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Journal of General Virology (2009), 90, 3022–3032 DOI 10.1099/vir.0.013862-0 Domain organization of the N-terminal portion of hordeivirus movement protein TGBp1 Valentin V. Makarov,13 Ekaterina N. Rybakova,13 Alexander V. Efimov,2 Eugene N. Dobrov,1 Marina V. Serebryakova,3 Andrey G. Solovyev,1,4 Igor V. Yaminsky,5 Michael E. Taliansky,6 Sergey Yu. Morozov1,7 and Natalia O. Kalinina1 Correspondence Natalia Kalinina 1 [email protected] 2 A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russia Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia 3 Institute of Physico-Chemical Medicine, Moscow 119828, Russia 4 Institute of Agricultural Biotechnology, Russian Academy of Agricultural Sciences, Moscow 127550, Russia 5 Physical Faculty, Moscow State University, Moscow 119992, Russia 6 Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland, UK 7 Department of Virology, Biological Faculty, Moscow State University, Moscow 119992, Russia Received 28 May 2009 Accepted 6 August 2009 Three ‘triple gene block’ proteins known as TGBp1, TGBp2 and TGBp3 are required for cell-tocell movement of plant viruses belonging to a number of genera including Hordeivirus. Hordeiviral TGBp1 interacts with viral genomic RNAs to form ribonucleoprotein (RNP) complexes competent for translocation between cells through plasmodesmata and over long distances via the phloem. Binding of hordeivirus TGBp1 to RNA involves two protein regions, the C-terminal NTPase/ helicase domain and the N-terminal extension region. This study demonstrated that the extension region of hordeivirus TGBp1 consists of two structurally and functionally distinct domains called the N-terminal domain (NTD) and the internal domain (ID). In agreement with secondary structure predictions, analysis of circular dichroism spectra of the isolated NTD and ID demonstrated that the NTD represents a natively unfolded protein domain, whereas the ID has a pronounced secondary structure. Both the NTD and ID were able to bind ssRNA non-specifically. However, whilst the NTD interacted with ssRNA non-cooperatively, the ID bound ssRNA in a cooperative manner. Additionally, both domains bound dsRNA. The NTD and ID formed low-molecular-mass oligomers, whereas the ID also gave rise to high-molecular-mass complexes. The isolated ID was able to interact with both the NTD and the C-terminal NTPase/helicase domain in solution. These data demonstrate that the hordeivirus TGBp1 has three RNA-binding domains and that interaction between these structural units can provide a basis for remodelling of viral RNP complexes at different steps of cell-to-cell and long-distance transport of virus infection. INTRODUCTION Transport of plant viruses from cell to cell occurs through plasmodesmata and requires virus-encoded movement proteins (Boevink & Oparka, 2005; Lucas, 2006; Epel, 2009). In the genus Hordeivirus, viral movement proteins are represented by three proteins encoded by a ‘triple gene block’ (TGB) and referred to as TGBp1, TGBp2 and TGBp3 (Solovyev et al., 1996). The TGB is a conserved 3These authors contributed equally to this work. 3022 module of overlapping genes found in a number of virus groups (Morozov & Solovyev, 2003). Hordeivirus TGBp1 binds viral genomic RNA forming a cell-to-cell transportcompetent complex (Brakke et al., 1988; Kalinina et al., 2001; Lim et al., 2008; Jackson et al., 2009). TGBp2 and TGBp3 are small membrane proteins necessary for intracellular transport of complexes containing TGBp1 and viral RNA to plasmodesmata (Morozov & Solovyev, 2003; Zamyatnin et al., 2004; Haupt et al., 2005; Jackson et al., 2009). Downloaded from www.microbiologyresearch.org by 013862 G 2009 SGM IP: 88.99.165.207 On: Mon, 15 May 2017 20:28:58 Printed in Great Britain Domain organization of hordeivirus TGBp1 Amino acid sequence analyses of TGB-encoded proteins, together with functional studies, have revealed two distinct TGB types termed ‘hordei-like’ and ‘potex-like’ TGB (Morozov & Solovyev, 2003). Viruses with hordei-like TGB, in contrast to viruses with potex-like TGB, require no coat protein for viral cell-to-cell and long-distance transport (Petty & Jackson, 1990; Morozov & Solovyev, 2003). Analysis of RNA–protein complexes isolated from plants infected with barley stripe mosaic virus (BSMV; the type member of the genus Hordeivirus) demonstrated that TGBp1 is the major if not the only protein that interacts with viral genomic and subgenomic RNAs in infected cells to form ribonucleoprotein (RNP) complexes (Brakke et al., 1988; Lim et al., 2008; Jackson et al., 2009). Such TGBp1–RNA complexes are considered to be a form of viral genome capable of cell-to-cell and long-distance transport in plants (Morozov & Solovyev, 2003; Lim et al., 2008). In potex-like TGBs, the whole TGBp1 sequence is represented by the NTPase/helicase domain (HELD) with seven conserved motifs of