Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, 117871 Moscow, Russia1
Department of Virology and Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow, Russia2
Department of Plant Virology, Microbiology and Biosafety, Federal Biological Research Centre for Agriculture and Forestry, Messeweg 11-12, D-38104 Braunschweig, Germany3
Institute for Plant Protection in Fruit Crops, Schwabenheimer Str. 101, D-69221 Dossenheim, Germany4
National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, 8600 Rockville Pike, Bethesda, MD 20894, USA5
Author for correspondence: Alexey Agranovsky. Fax +7 095 939 31 81. e-mail aaa{at}genebee.msu.su
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Abstract |
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Introduction |
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Although a picture is now emerging of the closterovirus replication-associated genes and their expression profile, little is known about the subcellular sites of virus replication. Closteroviruses cause a number of ultrastuctural effects in cells, of which the earliest to appear and the most characteristic are the BYV-type vesicles formed by membranes of unknown origin. These structures are either discrete vesicles of ca. 100 nm, each delimited by an inner and an outer membrane (and hence appearing as double-membrane bound), or clusters of single-membrane 100 nm vesicles surrounded by a common outer membrane (Esau & Hoefert, 1971 ; Lesemann, 1988
). The vesicle clusters are thought to arise by invaginations of the outer membrane into the lumen, as some 100 nm vesicles can be seen attached to the budding site by a narrow neck (Esau & Hoefert, 1971
; Lesemann, 1988
). The vesicles contain interior networks of fine fibrils interpreted as double-stranded nucleic acid. This, and observation of the virions and ribosomes in the cytoplasmic matrix between the aggregates of vesicles, prompted Esau & Hoefert (1971)
to propose that these may be the sites of BYV multiplication. Recently, the BYV-type vesicles were revealed in Nicotiana benthamiana protoplasts infected with LIYV RNA-1 transcripts encoding the 1a/1b products, suggesting that the closterovirus replicative proteins are responsible for vesicle induction in cells (Medina et al., 1998
).
Here, by using immunogold labelling of the BYV-infected specimens with the MAbs to the MT and HEL domains, we provide electron microscopic evidence that the closterovirus methyltransferase-like and helicase-like proteins are associated with the BYV-type vesicle membranes. Further, we mapped several MAb binding sites (epitopes) in the BYV MT and HEL domains, and predicted their state in the membrane-bound protein molecules.
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Methods |
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Preparation of plant tissue for electron microscopy.
Healthy or BYV-infected (2030 days post-infection) tissue of Tetragonia expansa was prepared for electron microscopy using two methods: (i) a standard protocol of Epon embedding after fixation with 2·5% glutaraldehyde in 0·1 M sodium phosphate buffer pH 7·0, with or without post-fixation in 0·5% osmium tetroxide (Bendayan & Zollinger, 1983 ; Riedel et al., 1998
), and (ii) low-temperature embedding with Monostep Lowicryl HM20 resin (Polysciences). For the latter, leaf tissue samples were fixed for 2 h at 4 °C in a mixture of 4% paraformaldehyde and 0·5% glutaraldehyde in 0·1 M cacodylate buffer pH 7·4 (CB). For cryoprotection, the samples were subjected to successive incubations in the 2·3 M sucroseCB mixtures (1:3, 1:2 and 1:1, v/v; 2 h for each step), and finally in 2·3 M sucrose (overnight at 4 °C). The samples were frozen in liquid nitrogen and dehydrated in a Balzers FSU 010 apparatus by freeze substitution with methanol (-90 °C for 30 h), followed by infiltration in Lowicryl HM20 at -45 °C. Polymerization of Lowicryl-embedded samples was done in BEEM hemihyperbolic capsules by UV irradiation (35 h at -45 °C and 48 h at 0 °C). Ultrathin sections (90110 nm) were cut with a diamond knife and placed on Formvar-carbon coated grids (75 mesh).
Immunogold labelling (IGL).
Grids were preblocked by incubation for 15 min in phosphate-buffered saline with 1% bovine serum albumin (PBS/BSA) or in CB/BSA, washed (3x20 drops) with CB, and incubated overnight at room temperature with primary antibody solutions, BYV antiserum (diluted 1:150 in PBS/BSA or CB/BSA) or individual MAbs (diluted to 2·510·0 µg/ml). After washing, the grids were incubated for 2 h at room temperature with goat anti-rabbit (GAR) or goat anti-mouse (GAM) IgG conjugated to 15 nm gold beads (Biocell), diluted 1/50 in PBS/BSA or CB/BSA, and washed with distilled water. Finally, the grids were stained with 1% uranyl acetate for 30 min and examined in a LEO EM-906 electron microscope.
Pepscan analysis.
