Complex molecular architecture of beet yellows particles

Valera V. Peremyslov*†, Igor A. Andreev†‡§, Alexey I. Prokhnevsky*, George H. Duncan‡, Michael E. Taliansky‡¶, and Valerian V. Dolja*¶

*Department of Botany and Plant Pathology and Center for Gene Research and Biotechnology, Oregon State University, Corvallis, OR 97331; ‡Gene Expression Programme, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, United Kingdom; and §Electronic Engineering and Physics Division, University of Dundee, Dundee DD1 4NH, United Kingdom

Communicated by Bryan D. Harrison, Scottish Crop Research Institute, Dundee, United Kingdom, January 14, 2004 (received for review November 17, 2003) Closteroviruses possess exceptionally long filamentous virus TMV, whose packaging signal is located at a distance of Ϸ1,000 particles that mediate protection and active transport of the nucleotides from the RNA 3Ј terminus, virions of the family genomic RNA within infected plants. These virions are composed and the genus Potexvirus start their assembly near of a long ‘‘body’’ and short ‘‘tail’’ whose principal components the RNA 5Ј terminus (11, 12). However, attempts to achieve are the major and minor capsid proteins, respectively. Here we efficient in vitro self-assembly of these filamentous virus particles use biochemical, genetic, and ultrastructural analyses to dissect were largely unsuccessful (7), suggesting that host factors may be the molecular composition and architecture of particles of beet required to aid assembly. yellows virus, a closterovirus. We demonstrate that the virion Virus species from the family have long tails encapsidate the 5؅-terminal, Ϸ650-nt-long, part of the viral filamentous virus particles that encapsidate the 15- to 20-kb RNA. In addition to the minor capsid protein, the viral Hsp70- positive-strand RNA genome (13, 14). The 15.5-kb genome of homolog, 64-kDa protein, and 20-kDa protein are also incorpo- (BYV), codes for at least 10 proteins rated into the virion tail. Atomic force microscopy of virions (Fig. 1A). Recent studies demonstrated that BYV virions revealed that the tail possesses a striking, segmented morphol- incorporate at least five of these proteins (15–18). The major ogy with the tip segment probably being built of 20-kDa protein. CP forms a long virion body that is analogous to ‘‘ordinary’’ The unexpectedly complex structure of closterovirus virions has filamentous virions. A minor capsid protein (CPm) is a CP important mechanistic and functional implications that may also homolog that assembles at one end of the virion to Ϸ5% of its apply to other virus families. length (15, 19). The third virion protein is a 64-kDa protein (p64) that harbors a CP-like C-terminal domain and a unique atomic force microscopy ͉ closterovirus ͉ RNA encapsidation ͉ virion N-terminal domain (17). structure ͉ virus transport Perhaps the most remarkable component of BYV virions is the virus-encoded homolog of cellular Hsp70 molecular chap- irus particles (virions) provide the functions of genome erones (Hsp70h) (16, 20). So far, only closteroviruses have been Vpackaging, protection, and transfer between host organisms, found to harbor a Hsp70 gene, although papovaviruses and a tissues, and cells. All virions possess a protein shell that sur- baculovirus code for Hsp70 cochaperones called J-domain pro- rounds the viral genome. In addition, enveloped acquire teins (10, 21). In addition to functions in DNA replication and a lipoprotein ‘‘overcoat’’ by budding through the cell membrane. tumorigenesis, the papovavirus J-domain protein was implicated Two types of shell architecture exist: icosahedral and helical. A in assembly of the icosahedral virions. The fifth virion protein of textbook example of helical architecture is provided by the rigid, BYV is a 20-kDa protein (p20) that interacts with Hsp70h (18). rod-shaped virions of tobacco mosaic virus (TMV) that are BYV CP, CPm, p64, and Hsp70h are each required for virion formed from a single RNA molecule and Ϸ2,000 copies of a assembly and subsequent cell-to-cell movement (17, 22–24), capsid protein (CP), with the RNA winding in a helical path that indicating that the formation of the ‘‘tailed’’ virions is a prere- follows the arrangement of the CP subunits (1, 2). Variations on