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An Unusual Structure at One End of Potato Potyvirus Particles
doi:10.1016/j.jmb.2005.12.021
J. Mol. Biol. (2006) 357, 1–8
C OMMUNICATION
An Unusual Structure at One End of Potato Potyvirus Particles Lesley Torrance1†, Igor A. Andreev1,2†, Rasa Gabrenaite-Verhovskaya3 Graham Cowan1, Kristiina Ma¨kinen3 and Michael E. Taliansky1* 1 Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK 2
School of Physics and Astronomy, University of Leeds Leeds LS2 9JT, UK 3
Department of Applied Biology University of Helsinki FIN-00014 Helsinki, Finland
The particles of potato virus Y (PVY) and potato virus A (PVA) potyviruses are helically constructed filaments that are thought to contain a single type of coat protein subunit. Examination of negatively stained virions by electron microscopy reveals flexuous rod-shaped particles with no obvious terminal structures. It is known that some helically constructed rod-shaped virus particles incorporate additional minor protein components, which form stable complexes that mediate particle disassembly, movement or transmission by vectors. Some of this information has been obtained using imaging techniques such as atomic force microscopy. The particles of PVY and PVA were examined by atomic force microscopy and immunogold labelling electron microscopy. Our results show that some of the potyvirus particles contain a protruding tip at one end of the virus particles, which is presumably associated with the 5 0 -end of viral RNA. The tip contains the virus-encoded proteins genome-linked protein and helper-component proteinase. The composition and possible roles of the terminal tip structures in virus biology are discussed. q 2005 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: potyviruses; HC-Pro; VPg; atomic force microscopy; immunogold labelling electron microscopy
The structure of small geometric viruses is based on two types of architecture: either icosahedral (spherical) or helical (rod-shaped). Icosahedral viruses may contain one or two types of designated coat protein (CP), whereas helical viruses are represented by rigid or flexuous (filamentous) rods and are believed to contain a single type of CP molecule. Although the principle governing assembly of helical particles from identical CP subunits, first formulated by Crick and Watson,1 remains valid, there is a growing body of information showing that some additional virus† L.T. and I.A.A contributed equally to this work. Abbreviations used: PVY, potato virus Y; PVA, potato virus A; AFM, atomic force microscopy; IGEM, immunogold labelling electron microscopy; CP, coat protein; HC-Pro, helper component-proteinase; CI, cylindrical inclusion protein; VPg, virus genome-linked protein; PVIP, potyvirus VPg-interacting protein; eIF4E, eukaryotic translation initiation factor 4E; PABP, poly(A) binding protein. E-mail address of the corresponding author: [email protected]
encoded protein molecules may interact with virions and form stable complexes. One of the examples of the complex architecture of helical viruses is provided by long filamentous closterovirus particles that encapsidate the 15–20 kb RNA genome. In addition to the designated CP, closterovirus particles contain four virus proteins that form a short (w70 nm) tail at one end of the virion.2–7 Such tails seem to be an integral part of the particles, playing a role in intercellular virus movement.6,7 Another example is the potato virus X (PVX) movement protein TGBp1, which attaches to one of the extremities of its filamentous virions and mediates their destabilization and disassembly in a 5 0 to 3 0 direction.8,9 The virus capsid readthrough protein of potato mop-top virus was also localized near one (concave or RNA 5 0 -end) extremity of the virus particles.10,11 The Potyvirus genus contains the largest number of virus species, with about 180 members, causing significant losses in a broad range of plants. The potyvirus genome consists of a single-stranded RNA molecule of about 10 kb, which is translated into a polyprotein that is proteolytically processed
0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
2
Molecular Architecture of Potyvirus Particles
Figure 1. AFM analysis of potato potyvirus particles. Virus particles were diluted to w5 ng/ml in 0.02 M phosphate buffer (pH 7.3), and 5–10 ml was placed onto freshly cleaved mica strips for 5–15 min. The strips were rinsed with deionized water and vacuum-dried at room temperature. Imaging of particles was done in the tapping mode7,19,20,22 (a)–(e) in air or (d) water at a frequency of 300–380 kHz on a NanoScope IIIa multimode scanning probe microscope (Digital Instruments, Santa Barbara, CA), using standard AFM silicon-nitride cantilevers with a length of 123 mm (Nanosensors GmbH, Neuchatel, Switzerland). Images, including 3-D representations were processed using Nanoscope
Molecular Architecture of Potyvirus Particles
by three virus-encoded proteinases, giving up to ten final protein products, many of which are multifunctional.12 Potyviruses are transmitted by aphids in a non-persistent manner and two potyvirusencoded proteins are involved in this process: CP and the helper component-proteinase (HC-Pro, which also acts as a suppressor of RNA silencing).13 At least four potyviral proteins, CP, the cylindrical inclusion protein (CI, an RNA helicase, which is also required for genome replication), the genomelinked protein (VPg) and 6K2 protein are involved in potyvirus movement.13,14 VPg forms a covalent linkage to the 5 0 end of the viral RNA being therefore an integral part of potyvirus particles.15,16 The location of VPg at the 5 0 -end of the viral RNA, in place of an mRNA cap structure may indicate another role in the initiation of translation.17 HCPro is able to interact with the virions, possibly serving as a bridge between virion CP and a putative receptor in the aphid mouthparts. 