or cytoskeleton components and endoplasmatic reticulum membranes. The Potex- and Potyvirus cell-to-cell movement is closely related to replication network. The PVX capsid protein and triple gene block protein system are responsible for efficient local transport. Potyviruses move through the plasmodesmata by involving viral encoded proteins but not specific movement proteins. While the Potyvirus is the biggest known plant virus genus, host components participating in or regulating directly its plasmodesmata-movement are still not clear.     

Replication of tobacco mosaic virus RNA

The replication of tobacco mosaic virus (TMV) RNA involves synthesis of a negative-strand RNA using the genomic positive-strand RNA as a template, followed by the synthesis of positive-strand RNA on the negative-strand RNA templates. Intermediates of replication isolated from infected cells include completely double-stranded RNA (replicative form) and partly double-stranded and partly single-stranded RNA (replicative intermediate), but it is not known whether these structures are double-stranded or largely single-stranded in vivo. The synthesis of negative strands ceases before that of positive strands, and positive and negative strands may be synthesized by two different polymerases. The genomic-length negative strand also serves as a template for the synthesis of subgenomic mRNAs for the virus movement and coat proteins. Both the virus-encoded 126-kDa protein, which has amino-acid sequence motifs typical of methyltransferases and helicases, and the 183-kDa protein, which has additional motifs characteristic of RNA-dependent RNA polymerases, are required for efficient TMV RNA replication. Purified TMV RNA polymerase also contains a host protein serologically related to the RNA-binding subunit of the yeast translational initiation factor, eIF3. Study of Arabidopsis mutants defective in RNA replication indicates that at least two host proteins are needed for TMV RNA replication. The tomato resistance gene Tm-1 may also encode a mutant form of a host protein component of the TMV replicase. TMV replicase complexes are located on the endoplasmic reticulum in close association with the cytoskeleton in cytoplasmic bodies called viroplasms, which mature to produce 'X bodies'. Viroplasms are sites of both RNA replication and protein synthesis, and may provide compartments in which the various stages of the virus mutiplication cycle (protein synthesis, RNA replication, virus movement, encapsidation) are localized and coordinated. Membranes may also be important for the configuration of the replicase with respect to initiation of RNA synthesis, and synthesis and release of progeny single-stranded RNA.     

The Tobacco mosaic virus 126-kDa protein associated with virus replication and movement suppresses RNA silencing

Systemic symptoms induced on Nicotiana tabacum cv. Xanthi by Tobacco mosaic virus (TMV) are modulated by one or both amino-coterminal viral 126- and 183-kDa proteins: proteins involved in virus replication and cell-to-cell movement. Here we compare the systemic accumulation and gene silencing characteristics of TMV strains and mutants that express altered 126- and 183-kDa proteins and induce varying intensities of systemic symptoms on N. tabacum. Through grafting experiments, it was determined that M(IC)1,3, a mutant of the masked strain of TMV that accumulated locally and induced no systemic symptoms, moved through vascular tissue but failed to accumulate to high levels in systemic leaves. The lack of M(IC)1,3 accumulation in systemic leaves was correlated with RNA silencing activity in this tissue through the appearance of virus-specific, approximately 25-nucleotide RNAs and the loss of fluorescence from leaves of transgenic plants expressing the 126-kDa protein fused with green fluorescent protein (GFP). The ability of TMV strains and mutants altered in the 126-kDa protein open reading frame to cause systemic symptoms was positively correlated with their ability to transiently extend expression of the 126-kDa protein:GFP fusion and transiently suppress the silencing of free GFP in transgenic N. tabacum and transgenic N. benthamiana, respectively. Suppression of GFP silencing in N. benthamiana occurred only where virus accumulated to high levels. Using agroinfiltration assays, it was determined that the 126-kDa protein alone could delay GFP silencing. Based on these results and the known synergies between TMV and other viruses, the mechanism of suppression by the 126-kDa protein is compared with those utilized by other originally characterized suppressors of RNA silencing.     

Isolation from tobacco mosaic virus-infected tobacco of a solubilized template-specific RNA-dependent RNA polymerase containing a 126K/183K protein heterodimer

The complete nucleotide sequence was determined for the putative RNA polymerase (183K protein) gene of tobacco mosaic virus (TMV) OM strain, which differed from the related strain, vulgare, by 51 positions in its nucleotide sequence and 6 residues in its amino acid sequence. Three segments of this 183K protein, each containing the sequence motif of methyltransferase (M), helicase (H), or RNA-dependent RNA polymerase (P), were expressed in Escherichia coli as fusion proteins with hexahistidine tags, and domain-specific antibodies were raised against purified His-tagged M and P polypeptides. By immunoaffinity purification, a template-specific RNA-dependent RNA polymerase containing a heterodimer of the full-length 183K and 126K (an amino-terminal-proximal portion of the 183K protein) viral proteins was isolated. We propose that the TMV RNA polymerase for minus-strand RNA synthesis is composed of one molecule each of the 183- and 126-kDa proteins, possibly together with two or more host proteins.     

Identification of a region of the tobacco mosaic virus 126- and 183-kilodalton replication proteins which binds specifically to the viral 3'-terminal tRNA-like structure

UV irradiation of a mixture of an isolated tobacco mosaic virus (TMV; tomato strain L [TMV-L]) RNA-dependent RNA polymerase complex and the TMV-L RNA 3'-terminal region (3'-TR) resulted in cross-linking of the TMV-L 126-kDa replication protein to the TMV-L 3'-TR. Using both Escherichia coli-expressed proteins corresponding to parts of the 126-kDa protein and mutants of the 3'-TR, the interacting sites were located to a 110-amino-acid region just downstream of the core methyltransferase domain in the protein and a region comprising the central core C and domain D2 in the 3'-TR. Mutation to alanine of a tyrosine residue at position 409 or a tyrosine residue at position 416 in the protein binding region abolished cross-linking to the 3'-TR, and corresponding mutations introduced into TMV-L RNA abolished its ability to replicate in tomato protoplasts, with no detectable production of either plus- or minus-strand RNA. The results are compatible with a model for initiation of TMV-L minus-strand RNA synthesis in which an internal region of the TMV-L 126-kDa protein first binds to the central core C and domain D2 region of the TMV-L 3'-TR and is then followed by binding of the 183-kDa protein to this complex and positioning of the catalytically active site of the polymerase domain close to the 3'-terminal CCCA initiation site.     