quisite for intercellular trafficking. In contrast, p20 is dispens- this theme are seen in many families of rod-shaped (3) and able for virion assembly and cell-to-cell movement, but is filamentous (4) plant RNA viruses. Based on amino acid se- necessary for BYV transport through the plant vascular system quence analyses, the CPs that assemble into helical capsids of (18). A CPm, Hsp70h, and p64 ortholog were also found in the plant viruses belong to two distinct protein families that might virions of lettuce infectious yellows virus (25) and were impli- have diverged from a single ancestor (5, 6). cated in assembly of (26). Both these viruses In the TMV, virions self-assemble under appropriate con- belong to the family Closteroviridae and are distantly related to ditions, because all required information is contained within BYV. the virion components (7); that is, the RNA possesses an origin Despite the rapid progress in closterovirus research, the exact of assembly or packaging signal, and the folding of the CP architecture of the virion has not been established. Here we molecule facilitates interactions with RNA and other CP determine the molecular composition of the virion tails and molecules. Although ‘‘automation’’ is used by virtually all reveal their complex morphology by using atomic force micros- viruses for assembly of the virions or parts thereof, the copy (AFM). Possible implications of our data for virion assem- complex architecture of some virions requires participation of bly and function in virus transport are discussed in relation to scaffolding or chaperone proteins that normally are not in- other plant viruses with helical capsids. corporated into the mature virion. Some of these proteins are

virus-encoded (8), whereas others are borrowed from the cell Abbreviations: CP, capsid protein; TMV, tobacco mosaic virus; BYV, beet yellows virus; CPm, molecular chaperone machinery. Among the latter, members minor capsid protein; p64, 64-kDa protein; Hsp70h, Hsp70 homolog; p20, 20-kDa protein; of Hsp70 family of proteins are recruited for virion formation AFM, atomic force microscopy; EM, electron microscopy. or transport of virion components by diverse RNA and DNA †V.V.P. and I.A.A. contributed equally to this work. viruses and (9, 10). ¶To whom correspondence may be addressed. E-mail: [email protected] or doljav@ Except for TMV, little is known about the assembly mecha- science.oregonstate.edu. nisms of helical plant viruses. It has been suggested that, unlike © 2004 by The National Academy of Sciences of the USA

5030–5035 ͉ PNAS ͉ April 6, 2004 ͉ vol. 101 ͉ no. 14 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0400303101 Downloaded by guest on September 25, 2021 analysis was performed according to standard procedures (16). Membranes were incubated with polyclonal antibodies to CP (1:10,000 dilution), CPm (1:2,000 dilution; ref. 23), Hsp70h (1:4,000 dilution; ref. 28), p64 (1:1,000 dilution; ref. 17), or p20 (1:1,000; ref. 18), followed by goat anti-rabbit IgG-horseradish peroxidase conjugate (1:5,000 dilution, Bio-Rad) and visual- ized by chemiluminescence (ECL Kit, Amersham Pharmacia).

Northern Hybridization Analyses. Total RNA was extracted from virion tail pellets or from intact BYV particles by using TRIzol (Invitrogen) according to the manufacturer’s protocol, and the hybridization analysis was carried out as described (27). To generate [32P]UTP-labeled, negative-sense RNA probes, 5Ј and 3Ј regions of the BYV genome were cloned downstream of the T7 RNA polymerase promoter, linearized to yield cDNA templates of 534 and 590 nucleotides, respectively, and tran- scribed in vitro. Prehybridization and hybridization steps were carried out at 65°C in NorthernMax Prehyb͞Hyb buffer (Am- bion, Austin, TX). To determine the size distribution of the derived from the fragmented virions, a specially made set of RNA size markers was designed. These positive-sense, 5Ј-coterminal BYV RNA fragments were derived in the same way as the RNA probes described above, except that the SP6 RNA polymerase promoter was introduced upstream of the 5Ј terminus of the BYV cDNA. In vitro transcription was used to generate a mixture of the RNAs corresponding to 5Ј-proximal 140, 270, 490, 840, 1,580, and 2,150 nucleotides of BYV RNA.