18 Collectively, these observations allow us to postulate a more complex molecular architecture of potyviral particles than just uniform flexuous rods. Here, we determine the complex morphology of the potyviruses potato virus Y (PVY) and potato virus A (PVA) particles using both atomic force microscopy (AFM) and immunogold labelling electron microscopy (IGEM). We demonstrate the presence of protrusions (tips) at one end of the particles that are attached to the virions, at least at some stages of the virus infection process. The possible implications of our data for the function of these tips as terminal protein complexes in virus replication, transport and aphid transmission are discussed in relation to other plant viruses with helical particles. To determine the fine details of PVY and PVA virion architecture, we employed AFM as a highresolution technique, which has been used in studies of nucleic acids, proteins and their complexes.19–22 Recent work demonstrated the ability of AFM to investigate the structure of virus particles,9,23,24 including those of closterovirus, beet yellows virus, containing three-segmented tails, without the need for staining.7 PVY particles were purified essentially as described,25 except that during ultracentrifugation the preparation was sedimented through 25% (w/v) sucrose cushions. Purification of PVA particles was done as described.26
3 AFM imaging of intact PVY virions readily revealed the polar nature of approximately 10% of particles (23/210 particles in a first virus sample and 13 of 132 particles in another sample) containing a protruding tip at one virion extremity (Figure 1(a), images 1, 2 and 3), indicating that this was not a chance occurrence. The tips were apparent as w40 (41G6) nm segments that were darker than the virion bodies. In AFM, a darker appearance indicates less height or, in the case of a filament, smaller diameter. Reconstructed 3-D images of the virion tips consistently showed a peculiar morphology with a tapered end (Figure 1(a), images 3 and 5). In contrast, the opposite end of the virion appeared to be blunt, lacking any fine structural features (Figure 1(a), images 4 and 6). However, in other particles both ends were blunt and did not differ in their appearance (Figure 1(b), suggesting that the tip structures are either unstable or that they are present in a sub-population of particles. It is well known that physical measurements made by AFM usually give heights that are reduced slightly by compression and widths that are increased greatly by the effects of AFM tip convolution.27–29 Since compression effects are much smaller than the effects of AFM tip convolution, we present here the height measurements. Statistical evaluation of the tail measurements in cross-sections (as shown in Figure 1(a), image 5) indicated that the tip had a diameter of w 2.5 (2.3G 0.5) nm. In contrast, the w9 (9.0G1.2) nm diameter of the virion body was virtually constant along its entire length. These measurements reveal differences in geometry and/or physical properties between the virion tips and the rest of the virus particles. Similar virion tip morphology (although sometimes represented by a two-segmented tail; Figure 1(c)) was observed in particles of PVA, indicating that the tip structure is conserved, at least in two members of the Potyvirus genus. Approximately 10% of PVA particles (24/253 particles) imaged in the air or liquid environment contained such a tip structure at one end (Figure 1(c) and (d)). It is worth noting that both ends of virus-like particles formed by Escherichia coli-expressed PVA CP (which forms virus-like particles in the absence of viral RNA and other virus-encoded proteins30) analyzed by
software and transferred to Adobe Photoshop for layout. Sample heights and lengths were measured automatically using the Nanoscope software. (a) Typical PVY particles containing a protruding tip at one virion extremity indicated by arrows (panels 1 and 2; the scale bar represents 250 nm). Panels 3, 4, 5 and 6 show 3-D images (panels 3 and 4) and the cross-sections (panels 5 and 6) of the protruding tip (panels 3 and 5) and the opposite blunt end (panels 4 and 6) of PVY particles. (b) PVY particles containing no protruding tips (panels 1 and 2; the scale bar represents 250 nm). Panels 3, 4, 5 and 6 show 3-D images (panels 3 and 4) and the cross-sections (panels 5 and 6) of the blunt ends of PVY particles. (c) and (d) Typical PVA particles containing a protruding tip at one virion extremity indicated by arrows, imaged in (c) air or (d) water (panel 1; bar, 250 nm). Panels 2, 3, 4 and 5 show 3-D images (panels 2 and 3) and the cross-sections (panels 4 and 5) of the protruding tip (panels 3 and 5) and the opposite blunt end (panels 2 and 4) of PVA particles. (e) Typical viruslike particles formed by Escherichia coli-expressed PVA CP in the absence of viral RNA and other virus-encoded proteins (panel 1; the scale bar represents 1000 nm). Panels 2, 3, 4 and 5 show 3-D images (panels 2 and 3) and the cross-sections (panels 4 and 5) of the blunt ends of PVA-like particles.
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Molecular Architecture of Potyvirus Particles
Figure 2. Immunogold labelling of potyvirus particles. Immunogold electron microscopy for PVY particles was done as described.33 Briefly, pyroxylin-filmed copper electron microscope grids coated with preparations of mAb 39 (2 mg/ml in PO buffer (0.07 M phosphate buffer, pH 7.0) were floated on drops of virus particles (diluted in PO buffer), incubated for 1 h then washed and incubated on drops of anti-HC-Pro antibody (2 mg/ml in PO buffer) for 1 h then washed and incubated on drops of anti-rabbit gold (15 nm) conjugate (Amersham Biosciences; 1/100 in PO buffer containing 0.1% (w/v) BSA and 0.05% (v/v) Tween 20) for 1 h. All incubations were at room temperature (w22 8C). Grids were stained with a few drops of sodium phosphotungstate (pH 6.5) before examination in a Jeol 1200 Ex electron microscope. PVA particles were labelled in essentially the same way, as described by.16 Purified polyclonal anti HC-Pro antibody produced against the native recombinant PVA HC-Pro was diluted 1:25 for immunogold labelling. (a) Panels 1–5 show individual particles of PVY labelled with gold at one extremity; (b) lower magnification image illustrating gold associated with particle ends (arrowed) and low levels of gold particles elsewhere on the grid; (c) low-magnification image showing PVY particles treated with pre-immune serum (negative control); (d) gold labelling of PVA particle at one extremity.
