Institute of Biotechnology, Program for Plant Molecular Biology, Viikki Biocenter, PO Box 56, FIN-00014 University of Helsinki, Finland1
Institute of Agricultural Biotechnology, Timiryazevskaya st. 42, Moscow 127550, Russia2
National Institute of Chemical Physics and Biophysics3 and Gene Technology Center, Tallinn Technical University4, Akadeemia tee 23, EE12618 Tallinn, Estonia
Author for correspondence: Kristiina Mäkinen. Fax +358 9 19159571. e-mail kristiina.makinen{at}helsinki.fi
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Abstract |
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Introduction |
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Several viruses from various virus groups have a protein (VPg) linked covalently at the 5'-end of the viral RNA. A model for the initiation of poliovirus RNA synthesis was proposed by Andino et al. (1993) . According to this model, uridinylated VPg (VPgpUpU) serves as a primer for 3D replicase. Both genomic and subgenomic RNAs of Southern bean mosaic virus (SBMV; genus Sobemovirus) contain a VPg of about 12 kDa covalently linked to their 5'-ends (Ghosh et al., 1979
, 1981
; van der Wilk et al., 1998
). The VPg of Southern cowpea mosaic virus (SCPMV; genus Sobemovirus) is about 10 kDa (Mang et al., 1982
). In the case of most of the single-stranded RNA viruses of which the VPg has been identified, the domains are arranged in the polyprotein in the following order: VPgproteinasepolymerase (Koonin & Dolja, 1993
). An increasing number of viruses, including SBMV and viruses related to sobemoviruses, have been shown to have a different polyprotein arrangement: proteinaseVPgpolymerase (van der Wilk et al., 1997
, 1998
; Revill et al., 1998
; Wobus et al., 1998
). Although the region encoding VPg is similarly located in the genomes of Potato leafroll virus (PLRV), SBMV, Pea enation mosaic virus (PEMV-1) and Mushroom bacilliform virus (MBV), no significant similarity has been reported in the amino acid sequence or in the size of the VPgs. Each of these viruses has been either shown or proposed to express its replicase by a -1 ribosomal frameshifting mechanism (Demler& de Zoeten, 1991
; Prüfer et al., 1992
; Kujawa et al., 1993
; Revill et al., 1994
). In SBMV, the N-terminal amino acid of VPg is threonine, which is preceded by a glutamic acid, indicating that an E/T processing site is used for VPg maturation (van der Wilk et al., 1998
). It is also known that no polyprotein processing of CfMV occurs in an in vitro translation reaction (Tamm et al., 1999
). No further information about the polyprotein processing of sobemoviruses exists.
In this work, a protein that appears to correspond to the CfMV VPg was isolated and its size and N-terminal amino acid sequence were determined. The proteolytic cleavage site at the VPg N terminus was identified, providing the first indication of how the CfMV polyprotein is processed in vivo. Immunoblotting of infected plant material showed that the polyprotein is processed at several additional sites; one of them was predicted by sequence analysis. Based on these data, a proteolytic cleavage scheme is proposed for the CfMV polyprotein.
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Methods |
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Inoculation.
For Western blot analysis, 12-day-old seedlings of Hordeum vulgare cv. Kustaa were inoculated mechanically by grinding 1 g of leaves of CfMV-infected barley cv. Lise with a mortar and pestle per 10 ml of distilled water and rubbing the sap onto the lowest leaf of each barley plant (each plant had a total of two to three leaves) dusted with carborundum. Western blot analysis was used for virus detection.
Preparation of total soluble protein samples from infected leaves.
Approximately 50 mg samples of CfMV-infected barley cv. Kustaa and the corresponding mock-inoculated leaves were collected 21 days post-inoculation and immediately frozen in liquid nitrogen. Frozen leaf tissues were ground with a pestle in a microcentrifuge tube and the homogenized material was mixed with 300 µl Laemmli sample buffer (Laemmli, 1970 ). After heating the samples for 5 min at 100 °C, the insoluble material was removed by centrifugation (14000 g) and the supernatant was loaded on the gel.