the superfamily 1 NTPases/helicases (Gorbalenya et al., 1989), whereas the hordei-like TGBp1 has an additional N-terminal ‘extension region’ (Morozov & Solovyev, 2003). Deletion analysis of BSMV TGBp1 has revealed that the protein has multiple RNA-binding sites, some of which map to the N-terminal extension region (Donald et al., 1997). As shown for TGBp1 of poa semilatent virus (PSLV; genus Hordeivirus), the Nterminal extension region expressed as a separate polypeptide binds RNA in a non-cooperative manner (Kalinina et al., 2001). The N-terminal 24 aa of TGBp1 of beet necrotic yellow vein virus (BNYVV; genus Benyvirus), another virus with a hordei-like TGB, also binds RNA (Bleykasten et al., 1996). Interestingly, BSMV and BNYVV TGBp1s are able to bind both ssRNA and dsRNA (Bleykasten et al., 1996; Donald et al., 1997). A double mutation in PSLV TGBp1 that disrupts two clusters of positively charged amino acids responsible for RNA binding blocks long-distance but not cell-to-cell transport of viral infection (Kalinina et al., 2001), whereas deletion of the 24 aa N-terminal region containing an RNA-binding site prevents BNYVV cell-to-cell transport (Bleykasten et al., 1996). Thus, the RNA-binding ability of the TGBp1 Nterminal extension region is necessary for viral transport in plants. In this paper, we showed that the N-terminal ‘extension region’ of the hordeivirus TGBp1 consists of two structurally and functionally distinct domains. METHODS Amino acid sequence analysis. For computer-assisted secondary structure prediction, the SCRATCH server (Cheng et al., 2005; available at http://www.igb.uci.edu/servers/psss.html), the FoldIndex server (Prilusky et al., 2005; available at http://bioportal.weizmann.ac.il/fldbin/findex) and the GOR IV server implementing an improved prediction algorithm (Garnier et al., 1978; available at http://cn.expasy.org/) were used. constructed for expression of the N-terminal domain (NTD) (aa 1– 190 of the 63K protein) and the internal domain (ID) (aa 190–290). The NTD-encoding DNA fragment was obtained by PCR using primers 59-TTGTGAGCGGATAACAATTTC-39 and 59-CCTCTCTAGATTATTTATCTTTCGTTTGCTTCT-39, whilst the ID-encoding DNA fragment was obtained with primers 59-AAAGGATCCGCTGAAGACTTAAATGCA-39 and 59-AAGCTCTAGATTATGTCTTCTTTAAGTGCTCCA-39. PCR products were digested with BamHI and XbaI, and ligated with similarly digested pQE30. E. coli strain M15 cells were transformed with plasmids pQE30-N63K, pQE30-NTD and pQE30-ID. Expression, purification and Western blot analysis were performed according to the method described by Leshchiner et al. (2006). Circular dichroism (CD) spectroscopy. CD spectra of the recombinant proteins in the far-UV region (198–250 nm) were measured in a modified Jobin–Jvon Mark V dichrograph interfaced with a computer, using RDA and Wtest programs developed in our laboratory. The spectra were measured at 25 uC in 1 mm cells at a protein concentration of 200 mg ml21. The spectra were calculated as [h] values per mole of amino acid residues. The concentration of the recombinant ID protein was measured spectrophotometrically using calculated absorption coefficients at 280 nm (E280, 1 cm, 0.1 %) of 0.24. In the case of the NTD, which does not contain Trp and Tyr, the concentration was determined by SDS-PAGE with Coomassie blue staining using BSA as a standard or by a Bradford assay. RNA-binding assays. North-Western analysis was carried out as described previously (Kalinina et al., 1996). For gel-shift assays, increasing concentrations of recombinant proteins were incubated with tobacco mosaic virus RNA (0.5 mg) at room temperature. Samples were analysed in ethidium bromide-containing 1 % agarose gels. For the dsRNA in gel-shift analysis, two annealed T7 transcripts complementary along their entire length (377 nt) were used. As templates for in vitro transcription, PCR products obtained from pCK-GFPC3 (Yelina et al., 2005) were used. PCR was carried out with two pairs of primers, C3-ds-T7-59 (59-GTAATACGACTCACTATAGGGAGAGGGTGAAGGTGATGCAAC-39) and C3-ds-39 (59-GGGCTGCCGTGATGTATACATTGTGT-39), and C3-ds-T7-39 (59-GTAATACGACTCACTATAGGGCTGCCGTGATGTATACATTGTGT-39) and C3-ds-59 (59-GGGAGAGGGTGAAGGTGATGCAAC-39). Ultracentrifugation in a sucrose concentration gradient. Protein preparations were layered onto a 10–40 % sucrose gradient prepared in buffer containing 50 mM Tris/HCl (pH 7.8), 100 mM NaCl, 5 mM MgCl2 and 2 mM dithiothreitol, and centrifuged at 4 uC for 21 h at 36 000 r.p.m. in an SW41 rotor in a Beckman L-2 ultracentrifuge. Proteins from the collected fractions were precipitated with 12 % trichloroacetic acid. The precipitates were collected by centrifugation, washed in acetone, dried, dissolved in Laemmli sample buffer and analysed by SDS-PAGE and immunoblotting. Dynamic laser light scattering (DLS). A DLS device (Zetasizer Nano ZS; Malvern Instruments) with a helium–neon laser (633 nm) was used. Measurements