AFM as controls, looked similar and did not contain any tip structures (Figure 1(e)). Previous work has demonstrated an interaction between potyviral HC-Pro and virus particles or CP.18,31,32 To investigate whether HC-Pro was associated with PVY particles, ELISA experiments were done following the method of Roudet-Tavert et al.,32 where virus particles were trapped from infected leaf extracts in ELISA plates coated with a monoclonal antibody (mAb) against the CP (mAb SCR 39). HC-Pro or virus CP were detected by antiHC-Pro or anti-CP antiserum (rabbit) followed by anti-rabbit alkaline phosphatase conjugate. In the tests with the HC-Pro antiserum, typical absorbance values (A405 nm) obtained with virus-infected extracts were approximately 0.27, indicating an association of HC-Pro with virus particles but the values obtained with anti-CP antiserum were typically O2.0, showing that many more CP molecules than HC-Pro molecules were associated with virus
particles (A405 nm values of control non-infected samples were subtracted and all values were recorded after incubation of the substrate for 1 h). To ascertain whether HC-Pro may comprise part of the potyviral tips revealed by AFM, we analyzed the structure of PVY particles by IGEM using the same antibodies as in the ELISA tests. In these experiments, PVY particles were trapped on grids with anti-CP MAb SCR 39 and then the grids were incubated with anti-HC-Pro antibody followed by anti-rabbit gold conjugate (following the methods of Roberts33). In these experiments, a few PVY particles (17 of 600 particles, that is about 3%) were labelled with gold at one extremity (Figure 2(a)). Although relatively small numbers of particles were labelled with gold, there was little background labelling and no gold associated elsewhere on the particles (Figure 2(b)). The pre-immune serum control did not label the particles at all (Figure 2(c)). Similar results were obtained when
Molecular Architecture of Potyvirus Particles
an antiserum to PVA HC-Pro was used in IGEM experiments with PVA particles. A few particles (2%; 4/200 particles in each of three experiments) were labelled at one extremity (Figure 2(d)). In control experiments, the pre-immune serum did not label the PVA particles at all, and the virus-like PVA particles formed by Escherichia coli-expressed PVA CP containing no HC-Pro30 were not labelled with anti-HC-Pro antibody. These results confirm the specificity of labelling the potyvirus particles with HC-Pro antibodies at one extremity. The protruding tips were not obvious when potyvirus particles were examined by electron microscopy (EM). One reason for this apparent difference in appearance could be because the PVY tip is less efficient at excluding the sodium phosphotungstate during negative staining: unlike EM, AFM does not require staining. Nevertheless, the lack of terminal tapered tips in some PVY (Figure 1(b)) and PVA particles as well as in all PVAlike particles produced in E. coli from the recombinant CP (Figure 1(d)) strongly confirm that the potyviral terminal tips visualized by AFM are not artefacts resulting from structural differences between concave and convex ends of virus helical rods. Moreover, both particle ends of another filamentous virus, PVX (in spite of the presence of TGBp1 protein at its 5 0 -extremity)9 or of the rodshaped tobacco mosaic virus (TMV) analyzed by AFM were blunt and did not differ in their appearance.7,9 The corollary of this work is that the potyviral protruding tips are located at one end of virus particles. Previously, both HC-Pro and VPg were shown to self-interact, and HC-Pro was shown to interact with VPg in the yeast two-hybrid system.34,35 VPg associated with the 5 0 -end of viral RNA in particles was shown to be available for protein–protein interactions on the surface of the particles.16 Taken together with our data, these observations suggest strongly that the HC-Pro may bind to the end of the virion containing a 5 0 -RNA terminus forming the tip either singly or as a dimer, in association with VPg (and possibly with some additional proteins). We could not employ IGEM to confirm this suggestion because of the inability of electron microscopy to resolve the potyviral protruding tips and, hence, we visualised complexes of PVY particles with anti-VPg and anti-HC-Pro antibodies using AFM. After incubation with non-specific preimmune serum or antibodies raised against proteins encoded by unrelated viruses such as coat proteins of PVX or TMV, the PVY particles were indistinguishable from non-treated particles, indicating that there was no non-specific binding between IgG and virions of PVY. In contrast, it was seen clearly that after incubation with specific anti-VPg antibody preparation, new and brighter “spots” appeared on the majority (about 70–80%) of protruding tips of PVY particles as shown in Figure 3(a). No spots were associated with blunt ends of tipped particles or distributed elsewhere
5
Figure 3. AFM images of PVY particles labelled with (a) anti-VPg and (b) anti-HC-Pro antibodies. Virus particles were diluted, immobilised on freshly cleaved mica strips and dried as described in the legend to Figure 1. Antibodies (IgG; 1.2 mg/ml) in 0.01 M phosphate buffer (pH 7.2) containing 0.05% (v/v) Tween 20 were applied to the antigen-covered mica surface and kept at room temperature for 30 min. The IgG solution was drawn off with a pipette, and the surface was washed three times with water. AFM imaging of virus–antibody complexes was done as described in the legend to Figure 1. Arrows indicate antibody “spots” on one of the ends of PVY particles (panels 1). Panels 2 and 3 show PVY particles at higher magnification, revealing association of antibody “spots” with protruding tips of the particles. The scale bars represent 200 nm (panels 1); 120 nm (panels 2); 60 nm (panels 3).
on the particles. The average height of the tip-associated bright spots was approximately 10 nm, which corresponds very well to the dimensions of individual complexes of the tips with antibody.36 However, the widths of some spots varied, reaching up to 80 nm. Such large spots probably correspond to side-to-side aggregation of IgG with up to five or six molecules per aggregate, which is consistent with a previous report that at high concentrations, antibodies tend to aggregate.36 Similar spots on the majority (approximately 70%) of protruding tips appeared after incubation of PVY particles with anti-HC-Pro antibody (Figure 3(b)) whereas no spots were observed on blunt ends or elsewhere on the particles. Collectively, these results confirm specificity of labelling of the PVY tips with anti-VPg and anti-HC-Pro antibodies.