Preparation of VPg antiserum.
The VPg-encoding region in the CfMV polyprotein gene was amplified by PCR from plasmid pORF2a/2b (Tamm et al., 1999 ) by using the oligonucleotides 5' CGCGGATCCAACAGTGAGTTATATCCC (contains a BamHI site and CfMV nucleotides 13851403) and 5' ACGCGTCGACTTATTCCTTCGTCACGCCAG (contains a SalI site, a UAA termination codon and sequence complementary to CfMV nucleotides 17081724). Base numbering refers to the CfMV genome as described in Mäkinen et al. (1995a
). The resulting fragment was cloned into pGEM-T (Promega) and sequenced. The correct fragment was subcloned into the BamHI and SalI sites of pQE30 as a 6-His fusion (Qiagen). The resulting expression plasmid was used to transform E. coli strain M15 (Qiagen). Expression and purification of the 6-HisVPg fusion were carried out according to the manufacturers protocol. Rabbit polyclonal antiserum was produced against the expressed 6-His fusion protein by using a denatured protein solution (in 4 M urea) for immunization.
Electrophoresis and Western blot analysis.
SDSPAGE was carried out on an RNase A-treated CfMV RNA-derived sample and the resulting gel was silver-stained. Western blot analysis was carried out on the same sample and on virus particles. Virus particles were prepared by disrupting them with 1% SDS prior to RNase A treatment. Proteins were separated in a 16·5% TricineSDSPAGE system (Schagger & von Jagow, 1987 ) and then electroblotted onto a nitrocellulose filter. The filter was blocked with 3% BSA. A specific antiserum produced in rabbits against ORF 2a-encoded recombinant protein of CfMV (P2a antiserum; Tamm et al., 1999
) was used at a 1:2000 dilution (Fig. 1
, lane 3). Anti-rabbit antibody conjugated with alkaline phosphatase (Sigma) was used as the second antibody.
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Iodination.
CfMV RNA (1340 µg depending on the experiment) was iodinated by the chloramine T method as described by Sainio et al. (1997) . The reaction contained 29 µl 0·5 M phosphate buffer, pH 7·5, 20 µl sample, 2·5 µl Na125I (250 µCi; Amersham) and 4 µl chloramine T (Serva; 2 mg in 1 ml 0·5 M phosphate buffer, pH 7·5). The chloramine T treatment lasted for 20 s and then 50 µl Na2S2O5 (2·4 mg in 1 ml 0·5 M phosphate buffer, pH 7·5) was added. After 30 s, 15 µl 0·1 M NaI and 50 µl 1% BSA in PBS were added to the reaction. After iodination, the free label was removed from the sample by using a Sephadex G-25 column (Pharmacia) in the presence of 1% BSA. The radioactivity of 1 µl samples from each fraction was counted by using a beta counter (Wallac). To eliminate excess BSA, the RNA sample was ethanol-precipitated prior to RNase A treatment (10 µg/ml, 1 h, 37 °C). Part of the sample was treated with proteinase K (50 µg/ml, 1 h, 37 °C) in addition to RNase A. Samples were analysed in a 15% SDSPAGE gel.
Immunoprecipitation.
Two hundred µl of the iodinated products (approximately 2·5x105 c.p.m. per sample) was immunoprecipitated (2 h, 0 °C) by using 5 µl antiserum raised against CfMV particles or ORF 2a- and ORF 2b-encoded proteins expressed in E. coli and thendiluted with 200 µl lysis buffer (Trisborate buffer, pH 7·5, 2 mM EDTA, 10% glycerol, 1% Nonidet P-40, 1% Triton X-100). Complexes were collected by adding 30 µl of a 50% suspension of Protein ASepharose granules (Pharmacia) in lysis buffer. Granules were previously treated with 0·2% BSA. The suspension was incubated for 1 h on a rotating wheel in a cold room (4 °C). Granules were washed four times with PBS/2% Triton X-100, 30 µl Laemmli sample buffer was added and the samples were boiled prior to separation by 15% SDSPAGE.
