Department of Virology and Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow, Russia1
Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, 117871 Moscow, Russia2
Federal Biological Research Centre for Agriculture and Forestry, Institute for Plant Protection in Fruit Crops, Schwabenheimer Str. 101, D-69221 Dossenheim, Germany3
Author for correspondence: Alexey Agranovsky. Fax +7 095 939 31 81. e-mail aaa{at}closter.genebee.msu.su
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Abstract |
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Introduction |
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In the genomes of closteroviruses, a group of insect-transmitted filamentous viruses of plants (Agranovsky, 1996 ; Agranovsky et al., 1994a
), the arrangement of replicative and proteinase domains is distinct. The MT and HEL domains and the POL domain are encoded in overlapping 5-terminal ORFs 1a and 1b, respectively, and the polymerase expression is likely to require +1 ribosomal frameshifting (reviewed in Agranovsky, 1996
). The PCP is located at the N terminus of the beet yellows closterovirus (BYV) ORF 1a product, and its cleavage, leading to the release of a 66 kDa leader protein from the rest of the polyprotein, has been confirmed experimentally by T7 transcription/translation and point mutagenesis in vitro (Agranovsky et al., 1994a
). A similar location of the PCP has been reported in the genomes of other closteroviruses, including citrus tristeza virus (CTV; Karasev et al., 1995
), lettuce infectious yellows virus (LIYV; Klaassen et al., 1995
), little cherry virus (Jelkmann et al., 1997
) and grapevine leafroll-associated virus-2 (Zhu et al., 1998
). Recently, the full-length cDNA clones of LIYV (Klaassen et al., 1996
) and BYV (Peremyslov et al., 1998
) were produced; it has been found that ORFs 1a and 1b are necessary and sufficient for closterovirus RNA replication, whereas the ORFs located in the 3-terminal part of the monopartite BYV genome and in RNA-2 of the bipartite LIYV genome are dispensable for replication. Interestingly, the leader PCP of BYV, apart from its proteolytic activity, had a function in enhancing viral RNA accumulation (Peremyslov et al., 1998
).
Isolation of virus replication-associated enzymes from infected eukaryotic cells (and their detection therein) is not an easy task, as the proteins normally accumulate to only low levels. The use of antibodies specific to recombinant or synthetic peptides has allowed the in vivo identification and functional dissection of several animal and plant viral replicases (Baron & Baltimore, 1982 ; Hayes et al., 1994
; Hills et al., 1987
; Manabe et al., 1994
; Scholthof et al., 1995
). In this study, monoclonal antibodies (MAbs) to the bacterially expressed N-and C-terminal fragments of the BYV 1a were produced and used for detection of the methyltransferase-like and helicase-like proteins in vivo.
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Methods |
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Serological tests.
Total protein was isolated by phenol extraction (Van Etten et al., 1979 ) from healthy and BYV-infected Tetragonia expansa leaf samples collected at 2, 4, 7, 11, 15, 20, 32 and 49 days post-inoculation (p.i.). Subcellular fractions S30 (cytoplasm), P30 (membranes), P1 (nuclei and large organelles) and CW (cell walls) were obtained from healthy and infected plants (1220 days p.i.) by filtering the sap through Miracloth followed by differential centrifugation at 1000 g and 30000 g (Niesbach-Klosgen et al., 1990
). The protein samples were separated on gradient (615% polyacrylamide) SDS gels (Laemmli, 1970
) and transferred to nitrocellulose membranes (Hybond-C extra, Amersham). Immediately after the transfer, the blots were UV-irradiated in Stratalinker-1800 (Stratagene); this treatment was found to greatly increase the sensitivity of protein detection (R. Zinovkin, unpublished observation). The blots were incubated with a purified MAb (3 µg/ml) or diluted ascitic fluid (1:1250), followed by goat anti-mouse IgGalkaline phosphatase conjugate (Sigma). Visualization was by treatment with BCIP/NBT substrate (Promega).
In vitro transcription and translation.
The cDNA sequence encoding a portion of the BYV 1a product between amino acids 589 and 1120 (60·2 kDa, containing the MT domain; Fig. 1 A) was amplified by PCR using the cDNA clone 515 142 (nt 14007 in the complete BYV genome sequence) as template, with the specific positive-sense primer 5 dTCGGATCCATGGAAGAAGAAGCTCCT 3 (introduced NcoI site is underlined) and M13 reverse primer (Promega). The NcoISacI fragment of the PCR product was cloned between the respective sites of the T7 expression vector pHIREScpGUS (gift of P. Ivanov; Ivanov et al., 1997
) to replace the GUS gene. The cDNA encoding the C-proximal 894 amino acids of BYV 1a (99·7 kDa, containing the HEL domain; Fig. 1A
) was amplified by reverse transcriptase PCR on the BYV virion RNA (Agranovsky et al., 1994b
) with the specific primers 5 dAGGGATCCCCTGCGAGCGAATC 3 and 5 dAACCACCATGGGCGTTTCG 3 (negative-sense and positive-sense, respectively; introduced BamHI and NcoI sites are underlined). The cDNA was digested with NcoI and BamHI and inserted between the respective sites of pHIREScpGUS. The resulting cDNAs, pSK-BYV.MT and pSK-BYV.HEL, were linearized with SacI. In vitro transcription with the T7 RNA polymerase and translation in the TNT Coupled Wheat Germ Extract system (Promega) were done according to the manufacturers protocol. The [35S]methionine-labelled products were separated on denaturing protein gels and visualized by autoradiography, or transferred to nitrocellulose for immunoblot analysis with the specific MAbs.