6 Although the reason for fewer virus particles labelled with anti-HC-Pro antibody in the IGEM experiments than in AFM experiments is not completely clear, a possible reason could be the reduced stability of PVY tips subjected to the IGEM procedure. It should be noted that the majority of PVY particles (about 60%) containing no apparent tips were also labelled at one end with anti-VPg antibody, whereas anti-HC-Pro antibody did not label non-tipped PVY particles at all. Taken together with the observation that VPg is covalently linked to the 5 0 -end of viral RNA,15,16 these results show that the potyviral protruding tips contain HC-Pro and are associated with VPg located at the RNA 5 0 terminus. It is worth noting that some other flexuous rod-shaped viruses belonging to distinct evolutionary and taxonomic groups such as closteroviruses,7 potexviruses,8,9 and pomoviruses,11 also contain additional virusencoded proteins or their complexes at the same particle extremity, demonstrating a general tendency among viruses sharing filamentous virus particle morphology and supporting the idea that the architecture of “simple” geometric viruses may be more “complex” than previously thought. The structural supplements at the virion end containing the 5 0 -end of RNA may potentially play essential roles in different virus-encoded functions, such as virus assembly/disassembly, movement and vector transmission. Interestingly, filamentous bacteriophages containing a circular single-stranded DNA genome also have a polar structure of virions composed of thousands of helically arranged copies of a single major CP with a few minor proteins at the tips that are essential for various bacteriophage functions.37 Taken together with the previous observations, current data allow us to propose the following working model showing a hypothetical functional role of the potyviral end containing the VPg protein covalently linked with the 5 0 terminus of viral RNA (Figure 4). It has been demonstrated that the VPg interacts with a number of distinct host proteins. Binding of the host translation factors eIF4E or eIF(iso)4E,38,39 as well as the poly(A)-binding protein (PABP)39 to the VPg may stimulate the initiation of translation of the viral RNA. For example, PABP attaching to VPg at the 5 0 terminus of viral RNA could interact also with its normal partner, the poly(A) tail at the 3 0 end (when it becomes available after virus disassembly), thus facilitating circularization of the RNA molecule and hence initiation of its translation.39 The eIF4E translation factor,40 and another host protein, the potyvirus VPg-interacting protein (PVIP)41 interacting with VPg assist potyvirus cell-to-cell movement. Whether these host proteins are in interaction with the tip structure of the particles remains to be determined. Finally, HC-Pro binding to VPg possibly forms a tip, which may serve as a bridge between virus particles and aphid mouthparts, determining virus transmission by aphids. The finding that HC-Pro may be a component of the
Molecular Architecture of Potyvirus Particles
Figure 4. Hypothetical model of a potyviral regulatory switch determining the involvement of the virus particles in different virus functions: (i) VPg attached to the 5 0 -end of virus particles interacts with HC-Pro and possibly neighbouring CP molecules to mediate aphid transmission; (ii) VPg interacts with host factors, possibly eIF4E, PVIP and/or other proteins to facilitate cell-to-cell movement; (iii) VPg interacts with eIF4E and host factors such as PABP to initiate uncoating and translation of viral RNA. The different VPg interactions may be regulated by the phosphorylation status of VPg or it is conceivable that binding of HC-Pro or another protein to VPg may control the interactions.
particle tip complex suggests a mechanism where the self-interaction of HC-Pro in particles with HC-Pro binding to aphid mouthparts facilitates transmission. Recent data on the structural analysis of HC-Pro supports the role of oligomeric forms in aphid transmission.42 The HC-Pro interacts also with the N terminus of the CP potyviral CP.18,31,32,43 Such an interaction is thought to be essential for virus transmission. 12,18,43 The N terminus is exposed in the assembled particle and the interaction with HC-Pro may take place during formation of the tip with the CP domain(s) at one (concave) extremity containing the 5 0 end of RNA, rather than along the entire length of the particles. Furthermore, we can postulate that all the proteins potentially interacting with VPg can compete with each other for such an interaction. Thus, the end of the potyviral particles containing VPg may be a regulatory switch modulating involvement of virus particles in different virus functions. The different terminal supplements may not be an integral part of the potyviral virions, but be associated with them at different stages of the virus infection. It seems conceivable that the switch may be controlled by, for example, either the phosphorylation status of VPg16 or through HC-Pro (and/or other proteins) located at the end of the virus particles (controlling the interactions with the various translation
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Molecular Architecture of Potyvirus Particles
factor(s), PABP or PVIP). One hypothetical reason for the relatively small proportion of tipped particles (approximately 10%) is that they may represent a sub-population of all the potyviral particles that are actively involved in the different virus-specific processes, as suggested above.
11.
12. 13.
Acknowledgements This work was supported by grants from the Leverhulme Trust (F/00766/A to M.E.T.) and INTAS (01-0045 to M.E.T., L.T. and K. M.). SCRI is grant-aided by the Scottish Executive Environment and Rural Affairs Department. The University of Helsinki group was supported by grants 53862 and 206870 from the Academy of Finland (to K. M.). We thank the Electron Microscopy unit of the Institute of Biotechnology, University of Helsinki for help with electron microscopy of PVA samples and Dr S. Butcher for valuable discussions.
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Molecular Architecture of Potyvirus Particles
26. Browning, I. A., Burns, R., George, E. L. & Darling, M. (1995). Development and evaluation of ELISA assays incorporating monoclonal antibodies for the detection of potato A potyvirus. EPPO Bull. 25, 259–268. 27. Keller, D. J. (1991). Reconstruction of STM and AFM images distorted by finite-size tips. Surf. Sci. 253, 353–364. 28. Fritz, M., Radmacher, M., Cleveland, J. P., Allersma, M. W., Stewart, R. J., Gieselman, R. et al. (1995). Imaging globular and filamentous proteins in physiological buffer solutions with tapping mode atomic force microscopy. Langmuir, 11, 3529–3535. 29. Bustamante, C. & Rivetti, C. (1996). Visualizing protein-nucleic acid interactions on a large scale with the scanning force microscope. Annu. Rev. Biophys. Biomol. Struct. 25, 395–429. 30. Ivanov, K. I., Puustinen, P., Garbrenaite, R., Vihinen, H., Ro¨nnstrand, L., Valmu, L. et al. (2003). Phosphorylation of the potyvirus capsid protein by protein kinase CK2 and its relevance for virus infection. Plant Cell, 15, 2124–2139. 31. Manoussopoulos, I. N., Maiss, E. & Tsagris, M. (2000). Native electrophoresis and western blot analysis (NEWeB): a method for characterization of different forms of potyvirus particles and similar nucleoprotein complexes in extracts of infected plant tissues. J. Gen. Virol. 81, 2295–2298. 32. Roudet-Tavert, G., German-Retana, S., Delauney, T., Dele´colle, B., Candresse, T. & Le Gall, O. (2002). Interaction between potyvirus helper componentproteinase and capsid protein in infected plants. J. Gen. Virol. 83, 1765–1770. 33. Roberts, I. M. (1986). Immunoelectron microscopy of extracts of virus-infected plants. In Electron microscopy of Proteins: 5. Viral Structure (Harris, J. R. & Horne, R. W., eds), 293–357, Academic Press, New York. 34. Yambao, M. L. M., Masuta, C., Nakahara, K. & Uyeda, I. (2003). The central and C-terminal domains of VPg of Clover yellow vein virus are important for VPg-HC-Pro and VPg-VPg interactions. J. Gen. Virol. 84, 2861–2869.