These RNA fragments were detected by hybridization with the MICROBIOLOGY 5Ј end, negative-sense RNA probe.

Atomic Force and Electron Microscopy. BYV virions or their frag- ments were diluted to Ϸ5ng͞␮l in 0.02 M phosphate buffer (pH 7.3), and 5–10 ␮l were placed onto freshly cleaved mica strips for 5–15 min. The strips were rinsed with deionized water and Fig. 1. (A) Functional map of the BYV genome showing the ORFs that code vacuum-dried at room temperature. Imaging of particles was for leader proteinase (L-Pro), replication proteins that possess methyltrans- done in the tapping mode (29–31) in air at a frequency of ferase (MET), RNA helicase (HEL), and RNA polymerase (POL) domains, 6-kDa protein (p6), Hsp70-homolog (Hsp70h), 64-kDa protein (p64), minor capsid 300–380 kHz on a NanoScope IIIa multimode scanning probe microscope (Digital Instruments, Santa Barbara, CA) by using protein (CPm), major capsid protein (CP), 20-kDa protein (p20), and 21-kDa ␮ protein (p21). (B) Immunoblot analyses of the intact or sonicated virions after standard AFM silicon-nitride cantilevers with a length of 123 m separation in a sucrose gradient. The fraction numbers from 1 (left) to 15 (Nanosensors, Neuchatel, Switzerland). Images, including 3D (right) are shown. The types of antibodies used for analysis are specified at the representations were processed with NANOSCOPE software and right. (C) Transmission EM analysis of the virion tails isolated from a sucrose transferred to PHOTOSHOP (Adobe Systems, Mountain View, gradient. CA) for layout. Sample heights and lengths were measured automatically with the NANOSCOPE software. Structural models of BYV tails were developed by averaging the dimensions Materials and Methods (heights and lengths) of the tail segments in cross sections (see Isolation and Immunoblot Analyses of the Virion Tails. Wild-type Fig. 5, which is published as supporting information on the PNAS virions of the BYV-California isolate were purified from web site). Data were mean Ϯ standard errors for 100 virus Nicotiana benthamiana plants as detailed (16). Mutation particles observed in three independent experiments. For elec- Nop20, which inactivated the start codon of the p20 ORF (27), tron microscopy, fragments of the BYV virions were negatively was introduced into the binary vector p35S-BYV-GFP har- stained with 2% uranyl acetate and photographed with a Philips boring a full-length cDNA copy of the BYV genome tagged by CM 10 electron microscope (Philips Electronic Instruments, insertion of the gene encoding the green fluorescent protein Eindhoven, The Netherlands). (BYV-GFP) (18). The resulting mutant was inoculated to N. benthamiana plants by the agroinfection protocol; because of Results the limited systemic spread of the p20-deficient virus, virions Molecular Composition of Virion Tails. Previous work demon- were purified from the green fluorescent areas (18). Wild-type strated that the BYV Hsp70h and p64 proteins are required for or mutant virions were resuspended in buffer 1 (20 mM sodium assembly of the virion tails, but not the bodies (17, 23), phosphate buffer, pH 7.2͞1 mM EDTA͞0.1% Triton X-100) suggesting that each of these proteins is an integral tail and treated with an Ultrasonic Processor XL (Heat Systems͞ component. It was also suggested that the tails are assembled Ultrasonics), at a setting of 20% power output, in 0.5-ml in the proximity of the RNA 5Ј terminus, although the length aliquots with eight 15-s bursts. The fragmented virions were of the RNA fragment encapsidated by the tails was not then loaded on the top of 20–60% sucrose gradients prepared determined (29). To ascertain the molecular composition of in buffer 1 and centrifuged at 100,000 ϫ g for16hina the tails, the virions were sonicated and the resulting random Beckman SW40 rotor at 4°C. Gradients were separated into 15 fragments were separated in sucrose gradients according to fractions; those fractions containing the peak of CPm were their buoyant density. Immunoblot analysis revealed that most pooled and diluted in buffer 1; virion fragments were recov- of the CP remained at the top of the gradient (fractions 1–3) ered by ultracentrifugation for6hat150,000 ϫ g. Immunoblot with a minor peak in fractions 6–8 (Fig. 1B). In contrast, when