were performed in 1 cm cells in 10 mM Tris/HCl (pH 7.5) at protein concentrations of 0.05–0.15 mg ml21. A Peltier thermostat system maintained the temperature at +25 uC. All curves were fitted using Dispersion Technology Software v5.10. RESULTS Construction of recombinant clones and expression in Escherichia coli. The vector pQE30-N63K used for expression of Limited proteolysis of recombinant PSLV TGBp1 in E. coli N63K, comprising aa 1–290 of the 63 kDa (63K) protein, has been described previously (Kalinina et al., 2001). Based on pQE30-N63K (Kalinina et al., 2001), plasmids pQE30-NTD and pQE30-ID were Expression of the full-length 63K protein in E. coli after IPTG induction (Kalinina et al., 2001) often resulted in its http://vir.sgmjournals.org Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 15 May 2017 20:28:58 3023 V. V. Makarov and others fragmentation into several cleaved products, the yield of which increased after prolonged induction. Such protein fragmentation may result from a depletion of media components that mimic minimal medium conditions known to enhance proteolytic attack by intracellular proteases (Schlotmann & Beyreuther, 1979). Two major proteolytic fragments co-purified with the full-length 63K protein during Ni-NTA agarose chromatography (Fig. 1a), showing that these fragments contained the 63K N terminus carrying the 6-His tag. To identify the cleavage sites in the 63K protein, purified 6His-tagged proteolytic fragments were analysed by mass spectrometry (MS). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis showed that the larger product that migrated in the gel as a 50 kDa protein corresponded to the N-terminal half of the 576 aa 63K protein with its C terminus located between aa 279 and 294. The smaller product that migrated in the gel as a 30 kDa protein represented the extreme N terminus of the 63K protein whose C terminus was located between aa 188 and 202. Thus, both of these 63K fragments, as well as the full-length 63K protein (Fig. 1a), had gel mobilities lower than expected based on their molecular masses. These reduced mobilities might be attributed to stretches of positively charged amino acid residues at their N terminus. In a total extract of E. coli expressing the 63K protein, one more proteolytic fragment of approximately 35 kDa was revealed by Western blotting with antibodies against the C-terminal region of the 63K protein comprising the HELD (Fig. 1b). This fragment therefore represented a C-terminal portion of the 63K protein and lacked the 6-His tag located at the N terminus of the recombinant 63K protein, preventing co-purification with the full-length polypeptide. It should be noted that in vitro protease treatment of multi-domain cellular and viral proteins containing RNA/DNA helicase domains also resulted in release of these domains as distinct protein fragments resistant to further proteolysis (O’Reilly et al., 1995; Bae et al., 2001). In E. coli cells grown on minimal medium, proteolytic attack presumably takes place during synthesis of the protein, and proteases preferentially attack inter-domain linkers in multi-domain polypeptides (Schlotmann & Beyreuther, 1979; Corchero et al., 1996). Thus, we hypothesized that the 63K protein contains at least three domains, namely the extreme NTD, an ID and the Cterminal HELD, which are separated by protease-sensitive linkers at aa 188–202 and 279–294. Interestingly, the ID could not be detected in our experiments as an individual polypeptide, presumably reflecting its low stability in bacteria. Secondary structure prediction for the TBGp1 extension domain The 63K amino acid sequence was analysed to predict secondary structure elements in the N-terminal extension domain (aa 1–300) preceding the conserved HELD. Strikingly, several algorithms of sequence analysis, such as those implemented in the web-based services SCRATCH (Cheng et al., 2005) and FoldIndex (Prilusky et al., 2005), confidently predicted that the N-terminal part of the 63K protein (from aa 1 to 210–223) represented a natively unfolded protein region (Fig. 2a). Therefore, according to this prediction, the NTD could be considered an unstructured protein region. Conversely, the region corresponding to the ID was predicted as the folded part of the 63K protein (Fig. 2a). Thus, the NTD and ID of PSLV TGBp1 that were proposed to be distinct protein domains based on proteolysis data appeared to have different predicted structural features. Further analysis of TGBp1 encoded by other viruses with hordei-like TGBs, namely BSMV, potato mop-top virus (PMTV; genus Pomovirus) and peanut clump virus (PCV; genus Pecluvirus), resulted in similar structure predictions for the N-terminal extension domains (Fig. 2b). Fig. 1. Spontaneous proteolysis of the recombinant 63K protein in E. coli. (a) Equal volumes of E. coli culture expressing 63K were taken at different time points (0.5–6 h) after induction