Peptide synthesis on a membrane support (Frank, 1992 ) was carried out on derivatized cellulose sheets using 20 Fmoc-L-amino acid active esters (SPOTs kit, Genosys). Each amino acid coupling step was controlled by spot colour change from blue to green or yellow. After the final cycle, the peptides were N-terminally acetylated by treatment with 0·4 M acetic anhydride (Sigma) in 1-methyl-2-pyrrolidinone, followed by side-chain deprotection in a mixture of dichloromethane, trifluoroacetic acid and triisobutylsilane (Sigma; 2:2:1, v/v), and successive washings with dichloromethane, dimethylformamide and methanol. Prior to the antibody binding assay, the membranes were washed with 0·1 M TBS pH 8·0 and incubated overnight at 4 °C with a blocking buffer (Genosys) diluted 1:10 in TBS with 0·05% Tween 20. This and further steps were followed by triple washing of the membranes with TBS/Tween. The membranes were incubated with MAb solution (5 µg/ml) or ascites fluid (1:1000) in blocking buffer for 2 h at room temperature, followed by treatment with anti-mouse goat antibodies conjugated with horseradish peroxidase (Imtek, Russia) for 1 h. The spots were visualized with tetramethylbenzidine substrate solution (Southern Biotechnology Associates, USA).
Computer analysis of protein sequences.
The non-redundant protein sequence database (National Center for Biotechnology Information, NIH, Bethesda) was searched using the BLASTP program (Altschul et al., 1997 ). Multiple alignments of protein sequences were constructed using the Clustal X program (Thompson et al., 1997
) and adjusted manually on the basis of the results of BLASTP searches and previous identification of conserved motifs. Protein secondary structure and solvent accessibility were predicted using the PHD program, with a multiple protein sequence alignment submitted as the input (Rost & Sander, 1994a
, b
).
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Results and Discussion |
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In view of the fact that the intensity of labelling of the vesicles with the HEL and MT MAbs was less than that with the BYV CP MAb by one to two orders of magnitude (Table 1), the significance of the IGL data was checked with several negative controls. MAbs 4A5, 4A2 and 1C4 did not react with ultrastructures of healthy cells; in infected cells, these MAbs did not label, or labelled weakly, ultrastructures other than BYV-type vesicles (Fig. 3a
, b
,c
and Table 1
). No labelling of infected cells was observed with the gold-conjugated secondary antibody alone (Fig. 3d
, Table 1
). A low labelling intensity (less than two to three particles per field) was recorded when the infected tissue sections were treated with MAbs to the particles of RGMV, WSMV or GLRaV-1 as primary antibodies (Table 1
). Hence, we considered as clearly positive the IGL reactions that were observed both with the Epon- and with the Lowicryl-embedded specimens, and exceeded significantly the background labelling threshold defined in the reactions with the heterologous MAbs. This confidently applies to the labelling of the BYV-type vesicle membranes with MAbs 4A5, 4A2 and 1C4.
Properties of MAbs and their binding sites in the BYV HEL and MT domains
To probe the epitopes in the MT and HEL domains, we synthesized sets of 71 and 87 octamer peptides spanning (with a two-residue offset) the amino acids 665813 and 24502630 in the BYV 1a product, i.e. the sequences that had been used as respective immunogens to raise the MAbs (Fig. 4a, b
; Erokhina et al., 2000
). In the MT MAb panel, 3B6, 3H4, 2D5, 2D10 and 2C10 gave no reaction with any peptide, whereas 3C5, 4C5, 4B4, 4A5, 4A2 and 2A4 reacted strongly with three distinct peptide groups (aa 686692, 750757 and 806813 in the BYV 1a protein; Fig. 4c
). MAbs 3C5, 4B4 and 4C5 recognized the peptide TMVTPGEL (with the latter MAb showing a weaker reaction with the neighbour peptide YLTMVTPG), 4A2 and 4A5 reacted with the SRLLENET peptide (the latter also showed weaker binding to LLENETLA) and 2A4 reacted with the SREQLVEA peptide (Fig. 4c
). In the HEL MAb panel, 1C4 reacted with three overlapping peptides, KFQEDDPF, QEDDPFRS and DDPFRSEN, whereas 1D1 reacted with only the latter two; the sequences DDPF and DDPFRS (aa 24932496 and 24932498) are thus the likely candidates for being the respective epitopes (Fig. 4c
and Table 2
). It should be noted, however, that the actual size of an epitope may be larger than revealed by peptide scanning analysis, and that the amino acids surrounding the key epitope in the native protein globule may influence its reactivity (van Regenmortel, 1992
; Commandeur et al., 1994
). The MAbs specific to the MT and HEL domains of BYV may thus be classed into two reaction groups: those that did not react with synthetic peptides, presumably because their epitopes are discontinuous or conformation-dependent, and those that apparently recognize linear epitopes (Table 2
).