Peremyslov et al. PNAS ͉ April 6, 2004 ͉ vol. 101 ͉ no. 14 ͉ 5031 Downloaded by guest on September 25, 2021 nonsonicated control preparations were centrifuged, the intact virions were concentrated in the bottom fractions 10–15, with only a small amount of CP being in fraction 1. Because the buoyant density of the protein–RNA complexes is propor- tional to their RNA content, these data suggest that ultrasound treatment resulted in dissociation of the CP from the RNA. Virtually no CPm was detected in the top gradient fractions, and most of it was concentrated in fractions 6–8 (Fig. 1B). Because CPm constitutes a major virion tail component, this result indicated that the tails withstood sonication and re- tained the encapsidated RNA. Electron microscopy (EM) analysis of fractions 6–8 revealed the presence of abundant virion fragments varying in length from 40 to 110 nm, with an average length of Ϸ70 nm. These fragments exhibited a peculiar polar morphology with a distinct staining pattern for a tapered segment present at one end (Fig. 1C). Remarkably, immunoblot analyses demonstrated that Hsp70h, p64, and p20 each peaked in the same fractions as CPm did (Fig. 1B). Colocalization of these proteins in the sucrose density gradient with a principal tail component, CPm, clearly suggests that virion tails incorporate Hsp70h, p64, and p20. Note that Hsp70h, p64, and p20 were undetectable in the top gradient fractions where the bulk of CP was present. Because the CP is a constituent the particle body, this result can be interpreted to mean that Hsp70h, p64, and p20 are not incorporated into bodies. The origin of the relatively minor amount of CP in fractions 6–8 remains unclear. One possibility is that short body segments remain attached to tails. Another Fig. 2. Analyses of the RNAs and proteins present in gradient fractions 6–8 possibility is the presence of ‘‘body-only’’ fragments in frac- after one or two cycles of centrifugation. (A) Northern hybridization analysis Ј tions 6–8. If this were the case, association of minor amounts of the RNA by using a probe complementary to the 5 -terminal 534 nucleotides of BYV RNA. (B) Northern hybridization analysis of the RNA by using a probe of Hsp70h, p64, and p20 with the particle bodies cannot be complementary to the 3Ј-terminal 590 nucleotides of BYV RNA. Note that the rigorously excluded. membrane exposure time in B was 2.5 times longer than that in A.(C and D) To reveal the nature of the RNA encapsidated in the tails, two Immunoblot analyses of the CPm and CP by using cognate antibodies, respec- consecutive cycles of sucrose gradient purification were con- tively. V, virion RNA marker; M, RNA size standards corresponding to 5Ј- ducted, and RNA was isolated from combined fractions 6–8of proximal segments of BYV RNA with their length in nucleotides indicated at each gradient. To determine which RNA terminal region (if any) the right; lanes 1 and 2, RNAs or proteins isolated after one or two cycles of is harbored by a tail, Northern hybridization analyses of these gradient fractionation, respectively. RNAs by using negative-sense RNA probes complementary to Ј Ј the 5 -terminal 534 nucleotides or to the 3 -terminal 590 nucle- Ϸ otides of the BYV genome were done. The 5Ј probe revealed a as 90-nm-long virion segments that were darker than the virion prominent RNA band of Ϸ650 nucleotides after the second cycle bodies. In AFM, a darker appearance indicates a lesser height or, of fractionation (Fig. 2A, lane 2). Much weaker signals were in the case of a filament, a lesser diameter. Reconstructed 3D detected by using the 3Ј-terminal probe (Fig. 2B). It is important images of the virion tails consistently showed a peculiar three- to emphasize that visualization of the signals in Fig. 2B required segment morphology with a tapered-tip segment (Fig. 3B). A 2.5 times longer exposure of the membrane than for the 5Ј- similar topography of segmented BYV tails was revealed by terminal probe. This is obvious from comparing the