Amino acid sequencing.
A CfMV RNA sample was isolated from the purified virus stock (13 mg/ml) by using the plant RNeasy Total RNA kit (Qiagen) according to the manufacturers instructions. Seventy µg RNA was hydrolysed in 10% trifluoroacetic acid (TFA) for 48 h at room temperature. The concentration of TFA was reduced by evaporation. The hydrolysed material was blotted onto a PVDF membrane by using a ProSorb cartridge. The PVDF-immobilized protein was sequenced directly by automated Edman degradation.
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Results |
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For further characterization of the 12 kDa protein, the RNA isolation method was changed to the plant RNeasy Total RNA kit (Qiagen). This was done in order to reduce the amount of contaminating non-covalently bound CP (Fig. 1, lane 1) in viral RNA samples. RNA obtained with the kit was labelled with 125I by the chloramine T method. The labelled samples were analysed by radiography of the polyacrylamide gel and immunoprecipitation (Fig. 2
). Two labelled products were observed in the 125I-labelled CfMV RNA sample treated with RNase A. The sizes of these proteins were 12 and 32 kDa (Fig. 2A
, lane 2). To confirm that the signals detected following SDSPAGE indeed originated from iodinated proteins, the RNase A-treated samples were treated with proteinase K. In this case, the 125I-labelled products disappeared, confirming that the iodinated targets were proteins (Fig. 2A
, lane 1). Much less of the 32 kDa CP was observed compared with the amount of the 12 kDa protein in the purified RNA sample isolated with the kit than in the corresponding sample isolated by NaClO4 extraction, showing that the kit yielded purer RNA.
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The sequence of the N-terminal 17 amino acids of the 12 kDa protein associated with the CfMV RNA was determined to be N*EL*PDQSSGPARELD. Amino acids represented by asterisks gave a signal that did not correspond to any residue used as a standard. According to the nucleotide sequence, they should be serine and tyrosine, respectively, and we suggest that these amino acids are probably modified in the mature 12 kDa protein. The other signals were unambiguous and the presence of trace amounts of the CP in the sample did not disturb the sequence analysis. The identified residues represent amino acids 320337 of the polyprotein (Mäkinen et al., 1995a ; Ryabov et al., 1996
). The N-terminal amino acid sequences of VPg molecules of other related viruses have been identified and studied by similar techniques (van der Wilk et al., 1997
, 1998
; Revill et al., 1998
; Wobus et al., 1998
). The data obtained taken together lead to the suggestion that the 12 kDa protein associated with the CfMV RNA represents the VPg molecule.
In order to analyse the size of the in vivo processing products of the CfMV polyprotein, Western blot analysis of CfMV-infected leaf material was carried out with P2a, P2b and P3 (CP) antisera (Fig. 3). The P2a antiserum recognized proteolytically processed proteins of 12, 18, 19, 20, 23, 24 and 30 kDa (Fig. 3A
). Occasionally, a protein of 8 kDa was also detected (data not shown). The major products recognized specifically by the P2b antiserum were 30 and 58 kDa (Fig. 3B
). The CP of CfMV, detected with the P3 antibody, is 30 kDa (Fig. 3C
, D
). Both the P2a and P2b antisera detected a protein that has the same mobility as the CP. Either a polypeptide that has the same mobility as the CP can be recognized by these two antibodies or a cross-reaction has occurred. The 1824 kDa polypeptides recognized by the P2a antiserum are not related to the CfMV CP (Fig. 3A
, C
).