with IPTG. Proteins were purified by Ni-NTA chromatography under denaturing conditions, separated by 15 % SDS-PAGE and stained with Coomassie blue. The sizes of the molecular mass markers are indicated on the left (kDa). Major degradation products were cut out of the gel and subjected to enzymic proteolysis and MS analysis on a MALDI-TOF MS system. The C-terminal amino acid sequences are shown on the right. (b) Total protein extract of E. coli cells after 2 h induction with IPTG (lane 1) and a preparation of the recombinant HELD (lane 2) were separated by 15 % SDS-PAGE and analysed by Western blotting with antibodies against the HELD. The sizes of the molecular mass markers are indicated on the left (kDa). HELD was termed C-63K in our previous study (Kalinina et al., 2001). 3024 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 15 May 2017 20:28:58 Journal of General Virology 90 Domain organization of hordeivirus TGBp1 expressed as separate polypeptides in bacteria, purified and renatured. The CD spectrum of the NTD had a strong minimum at 202 nm ([h]2025214 000u) and a weak signal at longer wavelengths (Fig. 3), typical for completely disordered proteins (Adler et al., 1973; Johnson, 1988; Uversky, 2002). The ID gave a spectrum characteristic of proteins with significant b-structure content and some fraction of a-helices (Fig. 3) (Sreerama & Woody, 2004). According to current opinion, only a-helix content can be determined quantitatively from the 200–250 nm CD spectra (Sreerama & Woody, 2004). Therefore, we estimated the a-helix content in the ID using the Greenfield–Fasman equation (Greenfield & Fasman, 1969). This calculation gave a value of 10–12 %. Thus, the CD spectra of TGBp1 deletion mutants confirmed the suggestion that the NTD and ID differ drastically in their structure: the NTD is unstructured whilst the ID contains a significant amount of b-structure elements and a fraction of a-helices. RNA-binding activity of NTD and ID domains The full-length 63K protein and its separated N-terminal extension region were shown previously by a NorthWestern assay to bind ssRNA efficiently and nonspecifically at NaCl concentrations ranging from 50 to 500 mM (Kalinina et al., 2001), which is in agreement with previous results on the RNA-binding properties of BSMV TGBp1 (Donald et al., 1997). To determine whether the proposed domains of the 63K N-terminal region exhibited a ssRNA-binding activity, the NTD and ID expressed in E. coli as separate polypeptides were immobilized on a nitrocellulose membrane and incubated with a non-specific 32 P-labelled RNA transcript. As a control, we used the TGBp1 deletion mutant N63K (comprising aa 1–290 of 63K) (Kalinina et al., 2001). Both the NTD and N63K bound ssRNA with a similar efficiency at NaCl concentrations ranging from 50 to 300 mM and exhibited no Fig. 2. Prediction of the multi-domain organization of the 63K protein and FoldIndex prediction for hordei-like TGBp1s. (a) Putative domain structure of the 63K protein of PSLV. Numbers indicate the borders of domains and possible inter-domain spacers. Domains are indicated by boxes. (b) FoldIndex prediction of folded and unfolded regions in TGBp1s encoded by BMSV (genus Hordeivirus), PMTV (genus Pomovirus) and PCV (genus Pecluvirus). The box indicates the HELD of the TGB1 proteins. CD spectra of TGBp1 deletion mutants To analyse the structural properties of the proposed domains of the 63K N-terminal extension region, the CD spectra in the far UV region (198–250 nm) were measured for the NTD (aa 1–190 of the 63K protein) and the central protein region comprising the ID (aa 190–290), which were http://vir.sgmjournals.org Fig. 3. CD spectra of the 63K NTD and ID expressed in E. coli as separate polypeptides. Far-UV CD spectra of the NTD and ID were recorded at room temperature. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 15 May 2017 20:28:58 3025 V. V. Makarov and others detectable binding at 500 mM NaCl, whereas the ID interacted with RNA only at 50–150 mM NaCl (Fig. 4a). Increases in NaCl concentration up to 300 mM blocked the ID RNA-binding activity (Fig. 4a). The ability of the NTD, ID and N63K to bind ssRNA was further compared in a gel-shift assay. In these experiments, the NTD, similarly to N63K, formed protein–RNA complexes migrating in the gel (Fig. 4b), whilst ID-formed complexes were unable to enter the gel (Fig. 4b). Application of a Hill transformation to quantified gel-shift data (Marcos et al., 1999) gave a Hill coefficient of approximately 2.9, strongly suggesting that the ID bound RNA in a cooperative manner. The ability of ID to bind RNA cooperatively was confirmed in a gel-shift assay using the ID region of BSMV TGBp1 (data not shown). As reported previously, N63K (which included both the NTD and ID) formed protein–RNA complexes capable of migrating into the gel (Fig. 4b; Kalinina et al., 2001). Thus, the ID RNA-binding activity