35. Guo, D., Rajamaki, M.-L., Saarma, M. & Valkonen, J. P. T. (2001). Towards a protein interaction map of potyviruses: protein interaction matrixes of two potyviruses based on the yeast two-hybrid system. J. Gen. Virol. 82, 935–939. 36. Browning-Kelley, M. E., Wadu-Mesthrige, K., Hari, V. & Liu, G. Y. (1997). Atomic force microscopy of specific antigen/antibody binding. Langmuir, 13, 343–350. 37. Marvin, D. A. (1998). Filamentous phage structure, infection and assembly. Curr. Opin. Struct. Biol. 8, 150–158. 38. Wittman, S., Chatel, H., Fortin, M. G. & Liberte, J.-F. (1997). Interaction of the viral protein genome linked of turnip mosaic potyvirus with the translational eukaryotic initiation factor (iso)4E of Arabidopsis thaliana using the yeast two-hybrid system. Virology, 234, 84–92. 39. Le´onard, S., Viel, C., Beauchemin, C., Daignealt, N., Fortin, M. G. & Laliberte´, J.-F. (2004). Interaction of VPg-Pro of turnip mosaic virus with the translation initiation factor 4E and poly(A)-binding protein in planta. J. Gen. Virol. 85, 1055–1063. 40. Gao, Z., Johansen, E., Eyers, S., Thomas, C. L., Ellis, T. H. N. & Maule, A. J. (2004). The potyvirus recessive resistance gene, sbm1, identifies a novel role for translation inititiation factor eIF4E in cell-to-cell trafficking. Plant J. 40, 376–385. 41. Dunoyer, P., Thomas, C., Harrison, S., Revers, F. & Maule, A. (2004). A cysteine-rich plant protein potentiates Potyvirus movement through an interaction with the virus genome-linked protein VPg. J. Virol. 78, 2301–2309. 42. Ruiz-Ferrer, V., Boskovic, J., Alfonso, C., Rivas, G., Llorca, O., Lo´pez-Abella, D. & Lo´pez-Moya, J. J. (2005). Structural analysis of tobacco etch potyvirus HC-Pro oligomers involved in aphid transmission. J. Virol. 79, 3758–3764. 43. Lo´pez-Moya, J. J., Wang, R. Y. & Pirone, T. (1999). Context of the coat protein DAG motif affects potyvirus transmissibility by aphids. J. Gen. Virol. 80, 3281–3288.
Edited by M. Moody (Received 4 November 2005; received in revised form 6 December 2005; accepted 6 December 2005) Available online 21 December 2005
J. Mol. Biol. (2006) 357, 1–8
C OMMUNICATION
An Unusual Structure at One End of Potato Potyvirus Particles Lesley Torrance1†, Igor A. Andreev1,2†, Rasa Gabrenaite-Verhovskaya3 Graham Cowan1, Kristiina Ma¨kinen3 and Michael E. Taliansky1* 1 Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK 2
School of Physics and Astronomy, University of Leeds Leeds LS2 9JT, UK 3
Department of Applied Biology University of Helsinki FIN-00014 Helsinki, Finland
The particles of potato virus Y (PVY) and potato virus A (PVA) potyviruses are helically constructed filaments that are thought to contain a single type of coat protein subunit. Examination of negatively stained virions by electron microscopy reveals flexuous rod-shaped particles with no obvious terminal structures. It is known that some helically constructed rod-shaped virus particles incorporate additional minor protein components, which form stable complexes that mediate particle disassembly, movement or transmission by vectors. Some of this information has been obtained using imaging techniques such as atomic force microscopy. The particles of PVY and PVA were examined by atomic force microscopy and immunogold labelling electron microscopy. Our results show that some of the potyvirus particles contain a protruding tip at one end of the virus particles, which is presumably associated with the 5 0 -end of viral RNA. The tip contains the virus-encoded proteins genome-linked protein and helper-component proteinase. The composition and possible roles of the terminal tip structures in virus biology are discussed. q 2005 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: potyviruses; HC-Pro; VPg; atomic force microscopy; immunogold labelling electron microscopy
The structure of small geometric viruses is based on two types of architecture: either icosahedral (spherical) or helical (rod-shaped). Icosahedral viruses may contain one or two types of designated coat protein (CP), whereas helical viruses are represented by rigid or flexuous (filamentous) rods and are believed to contain a single type of CP molecule. Although the principle governing assembly of helical particles from identical CP subunits, first formulated by Crick and Watson,1 remains valid, there is a growing body of information showing that some additional virus† L.T. and I.A.A contributed equally to this work. Abbreviations used: PVY, potato virus Y; PVA, potato virus A; AFM, atomic force microscopy; IGEM, immunogold labelling electron microscopy; CP, coat protein; HC-Pro, helper component-proteinase; CI, cylindrical inclusion protein; VPg, virus genome-linked protein; PVIP, potyvirus VPg-interacting protein; eIF4E, eukaryotic translation initiation factor 4E; PABP, poly(A) binding protein. E-mail address of the corresponding author: [email protected]
encoded protein molecules may interact with virions and form stable complexes. One of the examples of the complex architecture of helical viruses is provided by long filamentous closterovirus particles that encapsidate the 15–20 kb RNA genome. In addition to the designated CP, closterovirus particles contain four virus proteins that form a short (w70 nm) tail at one end of the virion.2–7 Such tails seem to be an integral part of the particles, playing a role in intercellular virus movement.6,7 Another example is the potato virus X (PVX) movement protein TGBp1, which attaches to one of the extremities of its filamentous virions and mediates their destabilization and disassembly in a 5 0 to 3 0 direction.8,9 The virus capsid readthrough protein of potato mop-top virus was also localized near one (concave or RNA 5 0 -end) extremity of the virus particles.10,11 The Potyvirus genus contains the largest number of virus species, with about 180 members, causing significant losses in a broad range of plants. The potyvirus genome consists of a single-stranded RNA molecule of about 10 kb, which is translated into a polyprotein that is proteolytically processed
0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
2
Molecular Architecture of Potyvirus Particles