signals from AFM in a liquid environment (data not shown). In contrast, the our standard, a genomic RNA that has equimolar amounts of the opposite end of the virion appeared to be blunt and lacked any 5Ј-terminal and 3Ј-terminal regions (Fig. 2 A and B, lanes V). fine structural features (Fig. 3C). Both particle ends of another Even more importantly, the signal from 3Ј-terminal fragments of filamentous virus, potato virus X, or of the rod-shaped TMV BYV genome present in fractions 6–8 was dramatically reduced analyzed by AFM as controls, were blunt and did not differ in after the second cycle of gradient separation (Fig. 2B, lanes 1 and their appearance (Fig. 6, which is published as supporting 2). The amount of CP was also reduced concomitantly (Fig. 2D). information on the PNAS web site). Thus, enrichment of the CPm content relative to CP in fractions The virion tails isolated after sonication exhibited same 6–8 (Fig. 2 C and D) resulted in higher prominence of the three-segment morphology as those seen in the intact virions Ϸ650-nt, 5Ј-terminal RNA segments and in significant depletion (Fig. 3D). In addition, two-segment tail fragments and small of the 3Ј-terminal RNA segments. These results strongly suggest virion fragments of undefined morphology were also occasion- that these 3Ј-terminal RNAs originate from body segments and ally seen in these preparations (not shown). that the CPm and possibly other tail proteins collectively en- Statistical evaluation of the tail measurements in cross sec- capsidate a 5Ј-terminal, Ϸ650-nt segment of the BYV RNA. tions (as shown in Fig. 5) allowed us to develop a model of the tail structure shown in Fig. 3E (Upper). This model indicates that AFM Reveals Complex Morphology of the Tails. The unusual mor- the tail has two Ϸ35-nm-long segments with a diameter of Ϸ8 phology of the virion tails revealed by the EM prompted us to nm, and a tip segment that is Ϸ25 nm long and has a diameter use AFM as an alternative, high-resolution technique, which has of Ϸ6 nm. In contrast, the Ϸ12-nm diameter of the virion body been used in studies of nucleic acids, proteins, and their com- is virtually constant along its entire length. This model likely plexes (30–32). Recent work demonstrated the ability of AFM to reflects differences in geometry and͞or physical properties resolve fine details of virion architecture without the need for between the segments and their connecting areas. It is well staining (33, 34). AFM imaging of intact BYV virions in air known that horizontal dimensions of objects are always overes- readily revealed their polar nature (Fig. 3A). Tails were apparent timated in AFM because of the effect of the geometry of the

5032 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0400303101 Peremyslov et al. Downloaded by guest on September 25, 2021 Fig. 4. (A) AFM-derived 3D image of the two-segmented virion tail and MICROBIOLOGY adjacent part of the body of the BYV mutant Nop 20. Horizontal bar ϭ 50 nm; vertical bar ϭ10 nm. (B) Protein composition of the virions isolated from plants infected with the parental BYV-GFP virus and its Nop20 mutant derivative. Fig. 3. (A–D) AFM images of the BYV virions. (A) A full-length virion. (B) Immunoblot analyses of the virions were done by using the specific antibodies A three-segmented virion tail. (C) An opposite blunt end of the virion. (D) indicated at the right of each blot. Three-segmented tail isolated after sonication of the virions. B–D are 3D images; horizontal bars ϭ 25 nm and vertical bars ϭ 5 nm. (E) Models of the tail regions for the wild-type virions (Upper) and p20-deficient virions Discussion (Lower). Here we describe the unusually complex molecular composi- tion and morphology of the BYV virion. We demonstrate that these filamentous virions are polar with a peculiar, tail-like cantilever (30–32). As a result, the objects appear broader and formation that encapsidates Ϸ650 bases of the 5Ј terminus of longer than their real dimensions. This appearance could explain the genomic RNA. The wild-type virion tails contain at least some differences in the lengths of virion tails measured by using four