resulting in formation of fully retarded RNA–protein complexes was suppressed when the ID was part of N63K. To analyse a possible mutual influence of the ssRNA-binding activities determined by the NTD and ID, these two separate protein fragments were mixed in an equimolar ratio and tested in a gel-shift assay. In contrast with N63K, where the NTD and ID were present as parts of a single polypeptide, the mixture of the separate NTD and ID gave rise to complexes that were fully retarded and that migrated into the gel (Fig. 4b). These data showed that when the ID is a part of N63K, the ID-specific RNA-binding activity is masked, probably due to conformational restrictions, rather than being outcompeted by the NTD-specific binding activity. Earlier in vitro analysis of BSMV TGBp1 RNA binding revealed its ability to bind dsRNA (Donald et al., 1997). Therefore, we tested the ability of the PSLV TGBp1 NTD and ID to bind dsRNA. In these experiments, two annealed non-specific in vitro transcripts complementary to each other along their entire length were used, so that ssRNA fragments were not present at the ends of the duplex (see Methods). One of these transcripts was used as a control ssRNA. In gel-shift experiments, both the NTD and ID were able to bind dsRNA (Fig. 5). Comparison of the protein : RNA ratio resulting in a degree of retardation that was similar for ssRNA and dsRNA revealed that both the NTD and ID bound the two types of RNA with a similar efficiency (Fig. 5). These data are in agreement with the results of BSMV TGBp1 deletion analysis demonstrating that dsRNA binding involves the N-terminal extension region (Donald et al., 1997) and showed that both the NTD and ID can be involved in such an interaction. Fig. 4. ssRNA-binding activity of the NTD and ID. (a) North-Western assay for RNA-binding activity of the NTD, ID and N63K protein. A nitrocellulose membrane with immobilized proteins was stained with Ponceau red and incubated with 32P-labelled RNA transcripts at different NaCl concentration. (b) Gel-shift assay for RNA-binding of the NTD, ID or an NTD/ID mixture. Increasing amounts of each recombinant protein or the NTD and ID mixed in an equimolar ratio were incubated with 0.5 mg tobacco mosaic virus RNA and subjected to electrophoresis on a 1 % agarose non-denaturing gel containing ethidium bromide. Protein : RNA molar ratios are indicated above each lane. The lane marked ‘RNA’ is the control without protein added. N63K was used as a control. 3026 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 15 May 2017 20:28:58 Journal of General Virology 90 Domain organization of hordeivirus TGBp1 Fig. 5. dsRNA-binding activity of the NTD and ID. Increased amounts of each recombinant protein were incubated with 0.2 mg of a 377 bp dsRNA or with 0.4 mg of a 377 nt control ssRNA and subjected to electrophoresis on a 1 % agarose non-denaturing gel containing ethidium bromide. Protein : RNA molar ratios are indicated above each lane. Protein–protein interactions analysed in sucrose gradients Previously, we found that the HELD region of the 63K protein is involved in homologous protein–protein interactions (Leshchiner et al., 2006). To test the self-interaction potential of the isolated NTD and ID, purified recombinant proteins were fractionated in a 10–40 % sucrose gradient and the resulting gradient fractions were analysed by Western blotting with antibodies against the 63K protein or its N- and C-terminal portions. Separately expressed HELD, used as a control, formed a dimer that was stable upon heating in SDS-containing sample buffer and subsequent SDS-PAGE, and also formed oligomers with a molecular mass of about 120–200 kDa (Fig. 6). Both the NTD and ID were found as oligomers of different orders. The majority of the NTD was found in the gradient fractions similar to those for HELD, whilst the ID predominantly formed oligomers of molecular masses up to 150 kDa (Fig. 6). In contrast to the NTD, the ID formed considerable amounts of high-molecular-mass complexes of more than 440 kDa. In addition, and similar to HELD, the ID was capable of forming a dimer, which was stable upon sample preparation and SDS-PAGE (Fig. 6). Interestingly, N63K, similar to the ID, was able to form high-molecular-mass multimers (Fig. 6), which can therefore be attributed to its ID moiety. Thus, the sucrose gradient experiments demonstrated that the NTD, ID and HELD are capable of self-interactions in solution. Importantly, only the ID and ID-containing NTD were able to form large multimeric complexes. We analysed further the potential interactions between TGBp1 domains. When an equimolar mixture of the NTD http://vir.sgmjournals.org and ID was analysed in a sucrose gradient, the ID sedimentation profile was similar to that in the experiment with the ID only, whereas the sedimentation profile of the NTD in the mixture was considerably changed compared with the experiment