Figure 1. AFM analysis of potato potyvirus particles. Virus particles were diluted to w5 ng/ml in 0.02 M phosphate buffer (pH 7.3), and 5–10 ml was placed onto freshly cleaved mica strips for 5–15 min. The strips were rinsed with deionized water and vacuum-dried at room temperature. Imaging of particles was done in the tapping mode7,19,20,22 (a)–(e) in air or (d) water at a frequency of 300–380 kHz on a NanoScope IIIa multimode scanning probe microscope (Digital Instruments, Santa Barbara, CA), using standard AFM silicon-nitride cantilevers with a length of 123 mm (Nanosensors GmbH, Neuchatel, Switzerland). Images, including 3-D representations were processed using Nanoscope
Molecular Architecture of Potyvirus Particles
by three virus-encoded proteinases, giving up to ten final protein products, many of which are multifunctional.12 Potyviruses are transmitted by aphids in a non-persistent manner and two potyvirusencoded proteins are involved in this process: CP and the helper component-proteinase (HC-Pro, which also acts as a suppressor of RNA silencing).13 At least four potyviral proteins, CP, the cylindrical inclusion protein (CI, an RNA helicase, which is also required for genome replication), the genomelinked protein (VPg) and 6K2 protein are involved in potyvirus movement.13,14 VPg forms a covalent linkage to the 5 0 end of the viral RNA being therefore an integral part of potyvirus particles.15,16 The location of VPg at the 5 0 -end of the viral RNA, in place of an mRNA cap structure may indicate another role in the initiation of translation.17 HCPro is able to interact with the virions, possibly serving as a bridge between virion CP and a putative receptor in the aphid mouthparts. 18 Collectively, these observations allow us to postulate a more complex molecular architecture of potyviral particles than just uniform flexuous rods. Here, we determine the complex morphology of the potyviruses potato virus Y (PVY) and potato virus A (PVA) particles using both atomic force microscopy (AFM) and immunogold labelling electron microscopy (IGEM). We demonstrate the presence of protrusions (tips) at one end of the particles that are attached to the virions, at least at some stages of the virus infection process. The possible implications of our data for the function of these tips as terminal protein complexes in virus replication, transport and aphid transmission are discussed in relation to other plant viruses with helical particles. To determine the fine details of PVY and PVA virion architecture, we employed AFM as a highresolution technique, which has been used in studies of nucleic acids, proteins and their complexes.19–22 Recent work demonstrated the ability of AFM to investigate the structure of virus particles,9,23,24 including those of closterovirus, beet yellows virus, containing three-segmented tails, without the need for staining.7 PVY particles were purified essentially as described,25 except that during ultracentrifugation the preparation was sedimented through 25% (w/v) sucrose cushions. Purification of PVA particles was done as described.26
3 AFM imaging of intact PVY virions readily revealed the polar nature of approximately 10% of particles (23/210 particles in a first virus sample and 13 of 132 particles in another sample) containing a protruding tip at one virion extremity (Figure 1(a), images 1, 2 and 3), indicating that this was not a chance occurrence. The tips were apparent as w40 (41G6) nm segments that were darker than the virion bodies. In AFM, a darker appearance indicates less height or, in the case of a filament, smaller diameter. Reconstructed 3-D images of the virion tips consistently showed a peculiar morphology with a tapered end (Figure 1(a), images 3 and 5). In contrast, the opposite end of the virion appeared to be blunt, lacking any fine structural features (Figure 1(a), images 4 and 6). However, in other particles both ends were blunt and did not differ in their appearance (Figure 1(b), suggesting that the tip structures are either unstable or that they are present in a sub-population of particles. It is well known that physical measurements made by AFM usually give heights that are reduced slightly by compression and widths that are increased greatly by the effects of AFM tip convolution.27–29 Since compression effects are much smaller than the effects of AFM tip convolution, we present here the height measurements. Statistical evaluation of the tail measurements in cross-sections (as shown in Figure 1(a), image 5) indicated that the tip had a diameter of w 2.5 (2.3G 0.5) nm. In contrast, the w9 (9.0G1.2) nm diameter of the virion body was virtually constant along its entire length. These measurements reveal differences in geometry and/or physical properties between the virion tips and the rest of the virus particles. Similar virion tip morphology (although sometimes represented by a two-segmented tail; Figure 1(c)) was observed in particles of PVA, indicating that the tip structure is conserved, at least in two members of the Potyvirus genus. Approximately 10% of PVA particles (24/253 particles) imaged in the air or liquid environment contained such a tip structure at one end (Figure 1(c) and (d)). It is worth noting that both ends of virus-like particles formed by Escherichia coli-expressed PVA CP (which forms virus-like particles in the absence of viral RNA and other virus-encoded proteins30) analyzed by
software and transferred to Adobe Photoshop for layout. Sample heights and lengths were measured automatically using the Nanoscope software. (a) Typical PVY particles containing a protruding tip at one virion extremity indicated by arrows (panels 1 and 2; the scale bar represents 250 nm). Panels 3, 4, 5 and 6 show 3-D images (panels 3 and 4) and the cross-sections (panels 5 and 6) of the protruding tip (panels 3 and 5) and the opposite blunt end (panels 4 and 6) of PVY particles. (b) PVY particles containing no protruding tips (panels 1 and 2; the scale bar represents 250 nm). Panels 3, 4, 5 and 6 show 3-D images (panels 3 and 4) and the cross-sections (panels 5 and 6) of the blunt ends of PVY particles. (c) and (d) Typical PVA particles containing a protruding tip at one virion extremity indicated by arrows, imaged in (c) air or (d) water (panel 1; bar, 250 nm). Panels 2, 3, 4 and 5 show 3-D images (panels 2 and 3) and the cross-sections (panels 4 and 5) of the protruding tip (panels 3 and 5) and the opposite blunt end (panels 2 and 4) of PVA particles. (e) Typical viruslike particles formed by Escherichia coli-expressed PVA CP in the absence of viral RNA and other virus-encoded proteins (panel 1; the scale bar represents 1000 nm). Panels 2, 3, 4 and 5 show 3-D images (panels 2 and 3) and the cross-sections (panels 4 and 5) of the blunt ends of PVA-like particles.