proteins, including CPm, which is the principal tail either EM or AFM. Alternatively, it is possible that AFM reveals component, and three additional constituents, Hsp70h, p64, the smallest tip tail segment, whereas EM does not. This and p20. The AFM analyses revealed that the tail is thinner outcome could be because the tail is less efficient at excluding than the rest of the virion and that it is subdivided into three the uranyl acetate during negative staining; unlike EM, AFM segments. Very similar tail morphology was observed for a does not require staining. BYV relative, grapevine leafroll-associated virus 2 (35) (Fig. 7, which is published as supporting information on the PNAS The p20 Is Required for Proper Tail Morphology. As mentioned in web site), indicating that the tail structure is conserved at least the introduction, all the virion components of BYV except for among two members of the genus Closterovirus. Because the p20 are required for assembly of cell-to-cell movement- p20-deficient virions contain all other virion proteins but lack competent virions. Because of this, inactivation of either the the pointed-tip segment, we propose that this segment is CP, CPm, p64, or Hsp70h limits virus accumulation in the formed by p20 (Fig. 3E). The fifth virion protein, the major CP, inoculated plants to single cells, thus precluding isolation of is a principal constituent of the long virion body that encap- the high-quality virion preparations required for AFM. How- sidates 96% of the BYV genome. The body morphology ever, we were able to isolate p20-deficient virions by using the appears to be uniform along its entire length and is similar to BYV mutant Nop20 (27) in which the p20 start codon was that described for many diverse filamentous plant viruses inactivated. AFM analysis of these virions revealed that most outside the Closteroviridae family. possess two-segmented tails (Fig. 4A). No virions containing We have recently developed an Agrobacterium-based, in planta all three tail segments were found in these preparations. As model system for investigation of BYV assembly. Using this expected, immunoblot analysis of the mutant virions revealed system, we isolated transport-deficient virions that were assem- normal amounts of the CP, CPm, Hsp70h, and p64, but no p20 bled in the presence of nonfunctional CPm (D. V. Alzhanova and (Fig. 4B). These data confirm our previous results with other V.V.D., unpublished data). These virions lacked not only CPm, p20 mutants (18) and extend the analysis to include p64. In but also Hsp70h and p64, suggesting that these proteins are addition, these data suggest that although p20 is not required coordinately incorporated into tails but not bodies, thus sup- for incorporation of other proteins into virions, it either forms porting conclusions reached in this study. the tip segment of the virion tail or is necessary for its How do the multicomponent BYV virions assemble? The fact assembly. that CPm and the other tail proteins encapsidate the 5Ј-terminal

Peremyslov et al. PNAS ͉ April 6, 2004 ͉ vol. 101 ͉ no. 14 ͉ 5033 Downloaded by guest on September 25, 2021 portion of the RNA strongly suggests that this region harbors a What could be the reason for the attachment of a complex packaging signal, which governs tail formation. Our preliminary transport device to the virions? One possibility is that the results with the trans-encapsidation of a BYV minireplicon by closterovirus virions are exceptionally long, and simply cannot ectopically expressed CPm indicate that this protein alone can be trafficked from cell to cell without an extra mechanism initiate virion assembly (A. J. Napuli and V.V.D., unpublished powered by the ATPase activity of Hsp70h. Plasmodesmal data). Moreover, a recent report on another closterovirus, citrus localization of Hsp70h (38) is also suggestive of a specific role tristeza virus, indicated that the CPm recognizes a packaging played by the thin tails in the entry of virions into the narrow Ј ʈ channels of plasmodesmata. A complementary possibility is a signal near the 5 end of the viral RNA. It is tempting to Ј Ј speculate