with NTD alone, and became similar to the ID sedimentation profile (Fig. 6). Similarly, sedimentation analysis of an equimolar mixture of HELD and ID revealed that, under these conditions, the HELD sedimentation pattern was changed and closely resembled that of the ID. These data demonstrate that the ID could interact with both the NTD and HELD in solution. Interactions between the NTD and HELD could not be analysed in these experiments as these TGBp1 domains have similar sedimentation profiles (Fig. 6). DLS analysis The DLS method is generally used to determine the size distribution of small particles dispersed in solution and is often applied to protein complexes (Schmitz, 1990; Barilla et al., 2005; Tönges et al., 2006). We employed DLS analysis to determine the hydrodynamic diameter, or size, of multimers formed by the NTD, ID and N63K. Analysis of the NTD preparations revealed protein particles of 15±3 nm diameter (Fig. 7). In ID preparations, the major population of particles ranged from 20 to 55 nm with a mean size of 40 nm. Distribution profiles found for the hydrodynamic diameter of N63K had a maximum at 60 nm and a range of 45–80 nm (Fig. 7). It should be emphasized that the detection of small-sized particles in the presence of high-molecular-mass multimers was impossible due to the technical restrictions of the DLS method (Schmitz, 1990). Thus, after treatment of ID and Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 15 May 2017 20:28:58 3027 V. V. Makarov and others Fig. 6. Fractionation of 63K protein deletion mutants by ultracentrifugation in sucrose density gradients. Preparations of the recombinant proteins were centrifuged in a 10–40 % sucrose density gradient. The proteins of the gradient fractions were analysed by SDSPAGE and Western blotting. The positions of marker proteins are indicated by arrows: BSA (67 kDa), aldolase (158 kDa), catalase (232 kDa), and apoferritin (440 kDa). D, Dimer; M, monomer. N63K preparations with SDS at a concentration as low as 0.01 %, a population of large-sized particles was not found, whereas small particles with sizes of 5.0±2.0 nm were easily detected (data not shown). Therefore, the DLS data demonstrated that the ID and N63K, unlike the NTD, are capable of forming large multimeric structures. Fig. 7. Size distribution of the hydrodynamic diameter of particles in the NTD, ID and N63K preparations by the DLS method. Renatured proteins were analysed by DLS. Size (nm) is the hydrodynamic diameter. Note, no values fell outside the range 10– 100 nm. Size distribution by number of particles (%) is shown for the NTD, ID and N63K preparations. 3028 DISCUSSION Hordeiviral genomic RNAs form RNP complexes containing TGBp1 as the major if not the sole protein component (Brakke et al., 1988; Lim et al., 2008; Jackson et al., 2009). As shown in vitro, two RNA-binding activities of hordeivirus TGBp1 can be involved in the formation of such RNPs, namely those specified by the C-terminal NTPase/helicase domain and the two clusters of positively charged amino acid residues in the protein N-terminal extension region (Donald et al., 1997; Kalinina et al., 2001). These clusters are crucial for both the in vitro RNA-binding ability of the extension region and the long-distance transport of virus during infection, but are not necessary for viral cell-to-cell movement (Kalinina et al., 2001). However, the role of the TGBp1 N-terminal region in viral vascular transport remains unknown. In this paper, we have reported that the N-terminal extension region of the 63 kDa PSLV TGBp1 consists of two domains termed the NTD and the ID. The NTD was found to be a disordered, or natively unfolded, protein domain. Intrinsically unstructured or natively unfolded proteins and protein domains are characterized by a lack of specific structure and represent a distinct protein element Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 15 May 2017 20:28:58 Journal of General Virology 90 Domain organization of hordeivirus TGBp1 involved, in most cases, in molecular interactions (Fink, 2005). It is generally accepted that functions of natively unfolded proteins or protein regions involve their binding to ligands such as proteins or nucleic acids, and that interaction with a target ligand often induces a transition from the previously disordered structure to a folded form, which represents the functional state of the polypeptide (Uversky, 2002; Fink, 2005). Two RNA-binding basic clusters of the 63K N-terminal extension region (Kalinina et al., 2001) fall into the natively unfolded NTD; therefore, one can speculate that NTD interaction with RNA can render this region structured and, as a consequence, competent for functional interactions involved, for example, in virus long-distance transport. On the other hand, natively unfolded domains are often found in proteins capable of self-assembly into large multimeric complexes such as viral