Laboratory of Plant Pathology, Department of Agricultural and Environmental Biology, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan1
Author for correspondence: Yutaka Takemoto. Fax +81 3 5841 5090. e-mail aa09301{at}mail.ecc.u-tokyo.ac.jp
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Abstract |
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Introduction |
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Caulimoviruses are members of the pararetroviruses. The genome of these viruses is composed of a circular double-stranded DNA molecule ca. 8 kbp in length containing one or more single-stranded discontinuities. The DNA is encapsidated into isometric virions, ca. 50 nm in diameter (for reviews see Hohn & Fütterer, 1997 ; Schoelz & Bourque, 1999
; Hohn, 1999
; Reddy & Richins, 1999
). Gene functions in caulimoviruses have been investigated, particularly in Cauliflower mosaic virus (CaMV), Figwort mosaic virus (FMV) and Peanut chlorotic streak virus (PClSV). The products of ORF I of CaMV and PClSV are involved in cell-to-cell movement (Thomas et al., 1993
; Ducasse et al., 1995
). ORF II of CaMV encodes an aphid transmission factor (Blanc et al., 1993
). ORF III of CaMV and ORF C of PClSV are essential for infectivity (Daubert et al., 1983
; Mushegian et al., 1995
). The CaMV ORF III product has DNA-binding activity (Mougeot et al., 1993
), associates with virus particles (Dautel et al., 1994
), forms a tetramer through an N-terminal coiled-coil (Leclerc et al., 1998
) and participates in aphid transmission (Leh et al., 1999
). In CaMV, ORF IV encodes the capsid protein (Daubert et al., 1982
), and ORF V protein possesses aspartic protease and reverse transcriptase activities (Torruella et al., 1989
; Takatsuji et al., 1986
). The ORF VI product of CaMV, a major constituent of the virus inclusion body matrix, is translated from monocistronic mRNA (Odell & Howell, 1980
) and plays an important role in host range determination and symptom development (Schoelz & Shepherd, 1988
; Baughman et al., 1988
; Goldberg et al., 1991
). A crucial function of the ORF VI products of CaMV, FMV and PClSV is translational transactivation to facilitate polycistronic translation by a reinitiation mechanism from pregenomic RNA (Bonneville et al., 1989
; Gowda et al., 1989
; Maiti et al., 1998
; Scholthof et al., 1992
). ORF VII is dispensable for PClSV infectivity (Mushegian et al., 1995
), but in FMV it is required as a cis-acting element for the expression of downstream genes on the pregenomic RNA (Gowda et al., 1991
).
The genus Soybean chlorotic mottle-like viruses, which includes SbCMV, PClSV and Cestrum yellow leaf curling virus, is distinguished from other caulimoviruses by the occurrence of three ORFs between ORF I and ORF IV instead of two (ORF II and III) (Hasegawa et al., 1989 ; Mushegian et al., 1995
; Stavolone et al., 1999
). These three ORFs, designated Ib, II and III in SbCMV or A, B and C in PClSV, show little or no similarity to the two ORFs of caulimoviruses. In the case of PClSV, ORF B was shown to be dispensable, whereas ORFs A and C were essential for systemic infection. Since ORF A contains a putative tRNA primer-binding site (PBS) for minus-strand DNA synthesis and additional cis-sequences required for efficient priming, it is not clear whether the protein product of ORF A is essential or not (Mushegian et al., 1995
). However these three ORFs of PClSV show no significant similarity to those of SbCMV.
In SbCMV, on the other hand, no analysis to determine the genes requisite for infection has been performed as yet, although based on amino acid sequence similarities in the core of each gene, ORFs Ia, IV, V and VI of SbCMV were predicted to encode a movement protein, capsid protein, protease/reverse transcriptase and inclusion body, respectively (Hasegawa et al., 1989 ). In this study, genes essential and non-essential for infection were determined by mutational analysis of a SbCMV 1·3-mer infectious DNA clone. Also the viral transcripts were characterized.
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Methods |
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Construction of an infectious 1·3-mer SbCMV DNA clone.
Cloning of an infectious 1·3-mer SbCMV DNA (pSbCMV1.3) clone was performed by the procedure described by Ducasse et al. (1995) for PClSV. A full-length clone of SbCMV DNA (pSbCMV1.0) was generated by cutting the viral DNA at the unique ClaI site and inserting it into the ClaI site of pBluescript II SK+ (Stratagene) in which the SalI site had been previously disrupted. The chloramphenicol resistance gene of pKF3 (Takara) was amplified by PCR using CmR-F primer (5' GAGCTCGTCGACGAATTTCTGCCATTCATCCG 3'; SalI site is underlined) and CmR-R primer (5' GAGCTCGTCGACACGGAAGATCACTTCGCAGA 3'; SalI site is underlined). The PCR product was inserted into the SalI site of pSbCMV1.0, and the plasmid obtained was designated pSbCMV1.0::CmR. To generate a 0·3-mer clone (pSbCMV0.3) which has a silent mutation in the SalI site of pSbCMV1.0, a ClaISnaBI fragment was prepared by overlap extension PCR (Ho et al., 1989
) using four primers (Table 1
), and the PCR product was digested with both ClaI and SnaBI, and then cloned into the ClaISmaI site of pBluescript II SK+ (Stratagene) in which the SalI site had been previously disrupted. Finally, to construct pSbCMV1.3, the larger ClaI fragment of pSbCMV1.0::CmR was inserted into the ClaI site of pSbCMV0.3 and then the chloramphenicol resistance gene was removed by SalI digestion followed by religation of the plasmid. pSbCMV1.3 includes a redundant part between the end of ORF V and the front half of ORF VII (nucleotide positions 5462 to 6747) (Fig. 1
).