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Discussion |
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The putative serine proteinase region in the ORF 2a-encoded part of the CfMV polyprotein resembles the picornavirus 3CPro proteinase (Mäkinen et al., 1995a ). It has been predicted that the luteovirus and sobemovirus polyproteins are cleaved at (Q,E)/(G,S,A) sites (Gorbalenya et al., 1988
). The finding that the N-terminal cleavage site of the CfMV VPg is between glutamic acid (E) and asparagine (N) was thus unexpected. The fact that another similar cleavage site was found in the polyprotein sequence at the beginning of the serine proteinase region gives some hints as to how the CfMV polyprotein may be processed. The calculated molecular masses of polypeptides cleaved from P2a using the E/N sites presented in Fig. 5
are 12·0 kDa for the protein preceding the putative proteinase region, 20·3 kDa for the putative serine proteinase and 26·7 kDa for the rest of P2a, including the VPg region. P2a antibody detected a 12 kDa protein in infected leaf material, which was either not detected or was detected faintly with the anti-VPg antibody from the same samples. Most of the 12 kDa protein detected by the P2a antibody probably corresponds to the polypeptide preceding the putative serine proteinase in the CfMV polyprotein. The polypeptide starting at asparagine-131 and ending at glutamic acid-319 would be 189 amino acids long. The number of amino acid residues in different picornavirus 3C proteins, which are closely related to sobemovirus serine proteinases, is close to 200 (Rueckert, 1996
). The 1823 kDa polypeptides recognized in infected leaf extracts by the P2a antiserum may represent differently modified, processed or degraded forms of the putative serine proteinase. Both the P2a and VPg antisera recognized a 24 kDa protein, which represents the part of P2a from the beginning of VPg to the C terminus of P2a. The 12 kDa protein, which appears to correspond to VPg, is a minor product in infected plants and can only be clearly detected by Western blot analysis of viral RNA-derived samples.
In PLRV-infected plants, a protein of 25 kDa is detected by P1 (ORF1) monoclonal and polyclonal antibodies (Prüfer et al., 1999 ). The N terminus of this protein is either identical to or located adjacent to the PLRV VPg. In a model suggested by Prüfer et al. (1999)
, it was proposed that the hydrophobic N-terminal region of PLRV polyprotein P1 targets the protein to cellular membranes and that the basic nucleic acid-binding domain at its C terminus interacts with the PLRV RNA. This membrane-bound complex can then serve as a proteolytic processing site for VPg maturation. Several aspects of this model could be applicable to the polyprotein processing of CfMV, since, because of similarities in the polyprotein sequence and arrangement, sobemoviruses and poleroviruses may share similar proteolytic processing strategies. The 60 N-terminal amino acids of P2a contain hydrophobic residues and have been proposed to form a transmembrane domain in the CfMV polyprotein (Ryabov et al., 1996
). The C-terminal part of P2a contains a strong basic region (amino acids 539552) in the P2a sequence according to Mäkinen et al. (1995a
) and may determine the RNA-binding property of P2a (Tamm & Truve, 2000
). No proteolytic processing of the CfMV polyprotein was observed in a cell-free translation system (Tamm et al., 1999
). Therefore, proteolytic processing of the CfMV polyprotein may require association with the replication complex and/or a proper cellular environment.
Interestingly, since the size of the CfMV VPg appears to be approximately 12 kDa, the coding region of the VPg probably overlaps the site in the CfMV polyprotein of the -1 frameshifting signal (Fig. 5). The antiserum against the ORF 2b product recognized a 58 kDa protein, which indicates that the fully processed replicase is either entirely or almost entirely encoded by ORF 2b. Two possibilities exist: the VPg attached to the CfMV RNA originates either from P2a or from the transframe protein. No suitably positioned E/N site, similar to the N-terminal VPg maturation site, is present downstream of the N terminus of the VPg sequence in either frame. Also, it is not possible to find a clearly conserved putative cleavage site by comparing the downstream sequences of sobemoviruses and poleroviruses. Therefore the C-terminal site used for maturation of CfMV VPg cannot be predicted. It will be interesting to determine the biological significance of the fact that VPg or a molecule N-terminally similar to VPg is produced in fusion with the replicase. In particular, this may be important for establishing the replication complex. Furthermore, -1 ribosomal frameshifting regulates the abundance of the various forms of the molecules related to the VPg as well as that of the replicase, which may contribute to the regulation of CfMV replication.
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Acknowledgments |
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References |
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Received 5 June 2000;
accepted 21 July 2000.