capsids and nucleocapsids (Namba, 2001). Natively unfolded domains both prevent unwanted spontaneous assembly of such proteins, as has been shown for the measles virus nucleoprotein (Longhi et al., 2003), and stabilize multimeric complexes due to folding upon interaction with the correct partners (Namba, 2001). The ID represents a protein region with a pronounced secondary structure. The ID is capable of self-interaction, resulting in the formation of stable dimers and highmolecular-mass multimeric complexes. Similar properties are found for the whole N-terminal extension region (N63K). Although comparative analysis of the ID sequence failed to reveal any known RNA-binding motifs, the ID appeared to bind ssRNA in a cooperative manner. Importantly, the ID-specific cooperative ssRNA binding was manifested only when the ID was analysed as a separate polypeptide, and was not detected when the ID was a part of a larger TGBp1 fragment comprising both the NTD and ID (Kalinina et al., 2001). Thus, the NTD and ID could not be considered as functionally independent domains, and one can speculate that the presence of the NTD either induces ID structural changes that are incompatible with RNA binding or binds to the ID to hinder its RNA-interacting interface. The latter hypothesis was confirmed by the observation that the isolated NTD and ID were able to interact in solution. This finding demonstrated that the ID can be considered as yet another NTD ligand that might induce an NTD conformational transition from the unfolded to an ordered state. This hypothesis implies that, in the native TGBp1 or in TGBp1-formed RNP complexes, the NTD could represent an ordered structure. Additionally, we found that the ID is capable of interaction with HELD. One can speculate that ID interaction with two other TGBp1 structural domains might be the basis for remodelling of the TGBp1-formed RNP complexes during different phases of cell-to-cell and longdistance transport. It should also be kept in mind that the TGBp1 NTPase/helicase domain has its own RNA-binding activity. Therefore, RNP formation by TGBp1 can involve RNA interaction with three different protein domains. Although TGBp1-formed RNP complexes isolated from BSMV-infected plants contain only virus-specific ssRNAs http://vir.sgmjournals.org of positive polarity (Lim et al., 2008), BSMV TGBp1 also appears to bind dsRNA in vitro (Donald et al., 1997). Moreover, both BSMV and PSLV TGBp1 are able to unwind RNA duplexes in vitro, thus demonstrating the RNA helicase activity (Kalinina et al., 2002). In this paper, we showed that both the NTD and ID had dsRNA-binding activity, which is in good agreement with the results of BSMV TGBp1 deletion analysis reported by Donald et al. (1997). Indeed, the truncated BSMV TGBp1 with the Nterminal 195 aa residues removed (the region including the NTD and most of the ID according to BSMV and PSLV sequence alignment) lost the ability to bind dsRNA (Donald et al., 1997). Interestingly, an internal deletion in BSMV TGBp1 spanning most of the ID sequence resulted in the inability of excess heterologous ssRNA or dsRNA to displace homologous ssRNA from complexes with such mutant protein in competition experiments (Donald et al., 1997), suggesting that the mutant lost – probably due to improper protein conformation – the native RNA-binding properties of the two other RNAbinding TGBp1 domains, the NTD and HELD. A similar effect was shown by an internal deletion in the NTPase/ helicase domain (Donald et al., 1997) that could potentially destroy its structural subdomain 2A (Morozov & Solovyev, 2003). These data suggest that the structural integrity of TGBp1 domains is required for their correct interactions and, in turn, their RNA-binding properties are dependent on such interactions. It has been suggested that the ability of TGBp1 to bind and unwind dsRNA can be important for unwinding of secondary structure elements in viral genomic RNAs to form RNP complexes capable of translocation through plasmodesmata (Morozov & Solovyev, 2003) or of vectorial diffusion, in a complex with TGBp2/TGBp3 rafts, along the endoplasmic reticulum (Epel, 2009). Many RNA chaperones – proteins able to bind nonspecifically to RNA and resolve its secondary structure in an ATP-independent manner (Rajkowitsch et al., 2007; Russell, 2008) – possess natively unfolded regions serving as molecular recognition elements typically required for interaction with RNA substrates (Tompa & Csermely, 2004). Upon such interaction, according to an entropy transfer model, unfolding of RNA secondary structure may be accompanied by folding of natively unfolded regions of RNA chaperones (Tompa & Csermely, 2004). Several wellstudied RNA chaperones such as YB-1, the major protein of messenger RNP complexes, and E. coli regulatory protein StpA have a bipartite structure composed of two domains, one of which is stably folded, exhibiting RNAbinding