Proc. Natl. Acad. Sci. USA Vol. 92, pp. 2470-2473, March 1995 Biochemistry "Rattlesnake" structure of a fiamentous plant RNA virus built of two capsid proteins ALEXEY A. AGRANOVSKY*, DIETRICH E. LESEMANNt, EDGAR MAIsst, ROGER HULLt, AND JOSEPH G. ATABEKOV*§ *A. N. Belozersky Institute of Physico-Chemical Biology and Department of Virology, Moscow State University, 119899 Moscow, Russia; tInstitute of Biochemistry and Plant Virology, Federal Biological Centre for Agriculture and Forestry, D-38104 Braunschweig, Germany; and tJohn Innes Centre, Colney, Norwich NR4 7UH, United Kingdom Communicated by Myron K Brakke, Crete, NE, December 12, 1994 (received for review September 19, 1994) ABSTRACT Elongated particles of simple RNA viruses the p24. Thus, closterovirus particles, unlike those of other of plants are composed of an RNA molecule coated with plant viruses with helical symmetry, apparently have a mor- numerous identical capsid protein subunits to form a phologically polar structure composed of two CPs, for which regular helical structure, of which tobacco mosaic virus is we propose the term "rattlesnake" structure. the archetype. Filamentous particles of the closterovirus beet yellows virus (BYV) reportedly contain '4000 identical MATERIALS AND METHODS 22-kDa (p22) capsid protein subunits. The BYV genome encodes a 24-kDa protein (p24) that is structurally related Production of the BYV p24-Specific Antiserum. The clone to the p22. We searched for the p24 in BYV particles by using for expressing the fusion antigen for As-Np24/p35 production immunoelectron microscopy with specific antibodies was based on the D22 plasmid (GenBank accession no. against the recombinant p24 protein and its N-terminal X73475). The BYV-specific cDNA insert in this plasmid peptide. A 75-nm segment at one end of the 1370-nm contains the Nco I site immediately downstream of and filamentous viral particle was found to be consistently in-frame with the sequence coding for the first 20 N-terminal labeled with both types of antibodies, thus indicating that amino acids of the BYV p24 protein. This target sequence was p24 is indeed the second capsid protein and that the amplified by the polymerase chain reaction with oligode- closterovirus particle, unlike those of other plant viruses oxynucleotide primers 24 and 8, as described (10), and theXba with helical symmetry, has a "rattlesnake" rather than I-linearized D22 as template. The amplification product was uniform structure. digested with Bgl II and Nco I and ligated into the similarly cut plasmid pGEM1521 (6) to yield an intermediate construct. The orthodox design of elongated RNA-containing plant The sequence of the 35-kDa protein gene ofred clover necrotic viruses, exemplified in molecular biology textbooks by the mosaic virus from the pRC2IG5'Nco plasmid (11) was in- structure of tobacco mosaic tobamovirus, is the helical array serted into this construct between Nco I and Xba I sites. The of an RNA molecule coated with numerous identical capsid insert was excised with Bgl II and ligated into the BamHI- protein (CP) subunits encoded by a single viral gene (1, 2). digested expression plasmid pQE-60 (Diagen, Dusseldorf, Beet yellows virus (BYV), a member of the closterovirus Germany). The recombinant Np24/p35 protein (the N- is terminal peptide of the BYV p24 fused to the 35-kDa protein group, transmitted by aphids semipersistently to a wide in which the C-terminal 39 amino acids were replaced with a range ofplant species and causes important losses in sugar beet tag of six histidines) was expressed in transformed Escherichia crops (3). The virus has very flexuous filamentous particles coli M15 cells and purified from cell lysates on nickel-charged 1250-1450 nm long, with a helix pitch of 3.75 nm and 8.5 CP nitrilotriacetic acid-agarose (12). The polyclonal antiserum subunits per helix turn (3, 4). Although evolutionarily related (As-Np24/p35) was raised in mice by three subcutaneous to the "tobamo-like" plant viruses, BYV has a genome that is injections (each with 0.1 mg of the protein), made at 2-week 2.5 times larger than tobacco mosaic virus (5, 6). This differ- intervals. Antiserum was drawn 3 weeks after the last injection. ence may be due, in part, to acquisition of foreign coding Rabbit antiserum to the purified BYV particles (As-BYV) was sequences via RNA recombination and to gene duplication in from the stock of the Federal Biological Research Centre closterovirus genomes (6, 7). The BYV genome contains a (Braunschweig). Preparation of the polyclonal antiserum to peculiar tandem of genes, one for the 22-kDa CP (p22) and the the full-sized recombinant BYV p24 (As-p24) was described other for a structurally related 24-kDa protein (p24). It has (10). been suggested that the p24 gene evolved by duplication of and Immunoelectron Microscopy (IEM). BYV (Ukrainian and subsequent divergence from the downstream CP gene (8). German isolates) was maintained on Tetragonia expansa plants Similar tandem arrangement of normal and aberrant CP genes and virions were isolated by differential centrifugation (13). is found in another aphid-transmissible closterovirus, citrus Purified virion preparations were kept in 10 mM sodium tristeza virus (CTV; ref. 8). Interestingly, the genes for CP and borate, pH 7.0/50% (vol/vol) glycerol at -20°C until use. its duplicate have diverged much more and are transposed in Crude sap extracts were prepared by homogenizing freshly the whitefly-transmissible lettuce infectious yellows clostero- infected T. expansa leaves with vein-clearing symptoms (10 virus compared to BYV and CTV (7). Heretofore, only a single days after inoculation) in 0.1 M sodium phosphate (pH 7.0; CP species has been found in closterovirus particles, and it has PB). For antibody decoration and immunogold labeling, the been speculated that the CP duplicates may be involved in BYV particles were allowed to adsorb onto electron micro- long-distance and cell-to-cell movement of viral infection scopic grids for 5 min, followed by washing with PB and rather than in virion formation (7-9). incubation with antisera for 30 min. For decoration, the We show here the presence of a distinct tail at one end of As-Np24/p35 and As-BYV were diluted 1:50 and 1:800, the BYV particles that is specifically labeled with antibodies to respectively. For some experiments, As-Np24/p35 was pread- The publication costs of this article were defrayed in part by page charge Abbreviations: BYV, beet yellows virus; CP, capsid protein; IEM, payment. This article must therefore be hereby marked "advertisement" in immunoelectron microscopy; CTV, citrus tristeza virus. accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 2470 Downloaded by guest on September 25, 2021 Biochemistry: Agranovsky et aL Proc. Nati Acad. Sci USA 92 (1995) 2471 sorbed with the purified full-sized p24 as described (10). Prior we raised an antiserum specific to the N-terminal peptide of to immunogold labeling with secondary goat antibody-gold the p24, which has no counterpart in the p22 sequence (8). To complexes, the antibody-labeled specimens were blocked with this end, a recombinant antigen (Np24/p35) was engineered to 0.5% bovine serum albumin and washed with PB containing include three heterologous parts, the N-terminal unique pep- 0.15 M NaCl and 0.05% Tween 20 (14). tide of the p24 as the key immunogen, an unrelated 35-kDa protein of red clover necrotic mosaic virus as a carrier, and a RESULTS AND DISCUSSION C-terminal tag of six histidines to purify the fusion protein on IEM, employed here for testing the presence of the second nickel-charged nitrilotriacetic acid-agarose (12). On Western structural protein in BYV, is sensitive enough to allow detec- blots, purified Np24/p35 showed clear positive reaction with the tion of single epitopes on viral particles (14). In pilot tests, we As-p24, and the polyclonal antiserum As-Np24/p35 selectively used polyclonal antisera to purified BYV and to the recom- recognized the full-sized recombinant p24 (data not shown). binant BYV p24 protein expressed in bacteria (10). Since the In IEM tests, As-Np24/p35 (Fig. 1A) and As-p24 (data not p22 and p24 of BYV may be serologically cross-reactive (10), shown) consistently labeled a 75-nm "tail" segment at one end, b s. .s, t \ X o A., W §0 FIG. 1. Demonstration of a distinct tail in BYV particles by IEM of crude extract of virus-infected T. expansa plants. (A) Labeling with mouse polyclonal antiserum to the N-terminal peptide of the BYV p24 protein (As-Np24/p35). (B) Labeling with rabbit anti-BYV serum (As-BYV) and immunogold labeling with secondary goat anti-rabbit 10-nm gold conjugate. (C) Unlabeled (uranyl acetate stained) particle with a distinct terminal structure. (D) The terminal structure of the particle shown in C but at higher magnification. Arrows tag the distinct virion tail. (Bars: A-C, 300 nm; D, 100 nm.) Downloaded by guest on September 25, 2021 2472 Biochemistry: Agranovsky et at Proc. Natl. Acad Sci USA 92 (1995) but not the main part, of the viral particles in crude sap extracts assisted predictions of its structure. Thus, the amino acid of BYV-infected plants and in purified virion preparations. sequence of the p24 fits the general alignment of the CPs of Preadsorbtion of the As-Np24/p35 with the full-sized recom- filamentous plant viruses and specifically retains the con- binant p24 rendered it ineffective in labeling the tail (data not served hydrophobic and charged amino acid residues that are shown), thus indicating that the tail labeling was due to the thought to ensure the proper folding of these proteins (8). The antibodies specific to the N-terminal peptide of the BYV p24 fact that As-Np24/p35 specifically decorated the BYV parti- but not to those specific to the 35-kDa protein of red clover cles (Fig.