The Complete Nucleotide Sequence of Odontoglossum Ringspot Virus (Cy-1 Strain) Genomic RNA

The complete nucleotide sequence of the genomic RNA of odontoglossum ringspot virus Cy-1 strain (ORSV Cy-1) was determined using cloned cDNA. This sequence is 6611 nucleotides long containing four open reading frames, which correspond to 126 K, 183 K, 31 K, and 18 K proteins. Its genomic organization is similar to other tobamoviruses, TMV-V(vulgare), TMV-L (tomato strain), tobacco mild green mosaic virus (TMGMV) and cucumber green mottle mosaic virus (CGMMV). The 5′ non-coding regions of ORSV Cy-1 is 62 nucleotides. The ORFs encoded a 126 K polypeptide and a 183 K read-through product in which helicase-sequence and polymerase-sequence motifs are found. The ORFs encoding the 126 K and 183 K proteins have 61% and 63% identities with those of TMV-V. The third ORF encoded a 31 K protein homologous to TMV cell-to-cell movement protein. It has 63% identities with that of TMV-V. The fourth ORF encoded an 18 K coat protein. The 5′ non-coding region, which extends from base 1 to 62 has 2 G residues and a ribosome binding site (AUU). The 3′ non-coding region, 414 nucleotides in length, is entirely different from that of other tobamoviruses.     

The interaction of soybean reticulon homology domain protein (GmRHP) with Soybean mosaic virus encoded P3 contributes to the viral infection

Soybean mosaic virus (SMV), a member of the Potyvirus genus, is a prevalent and devastating viral pathogen in soybean-growing regions worldwide. Potyvirus replication occurs in the 6K2-induced viral replication complex at endoplasmic reticulum exit sites. Potyvirus-encoded P3 is also associated with the endoplasmic reticulum and is as an essential component of the viral replication complex, playing a key role in viral replication. This study provides evidence that the soybean (Glycine max) reticulon homology domain protein (designated as GmRHP) interacts with SMV-P3 by using a two-hybrid yeast system to screen a soybean cDNA library. A bimolecular fluorescence complementation assay further confirmed the interaction, which occurred on the cytomembrane, endoplasmic reticulum and cytoskeleton in Nicotiana benthamiana cells. The transient expression of GmRHP can promote the coupling of Turnip mosaic virus replication and cell-to-cell movement in N. benthamiana. The interaction between the membrane protein SMV-P3 and GmRHP may contribute to the potyvirus infection, and GmRHP may be an essential host factor for P3's involvement in potyvirus replication.     

Beneficial effects of fenofibrate in pulmonary hypertension in rats

Viruses depend on cellular machinery to efficiently replicate. The host cytoskeleton is one of the first cellular systems hijacked by viruses in order to ensure their intracellular transport and promote the development of infection. Our previous results demonstrated that stable microfilaments and microtubules interfered with human influenza A/NWS/33 virus (H1N1) infection in semi-permissive LLC-MK2 cells. Although formins play a key role in cytoskeletal remodelling, few studies addressed a possible role of these proteins in development of viral infection. Here, we have demonstrated that mammalian Diaphanous-related formin-1 (mDia1) is involved in the control of cytoskeleton dynamics during human influenza A virus infection. First, by employing cytoskeleton-perturbing drugs, we evidenced a cross-talk occurring between microtubules and microfilaments that also has implications on the intracellular localization of mDia1. In influenza A/NWS/33 virus-infected LLC-MK2 cells, mDia1 showed a highly dynamic intracellular localization and partially co-localized with actin and tubulin. A depletion of mDia1 by RNA-mediated RNA interference was found to improve the outcome of influenza A/NWS/33 virus infection and to increase the dynamics of microfilament and microtubule networks in LLC-MK2 cells. Consistent with these findings, observations made in epithelial respiratory cells from paediatric patients with acute respiratory disease assessed that the expression of mDia1 is stimulated by influenza A virus but not by respiratory syncytial virus. Taken together, the obtained results suggest that mDia1 restricts the initiation of influenza A/NWS/33 virus infection in LLC-MK2 cells by counteracting cytoskeletal dynamics.     

Control of myofibroblast differentiation and function by cytoskeletal signaling

The cytoskeleton consists of three distinct types of protein polymer structures–microfilaments, intermediate filaments, and microtubules; each serves distinct roles in controlling cell shape, division, contraction, migration, and other processes. In addition to mechanical functions, the cytoskeleton accepts signals from outside the cell and triggers additional signals to extracellular matrix, thus playing a key role in signal transduction from extracellular stimuli through dynamic recruitment of diverse intermediates of the intracellular signaling machinery. This review summarizes current knowledge about the role of cytoskeleton in the signaling mechanism of fibroblast-to-myofibroblast differentiation–a process characterized by accumulation of contractile proteins and secretion of extracellular matrix proteins, and being critical for normal wound healing in response to tissue injury as well as for aberrant tissue remodeling in fibrotic disorders. Specifically, we discuss control of serum response factor and Hippo signaling pathways by actin and microtubule dynamics as well as regulation of collagen synthesis by intermediate filaments.