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Some peptide sequences established as MAb binding sites in this work are conserved in the 1a proteins of CTV (Karasev et al., 1995 ) and Grapevine leafroll-associated virus 2 (GRLaV-2; Zhu et al., 1998
), the closteroviruses most closely related to BYV. Thus, the sequence SRLLENET is completely conserved in the GRLaV-2 1a protein, but contains three substitutions in that of CTV (SRMLENHL). It would be interesting to find out whether this stretch is a common immunoreactive epitope in the MT domains of these viruses. The epitope DDPF is only partially conserved in the proteins of CTV, GRLaV-2 and Beet yellow stunt virus (for the latter, a partial sequence of ORF 1a is available; Karasev et al., 1996
). Failure of MAb 1C4 to detect on immunoblots any proteins specific to the citrus tissue infected with CTV (Erokhina et al., 2000
) may be due to the change of DDPF to DTPF, or to additional mutations in the amino acid sequences surrounding this key epitope, in the CTV HEL domain.
BYV-type vesicles as possible sites for closterovirus replication in cells
In many, if not all, positive-stranded RNA virus systems, replication is associated with cell membranes (reviewed in Buck, 1996 ). For assembly of the replicative complexes, some viruses employ the pre-existing membrane organelles, whereas others induce their drastic modification leading to formation of cytopathic ultrastructures (De Graaf & Jaspars, 1994
; Buck, 1996
). Cytopathological studies revealed that many positive-strand RNA viruses of plants and animals induce similar modifications of host cell membranes, leading to formation of vesicles 50100 nm in diameter (Lesemann, 1991
). The vesicles originate by budding from membranes of various host organelles, such as endosomes and lysosomes (alphaviruses), tonoplast (tobamoviruses and cucumoviruses), endoplasmic reticulum (bromoviruses), chloroplasts (tymoviruses), perinuclear membranes (luteoviruses) or peroxisomes and mitochondria (tombusviruses and pecluviruses) (Francki et al., 1985
; Martelli & Russo, 1985
; Froshauer et al., 1988
; Martelli et al., 1988
; Lesemann, 1991
). The lumen of the vesicles often contains a network of fibrils resembling nucleic acid (in some cases cytochemically identified as dsRNA), implying involvement of these ultrastructures in viral RNA replication (Hatta & Francki, 1981
; Martelli et al., 1988
). Being the distinct ultrastructures suitable for diagnosis of closterovirus infections, the BYV-type vesicles may belong to the same morphological class as the above examples (Esau & Hoefert, 1971
; Lesemann, 1988
).
Our IGL data clearly indicated that the BYV methyltransferase-like (p63) and helicase-like (p100) proteins co-localize at the membranes of the vesicle clusters. This IGL profile agrees with the previous finding that p63 and p100 bulk in the crude membrane fractions of the infected plants (Erokhina et al., 2000 ). The involvement of the closterovirus MT and HEL domains in replication is supported by several lines of experimental and sequence-derived evidence: (i) the closterovirus 1a and 1b proteins are the principal viral products responsible for the RNA replication and transcription in vivo (Klaassen et al., 1996
; Peremyslov et al., 1998
; Satyanarayana et al., 1999
; Yeh et al., 2000
); (ii) MT and HEL are the only domains conserved in the closterovirus 1a proteins and in the other Sindbis-like superfamily virus replicases (Koonin & Dolja, 1993
; Agranovsky, 1996
); and (iii) in other viruses, the MT and HEL domains have established functions in RNA capping, plus- and minus-strand RNA synthesis, and duplex unwinding (reviewed in Strauss & Strauss, 1994
; Buck, 1996
; Ahola et al., 1999
). Hence, given that the MT- and HEL-containing proteins are essential components of the replicative complex, our IGL data strongly indicate that the BYV-type vesicle membranes are the specific sites of closterovirus replication. Formally, it cannot be excluded that the presence of MT and/or HEL at the vesicles reflects a mere deposition of excess proteins involved in replication. However, this possibility is less likely in view of the fact that BYV MT and HEL are expressed to only low levels in vivo (Erokhina et al., 2000
), and that in LIYV replicative genes are responsible for induction of the vesicles from early stages of viral infection in protoplasts (Medina et al., 1998
).
According to our observations, the single vesicles and the vesicle aggregates are predominant at the early and the late stages of the BYV infection, respectively, thus suggesting that the vesicle inclusions undergo kinetic changes. It is possible that continuous production of the single vesicles by budding from host membranes, along with their transformation into the multivesicular clusters by invaginations of the outer membrane, serve to increase the number of replication sites during the progression of the infection.
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Acknowledgments |
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References |
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Received 31 January 2001;
accepted 12 April 2001.