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Results |
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In order to obtain recombinant immunogens representing the central portion of the 1a protein, we constructed pQE-based expression vectors with BYV cDNAs overlapping the respective genomic region (nucleotides 39974323, 43234837, 39974837 and 48375220). However, none of these constructs provided detectable expression of the BYV-specific products in M15 and SG13009 bacterial strains. In our experience, some other coding regions of BYV (particularly, those for the PCP and POL domains) are also non-expressible in E. coli, presumably because of interference of the virus-specific RNA and/or protein with bacterial growth.
Detection of the BYV methyltransferase-like and helicase-like proteins in vivo
Upon immunoblot analysis of the subcellular fractions of the BYV-infected and healthy T. expansa plants, MAbs 3B6, 3C5, 2D5 and 4A5 visualized a single band specific to the virus-infected tissue, corresponding to a protein with an apparent molecular mass of 63 kDa (p63), and gave virtually no cross-reaction with healthy plant proteins (Fig. 2A and data not shown). The seven other antibodies in the MT MAb panel did not detect p63 or any other protein specific to the BYV-infected tissue. The bulk of p63 was in fractions P1 (containing nuclei, chloroplasts and associated membranes) and P30 (membranes from the endoplasmic reticulum and dissociated organelles), with only a trace of it found in the CW fraction (Fig. 2A
).
In the immunoblot lanes corresponding to the subcellular fractions of the BYV-infected, but not healthy, plants, antibody 1C4 from the HEL MAb panel readily recognized a protein with an apparent molecular mass of 100 kDa (p100) (Fig. 2B). Like p63, p100 was found to be predominantly associated with fractions P1 and P30. MAb 1D1 also detected the p100 band, albeit with lower efficiency compared with 1C4, whereas 1A2, 2C6 and 2B5 showed no detectable reaction with any protein on immunoblots (data not shown). MAb 1C4 did not show a positive reaction to any protein specific to the citrus leaf tissue infected with CTV, a closterovirus closely related to BYV (M. Bar-Joseph, personal communication), thus suggesting its specificity to a unique epitope(s) in the BYV helicase-like protein.
The total protein from T. expansa leaf samples taken at different times after aphid inoculation was analysed with MAbs 3B6 and 1C4 and MAb 1470 (specific to the major BYV CP). All three BYV proteins p22 coat protein, p63 and p100 were detectable from 11 days p.i. (at the onset of leaf symptoms development) to 49 days p.i., although the levels of p63 and p100 declined in older leaves collected after 20 days p.i. (Fig. 3).
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Discussion |
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Formally, it cannot be excluded that p100 contained both the HEL and POL domains in a fusion produced by the ORF1a/1b ribosomal frameshifting (Agranovsky et al., 1994a ). However, the fact that the MAbs specific to the C-terminal part of the BYV 1a protein detected a single protein band makes this possibility very unlikely, unless nearly all the translating ribosomes negotiated the stop codon in ORF 1a to produce the fusion. In fact, the efficiency of the closterovirus frameshifting signals in vitro was found to be less than 2% (Agranovsky, 1996
; ten Dam, 1995
; B. Renecke & W. Jelkmann, unpublished data).
The observed distribution of the BYV methyltransferase-like and helicase-like proteins in the subcellular fractions agrees well with that of other RNA virus replication-associated enzymes, which are most often found in the membrane compartments (Hills et al., 1987 ; Scholthof et al., 1995
; reviewed in David et al., 1992
). Closterovirus infection is accompanied by the induction of characteristic membranous ultrastructures, the so-called BYV-type vesicles, which have been implicated in virus replication (reviewed in Coffin & Coutts, 1993
). It would be interesting to see if these structures do accumulate the viral replicative proteins and are indeed the specific sites of closterovirus replication. Conceivably, p63 and p100 of BYV should have membrane-binding domain(s), unless they are retained in a membrane by proteinprotein interactions. It should be noted, however, that the
a protein of barley stripe mosaic hordeivirus (carrying the related MT and HEL domains) is found predominantly in the soluble protein fraction (Donald et al., 1993
). This implies that the variable amino acid contexts of the MT and HEL, rather than the conserved domains per se, may influence the intracellular destination of the viral proteins.
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Acknowledgments |
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References |
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Received 6 September 1999;
accepted 29 October 1999.