and/or protein oligomerization activity, whereas another is natively unfolded, containing sites of RNA chaperone activity (Matsumoto & Wolffe, 1998; Kloks et al., 2002; Mayer et al., 2007; Rajkowitsch et al., 2007). The PSLV TGBp1 N-terminal extension region is organized in a similar way, with RNA binding and protein oligomerization functions associated with the structured ID and another RNA-binding activity associated with the natively Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 15 May 2017 20:28:58 3029 V. V. Makarov and others unfolded NTD. Additionally, similarly to YB-1, which has been found to form homomultimeric complexes with molecular masses of up to 800 kDa (Evdokimova et al., 1995), N63K forms high-molecular-mass multimers. Taken together, the available data allow us to propose that the PSLV TGBp1 N-terminal extension region can share some properties with RNA chaperones. Further research is required to verify this hypothesis and to determine the step(s) of viral transport in plants at which this activity is required. Like many plant viruses, most TGB-containing virus species require viral coat protein (CP) for cell-to-cell and vascular movement. Comparisons of virus-coded protein arrays required for transport of different TGB-containing viruses has revealed an inverse correlation between the size/ complexity of the TGBp1 N-terminal extension region and the necessity for viral CP in viral spread in plants. One side is represented by potexviruses, where TGBp1 contains no N-terminal extension region and the CP is essential for both cell-to-cell and long-distance transport (Beck et al., 1991; Chapman et al., 1992). It is assumed that virions rather than viral RNA or a viral RNP complex is the transport form of the virus genome (Santa Cruz et al., 1998; Morozov & Solovyev, 2003), which is in agreement with the discovery of specific complexes between TGBp1 and potexvirus virions (Atabekov et al., 2000; Kiselyova et al., 2003; Rodionova et al., 2003; Karpova et al., 2006). The opposite pole is occupied by hordeiviruses characterized by the longest known TGBp1 N-terminal extension region. In this case, the CP is dispensable for both cell-tocell and systemic viral movement, which is believed to occur in the form of RNP complexes containing TGBp1 and genomic RNAs (Brakke et al., 1988; Petty & Jackson, 1990; Lim et al., 2008; Jackson et al., 2009). Between the two extremes are BNYVV (genus Benyvirus) and PCV (genus Pecluvirus) with TGBp1 characterized by a shorter N-terminal extension region comprising the ID and a truncated NTD (Morozov & Solovyev, 2003). In BNYVV and PCV, the CP is dispensable for viral cell-to-cell movement (as in hordeiviruses) but is required for vascular transport (as in potexviruses) (Schmitt et al., 1992; Tamada et al., 1996; Herzog et al., 1998). Apparently, transport forms of BNYVV/PCV genomes involved in local and systemic movement are structurally different. One can presume that the hordeivirus NTD may play the role of RNA chaperone specifically involved in phloem transport by stabilizing/protecting genomic RNAs, therefore functioning similarly to the CP in other viruses. However, the CP and NTD fall into distinct categories in spite of a common function in virus spread: CPs stabilize RNA folding via specific binding and formation of a stable coat, whereas the NTD, like other RNA chaperones, may unfold RNA by means of transient and non-specific binding. Another rather unusual example of an intermediate TGB transport system is provided by PMTV. PMTV TGBp1 has an NTD similar in size to that of PCV (Morozov & Solovyev, 2003). Nevertheless, PMTV TGBp1 is able to 3030 function similarly to hordeivirus TGBp1: two PMTV genomic RNAs coding for a replicase and TGB proteins can move locally and systemically in the absence of the viral CP, presumably in the form of TGBp1-formed RNP complexes (Savenkov et al., 2003). However, PMTV TGBp1 has an additional function in viral systemic spread: in wild-type infections, the genomic RNA component encoding the CP and a CP-readthrough moves long distances in the form of a virus particle, one end of which is believed to be associated with the CP-readthrough and TGBp1 (Torrance et al., 2009). Thus, PMTV TGBp1 can function in systemic transport in two ways, either forming RNP complexes like hordeivirus TGBp1 or interacting with one end of the virion similar to potexvirus TGBp1. Conceivably, in the latter mode, the PMTV TGBp1 Nterminal extension region can function as a protein region responsible for protein–protein interaction, possibly due to the natively unfolded NTD. ACKNOWLEDGEMENTS The work was supported by grant no. 07-04-00061-a of the Russian Foundation for Basic Research, the Royal Society (M. E. T. and N. O. K.) and the Scottish Government (M. E. T.). REFERENCES Adler, A. J., Greenfield, N. J. & Fasman, G. D. (1973). 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