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Infectivity assay, dot blot hybridization assay and immunoselection assay.
P. vulgaris cv. Honkintoki, which is highly susceptible to SbCMV, was grown from seeds in a greenhouse. Two primordial leaves were dusted with Celite abrasive and inoculated with 25 µl DNA (500 µg/ml) in 0·05 M phosphate buffer (pH 7·0) by rubbing with a fingerstall. The inoculated leaves were rinsed immediately with water, and plants were transferred to a growth chamber at 70% relative humidity at 32 °C during the 14 h light period (ca. 20000 lx) and at 28 °C during the 10 h dark period. Symptoms were scored visually at various periods after inoculation. The accumulation of viral DNA in the infected leaves was verified by a dot blot hybridization assay (Maule et al., 1983 ) 14 or 30 days after inoculation in the case of the inoculated or upper leaves, respectively, using full-length SbCMV DNA prepared from pSbCMV1.0 as a probe. To detect virus infection restricted to the initially infected cells, 10 g of inoculated leaves was subjected to immunoselection PCR (ISPCR) 12 to 15 days after inoculation according to the protocol described by Ducasse et al. (1995)
using anti-SbCMV antiserum, Ia-F primer (5' 6909-CCAAACGGAATATAGGATGG-6928 3') and Ia-R primer (5' 7894-GAGTTTGTCCAGTTATCTCC-7875 3').
Sequencing of the capsid protein.
The purified virus was dissolved in 30 µl of 62·5 mM TrisHCl (pH 6·8) containing 3·5% SDS, 100 mM dithiothreitol and 10% glycerol, and then heated at 100 °C for 5 min. The protein sample was partially digested with 2 µg of V8 protease (PIERCE) for 1 h at room temperature, electrophoresed through an SDSpolyacrylamide gel (17%) by the method of Cleveland et al. (1977) , and then blotted onto an Immobilon P membrane (Millipore) (Hirano, 1989
). The appropriate bands were excised from the membrane and sequenced directly by sequential Edman degradation using a protein sequencer (ABI 492).
Analysis of viral transcripts.
Total RNA was extracted from young infected leaves of P. vulgaris by the phenol/SDS method and then purified by CsCl equilibrium density gradient centrifugation. The purified total RNA was fractionated by oligo(dT)-cellulose chromatography according to the protocol of Ausubel et al. (1995) . The polyadenylated RNA fraction was collected as mRNA. Approximately 2 µg of mRNA was electrophoresed in a 1% agarose gel containing 2·2 M formaldehyde and then transferred onto a BIODYNE PLUS membrane (Pall), and hybridized with several kinds of probes as shown in Fig. 1
using the ECL system (Amersham Pharmacia).
To map the 5'-end of the SbCMV pregenomic RNA, 5' RACE PCR was performed. cDNA was synthesized with VII-R primer (5' 7142-AACTTCTCCTGTCATTAGTC-7123 3') and incubated with terminal deoxynucleotidyl transferase (TdT) and dCTP. The dCTP-tailed cDNA was amplified by PCR using Sb-GSP primer (5' 6305-TAGAGTCTCTCGACTTTGCCGTCC-6281 3') and oligo(dG) anchor primer [5' GGCCACGCGTCGACTAGTAC(G)16 3']. For accurate identification of the 5'-end, the first strand cDNA was incubated with TdT and dATP, and then dATP-tailed cDNA was amplified by PCR using Sb-GSP primer and oligo(dT)Sph anchor primer [5' AACTGGAAGAATTGCATGCAGGAA(T)18 3'; SphI site is underlined]. To determine the poly(A) site, 3' RACE PCR was performed. cDNA was synthesized with oligo(dT)Sph anchor primer and the 3' end sequence was amplified by PCR using VI-F primer (5' 4687-GGAGGGAGGAAGTTACAAAAGG-4708 3') and TSP primer [5' TGGAAGAATTGCATGCAGGAA 3'; homologous to the anchor sequence of oligo(dT)Sph anchor primer] and sequenced.
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Results and Discussion |
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The direct sequencing of PCR products of the progeny viral DNA extracted from the upper leaves showed that the mutation was conserved completely (data not shown).
