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Complex Molecular Architecture of Beet Yellows Virus Particles

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Complex Molecular Architecture of Beet Yellows Virus Particles Complex molecular architecture of beet yellows virus 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 Potyviridae 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 Closteroviridae 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- beet yellows virus (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 viruses 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 citrus tristeza virus (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 retroviruses (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 RNAs 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.
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