4
Molecular Architecture of Potyvirus Particles
Figure 2. Immunogold labelling of potyvirus particles. Immunogold electron microscopy for PVY particles was done as described.33 Briefly, pyroxylin-filmed copper electron microscope grids coated with preparations of mAb 39 (2 mg/ml in PO buffer (0.07 M phosphate buffer, pH 7.0) were floated on drops of virus particles (diluted in PO buffer), incubated for 1 h then washed and incubated on drops of anti-HC-Pro antibody (2 mg/ml in PO buffer) for 1 h then washed and incubated on drops of anti-rabbit gold (15 nm) conjugate (Amersham Biosciences; 1/100 in PO buffer containing 0.1% (w/v) BSA and 0.05% (v/v) Tween 20) for 1 h. All incubations were at room temperature (w22 8C). Grids were stained with a few drops of sodium phosphotungstate (pH 6.5) before examination in a Jeol 1200 Ex electron microscope. PVA particles were labelled in essentially the same way, as described by.16 Purified polyclonal anti HC-Pro antibody produced against the native recombinant PVA HC-Pro was diluted 1:25 for immunogold labelling. (a) Panels 1–5 show individual particles of PVY labelled with gold at one extremity; (b) lower magnification image illustrating gold associated with particle ends (arrowed) and low levels of gold particles elsewhere on the grid; (c) low-magnification image showing PVY particles treated with pre-immune serum (negative control); (d) gold labelling of PVA particle at one extremity.
AFM as controls, looked similar and did not contain any tip structures (Figure 1(e)). Previous work has demonstrated an interaction between potyviral HC-Pro and virus particles or CP.18,31,32 To investigate whether HC-Pro was associated with PVY particles, ELISA experiments were done following the method of Roudet-Tavert et al.,32 where virus particles were trapped from infected leaf extracts in ELISA plates coated with a monoclonal antibody (mAb) against the CP (mAb SCR 39). HC-Pro or virus CP were detected by antiHC-Pro or anti-CP antiserum (rabbit) followed by anti-rabbit alkaline phosphatase conjugate. In the tests with the HC-Pro antiserum, typical absorbance values (A405 nm) obtained with virus-infected extracts were approximately 0.27, indicating an association of HC-Pro with virus particles but the values obtained with anti-CP antiserum were typically O2.0, showing that many more CP molecules than HC-Pro molecules were associated with virus
particles (A405 nm values of control non-infected samples were subtracted and all values were recorded after incubation of the substrate for 1 h). To ascertain whether HC-Pro may comprise part of the potyviral tips revealed by AFM, we analyzed the structure of PVY particles by IGEM using the same antibodies as in the ELISA tests. In these experiments, PVY particles were trapped on grids with anti-CP MAb SCR 39 and then the grids were incubated with anti-HC-Pro antibody followed by anti-rabbit gold conjugate (following the methods of Roberts33). In these experiments, a few PVY particles (17 of 600 particles, that is about 3%) were labelled with gold at one extremity (Figure 2(a)). Although relatively small numbers of particles were labelled with gold, there was little background labelling and no gold associated elsewhere on the particles (Figure 2(b)). The pre-immune serum control did not label the particles at all (Figure 2(c)). Similar results were obtained when
Molecular Architecture of Potyvirus Particles
an antiserum to PVA HC-Pro was used in IGEM experiments with PVA particles. A few particles (2%; 4/200 particles in each of three experiments) were labelled at one extremity (Figure 2(d)). In control experiments, the pre-immune serum did not label the PVA particles at all, and the virus-like PVA particles formed by Escherichia coli-expressed PVA CP containing no HC-Pro30 were not labelled with anti-HC-Pro antibody. These results confirm the specificity of labelling the potyvirus particles with HC-Pro antibodies at one extremity. The protruding tips were not obvious when potyvirus particles were examined by electron microscopy (EM). One reason for this apparent difference in appearance could be because the PVY tip is less efficient at excluding the sodium phosphotungstate during negative staining: unlike EM, AFM does not require staining. Nevertheless, the lack of terminal tapered tips in some PVY (Figure 1(b)) and PVA particles as well as in all PVAlike particles produced in E. coli from the recombinant CP (Figure 1(d)) strongly confirm that the potyviral terminal tips visualized by AFM are not artefacts resulting from structural differences between concave and convex ends of virus helical rods. Moreover, both particle ends of another filamentous virus, PVX (in spite of the presence of TGBp1 protein at its 5 0 -extremity)9 or of the rodshaped tobacco mosaic virus (TMV) analyzed by AFM were blunt and did not differ in their appearance.7,9 The corollary of this work is that the potyviral protruding tips are located at one end of virus particles. Previously, both HC-Pro and VPg were shown to self-interact, and HC-Pro was shown to interact with VPg in the yeast two-hybrid system.34,35 VPg associated with the 5 0 -end of viral RNA in particles was shown to be available for protein–protein interactions on the surface of the particles.16 Taken together with our data, these observations suggest strongly that the HC-Pro may bind to the end of the virion containing a 5 0 -RNA terminus forming the tip either singly or as a dimer, in association with VPg (and possibly with some additional proteins). We could not employ IGEM to confirm this suggestion because of the inability of electron microscopy to resolve the potyviral protruding tips and, hence, we visualised complexes of PVY particles with anti-VPg and anti-HC-Pro antibodies using AFM. After incubation with non-specific preimmune serum or antibodies raised against proteins encoded by unrelated viruses such as coat proteins of PVX or TMV, the PVY particles were indistinguishable from non-treated particles, indicating that there was no non-specific binding between IgG and virions of PVY. In contrast, it was seen clearly that after incubation with specific anti-VPg antibody preparation, new and brighter “spots” appeared on the majority (about 70–80%) of protruding tips of PVY particles as shown in Figure 3(a). No spots were associated with blunt ends of tipped particles or distributed elsewhere
5
Figure 3. AFM images of PVY particles labelled with (a) anti-VPg and (b) anti-HC-Pro antibodies. Virus particles were diluted, immobilised on freshly cleaved mica strips and dried as described in the legend to Figure 1. Antibodies (IgG; 1.2 mg/ml) in 0.01 M phosphate buffer (pH 7.2) containing 0.05% (v/v) Tween 20 were applied to the antigen-covered mica surface and kept at room temperature for 30 min. The IgG solution was drawn off with a pipette, and the surface was washed three times with water. AFM imaging of virus–antibody complexes was done as described in the legend to Figure 1. Arrows indicate antibody “spots” on one of the ends of PVY particles (panels 1). Panels 2 and 3 show PVY particles at higher magnification, revealing association of antibody “spots” with protruding tips of the particles. The scale bars represent 200 nm (panels 1); 120 nm (panels 2); 60 nm (panels 3).
on the particles. The average height of the tip-associated bright spots was approximately 10 nm, which corresponds very well to the dimensions of individual complexes of the tips with antibody.36 However, the widths of some spots varied, reaching up to 80 nm. Such large spots probably correspond to side-to-side aggregation of IgG with up to five or six molecules per aggregate, which is consistent with a previous report that at high concentrations, antibodies tend to aggregate.36 Similar spots on the majority (approximately 70%) of protruding tips appeared after incubation of PVY particles with anti-HC-Pro antibody (Figure 3(b)) whereas no spots were observed on blunt ends or elsewhere on the particles. Collectively, these results confirm specificity of labelling of the PVY tips with anti-VPg and anti-HC-Pro antibodies.