that the assembly of the virion body from CP involves need for a directional, 5 to 3 transport of the viral RNA. a second packaging signal. Alternatively, the CP could assemble Because the large BYV genome needs to be protected from degradation by the host machinery used in RNA silencing (39, by attachment of the subunits to the surface at one end of a 40), particle disassembly on arrival in the adjacent cells should preformed tail. The roles of Hsp70h and p64 in tail formation are be tightly regulated and coupled to primary translation. One less clear. It seems that, collectively, these proteins may act as of the tail functions could be control of virion stability and molecular rulers for determining the tail’s lengthʈ or as connec- ͞ disassembly. tors between tail and body and or individual tail segments. The evidence suggests that directional transport and virus Perhaps the most remarkable aspect of this study is the particle disassembly could also be used by other elongated demonstration that Hsp70h is an integral component of the viruses. Such functions could be performed by the potyviral VPg, virion tail. Normally, Hsp70 and other protein chaperones are which is attached to the 5Ј terminus of the viral genome and is not incorporated into the final products of chaperone-mediated exposed at the virion’s end (41). VPg has been implicated in processes (36). Thus, closteroviral Hsp70h violates a chaperone potyvirus transport (42, 43). Another possible example is pro- dogma. vided by the potexviral movement protein TGBp1, which at- What are the possible functional implications of the complex taches to virions and mediates their destabilization and disas- architecture of BYV virions? It was demonstrated that, in the sembly in a 5Ј to 3Ј direction (44, 45). Given that closteroviruses, absence of functional CPm, CP is capable of encapsidating the potexviruses, and potyviruses belong to distinct evolutionary entire BYV genome (23), suggesting that the tail has not lineages of positive-strand RNA viruses (46), it seems conceiv- evolved merely for RNA protection. Because the Hsp70h, p64, able that directional intercellular transport could be a general and CPm are each required for BYV cell-to-cell movement, it tendency among viruses sharing filamentous virus particle mor- was proposed that the tail represents a viral transport device phology. (17, 23). This interpretation seems to be even more likely in Note Added in Proof. relation to the tip segment. The only function assigned to the After submission of this manuscript, Satyanaray- ana et al. (47) demonstrated that, in the absence of CP, CPm of citrus tip-forming p20 is long-distance transport of the virions by the tristeza closterovirus recognizes the packaging signal near the 5Ј termi- plant phloem (18). Therefore, it seems plausible that the tip nus of viral RNA. Moreover, in the presence of Hsp70h and p61 segment mediates transport of the virions by plasmodesmata (ortholog of BYV p64), CPm encapsidates Ϸ630 5Ј-terminal nucleotides interconnecting companion cells and sieve elements and͞or of the viral RNA. These results are in agreement with our analyses of the stabilizes the virion tails during their passage through the tails derived from intact BYV particles. hostile phloem environment (37). The latter possibility is We thank Stuart MacFarlane for critical reading of the manuscript. supported by the lesser stability of the p20-deficient virions This work was supported by National Institutes of Health Grant compared with that of the wild-type virions (I.A.A. and R1GM53190B and U.S. Department of Agriculture, National Re- M.E.T., unpublished data). search Institute, Cooperative State Research, Education, and Exten- sion Service Grant 2001-35319-10875 (to V.V.D.) and by a grant-in-aid from the Scottish Executive Environment and Rural Affairs Depart- ʈSatyanarayana, T., Gowda, S. & Dawson, W. O. 22nd Annual meeting of American Society ment and Research Grant F͞00766͞A from the Leverhulme Trust (to for Virology, July 12–16, 2003, University of California, Davis, CA, abstr. 143. M.E.T.).

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