6 Although the reason for fewer virus particles labelled with anti-HC-Pro antibody in the IGEM experiments than in AFM experiments is not completely clear, a possible reason could be the reduced stability of PVY tips subjected to the IGEM procedure. It should be noted that the majority of PVY particles (about 60%) containing no apparent tips were also labelled at one end with anti-VPg antibody, whereas anti-HC-Pro antibody did not label non-tipped PVY particles at all. Taken together with the observation that VPg is covalently linked to the 5 0 -end of viral RNA,15,16 these results show that the potyviral protruding tips contain HC-Pro and are associated with VPg located at the RNA 5 0 terminus. It is worth noting that some other flexuous rod-shaped viruses belonging to distinct evolutionary and taxonomic groups such as closteroviruses,7 potexviruses,8,9 and pomoviruses,11 also contain additional virusencoded proteins or their complexes at the same particle extremity, demonstrating a general tendency among viruses sharing filamentous virus particle morphology and supporting the idea that the architecture of “simple” geometric viruses may be more “complex” than previously thought. The structural supplements at the virion end containing the 5 0 -end of RNA may potentially play essential roles in different virus-encoded functions, such as virus assembly/disassembly, movement and vector transmission. Interestingly, filamentous bacteriophages containing a circular single-stranded DNA genome also have a polar structure of virions composed of thousands of helically arranged copies of a single major CP with a few minor proteins at the tips that are essential for various bacteriophage functions.37 Taken together with the previous observations, current data allow us to propose the following working model showing a hypothetical functional role of the potyviral end containing the VPg protein covalently linked with the 5 0 terminus of viral RNA (Figure 4). It has been demonstrated that the VPg interacts with a number of distinct host proteins. Binding of the host translation factors eIF4E or eIF(iso)4E,38,39 as well as the poly(A)-binding protein (PABP)39 to the VPg may stimulate the initiation of translation of the viral RNA. For example, PABP attaching to VPg at the 5 0 terminus of viral RNA could interact also with its normal partner, the poly(A) tail at the 3 0 end (when it becomes available after virus disassembly), thus facilitating circularization of the RNA molecule and hence initiation of its translation.39 The eIF4E translation factor,40 and another host protein, the potyvirus VPg-interacting protein (PVIP)41 interacting with VPg assist potyvirus cell-to-cell movement. Whether these host proteins are in interaction with the tip structure of the particles remains to be determined. Finally, HC-Pro binding to VPg possibly forms a tip, which may serve as a bridge between virus particles and aphid mouthparts, determining virus transmission by aphids. The finding that HC-Pro may be a component of the
Molecular Architecture of Potyvirus Particles
Figure 4. Hypothetical model of a potyviral regulatory switch determining the involvement of the virus particles in different virus functions: (i) VPg attached to the 5 0 -end of virus particles interacts with HC-Pro and possibly neighbouring CP molecules to mediate aphid transmission; (ii) VPg interacts with host factors, possibly eIF4E, PVIP and/or other proteins to facilitate cell-to-cell movement; (iii) VPg interacts with eIF4E and host factors such as PABP to initiate uncoating and translation of viral RNA. The different VPg interactions may be regulated by the phosphorylation status of VPg or it is conceivable that binding of HC-Pro or another protein to VPg may control the interactions.
particle tip complex suggests a mechanism where the self-interaction of HC-Pro in particles with HC-Pro binding to aphid mouthparts facilitates transmission. Recent data on the structural analysis of HC-Pro supports the role of oligomeric forms in aphid transmission.42 The HC-Pro interacts also with the N terminus of the CP potyviral CP.18,31,32,43 Such an interaction is thought to be essential for virus transmission. 12,18,43 The N terminus is exposed in the assembled particle and the interaction with HC-Pro may take place during formation of the tip with the CP domain(s) at one (concave) extremity containing the 5 0 end of RNA, rather than along the entire length of the particles. Furthermore, we can postulate that all the proteins potentially interacting with VPg can compete with each other for such an interaction. Thus, the end of the potyviral particles containing VPg may be a regulatory switch modulating involvement of virus particles in different virus functions. The different terminal supplements may not be an integral part of the potyviral virions, but be associated with them at different stages of the virus infection. It seems conceivable that the switch may be controlled by, for example, either the phosphorylation status of VPg16 or through HC-Pro (and/or other proteins) located at the end of the virus particles (controlling the interactions with the various translation
7
Molecular Architecture of Potyvirus Particles
factor(s), PABP or PVIP). One hypothetical reason for the relatively small proportion of tipped particles (approximately 10%) is that they may represent a sub-population of all the potyviral particles that are actively involved in the different virus-specific processes, as suggested above.
11.
12. 13.
Acknowledgements This work was supported by grants from the Leverhulme Trust (F/00766/A to M.E.T.) and INTAS (01-0045 to M.E.T., L.T. and K. M.). SCRI is grant-aided by the Scottish Executive Environment and Rural Affairs Department. The University of Helsinki group was supported by grants 53862 and 206870 from the Academy of Finland (to K. M.). We thank the Electron Microscopy unit of the Institute of Biotechnology, University of Helsinki for help with electron microscopy of PVA samples and Dr S. Butcher for valuable discussions.
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Edited by M. Moody (Received 4 November 2005; received in revised form 6 December 2005; accepted 6 December 2005) Available online 21 December 2005