Subsequently, to confirm that the putative PBS is essential for viral DNA replication, we constructed two clones with base substitution mutations in the PBS sequence: pDISPBS1, in which the amino acid sequence encoded was unaltered, and pDISPBS2, in which the coding sequence was changed to result in substitutions of two amino acid residues (Leu-104 to His, Val-105 to His) (Fig. 1). Both mutations in PBS were found to abolish virus infectivity (Table 2
). Also, no viral DNA could be amplified from the inoculated leaves by ISPCR in the case of either clone (Fig. 3
). These results indicate that the ORF Ib product is not essential for infection whereas the PBS in this ORF is essential for virus replication, as previously shown for a similar PBS located in ORF A of PClSV (Mushegian et al., 1995
).
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ORFs II, III, IV and V were found to be required for virus replication or assembly. The in-frame deletion mutants of ORF II (pII 1 and 2) were lethal, although no homologue of SbCMV ORF II is found in other caulimoviruses. These deletions did not change the reading frame and therefore would not interfere with the continuity of translation of downstream ORFs of the pregenomic RNA. We could not predict the function of the product of ORF II because no significant homology to any known protein was detected in the DNA and protein databases. Further research is necessary to determine the function of this gene product in the life-cycle of SbCMV. Like ORF C of PClSV (Mushegian et al., 1995
), ORF III of SbCMV might correspond to ORF III of the other caulimoviruses as it was found to be indispensable for infection and its product contains a basic/proline-rich domain in the C-terminal region and a putative coiled-coil domain in the N-terminal region (Leclerc et al., 1998
). In construction of p
III, the N-terminal coiled-coil domain was deleted. The deletion of 20 amino acid residues in the coiled-coil of CaMV ORF III was shown to lead to a non-infectious virus (Jacquot et al., 1998
). Our data confirm that this domain is important for infectivity of SbCMV. ORF IV was identified as a capsid protein because the determined sequences of various peptides derived from the capsid protein were identical to parts of the deduced amino acid sequence of this ORF, as described below. This ORF was found to be essential for virus multiplication. In construction of p
IV, the zinc finger motif conserved among all pararetroviruses was deleted. It has been previously shown in the case of CaMV and FMV that a mutation altering the zinc finger motif abolishes the infectivity of these viruses (Scholthof et al., 1993
; Guerra-Peraza et al., 2000
). The loss of infectivity of p
IV appears to be dependent on the lack of the zinc finger motif. The deletion of ORF V, which encodes a reverse transcriptase, completely abolished virus replication, as expected.
We could not analyse the functions of ORFs VI and VII in this study because it was difficult to construct mutants of these two ORFs as they are duplicated in pSbCMV1.3. But as the resequenced data of the full-length SbCMV genome (revised data are shown in DDBJ, EMBL and GenBank nucleotide sequence databases) showed the existence of a translational transactivator (TAV) motif in ORF VI, as already mentioned by De Tapia et al. (1993) , the ORF VI product of SbCMV appears to have the role of TAV in the other caulimoviruses.
ORF IV encodes a capsid protein
ORF IV of SbCMV was presumed to encode a capsid protein because it has one copy of a zinc finger motif conserved in the capsid proteins of other caulimoviruses (Hasegawa et al., 1989 ). However, strict identification of this ORF has not been performed previously. To confirm the identity of this ORF, N-terminal amino acid sequencing of the SbCMV capsid protein (56 kDa) was attempted, but the sequence could not be determined because the N terminus of the capsid protein appeared to be blocked. Therefore, the capsid protein was partially cleaved with V8 protease, and three of the resulting peptide fragments (24 kDa, 23 kDa and 14 kDa) were partially sequenced. The determined peptide sequences were identical to the deduced amino acid sequence of ORF IV at the amino acid positions 203 to 219 (SEEAFSQNNYYKLINLE), 220 to 234 (ICNMCYLENFLCEFQ) and 72 to 90 (AETSNKRKFDKNPEFTRFK).
Identification of viral transcripts
To investigate the mode of SbCMV gene expression, Northern blot analysis of viral transcripts was performed. The results indicated that two major virus-specific transcripts were produced (Fig. 4). The larger transcript (ca. 8200 nt) was found to be a pregenomic RNA as all of the six different probes covering all ORFs of SbCMV DNA hybridized with this RNA. The smaller transcript (ca. 1800 nt) hybridized only with a probe covering the ORF VI region of the genome, suggesting that it corresponds to a monocistronic mRNA like the 19S RNA of CaMV. In CaMV and FMV, the ORF VI product has been shown to possess TAV activity, causing reinitiation of translation (Bonneville et al., 1989
; Gowda et al., 1989
; Scholthof et al., 1992
). Therefore, the expression of all of the genes on the pregenomic RNA is thought to be efficiently activated by the ORF VI product translated from a monocistronic mRNA in these viruses. Since ORF VI of SbCMV has the TAV motif, as mentioned above, gene expression might take place in the same manner.
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Since the VI-F primer for 3' RACE PCR was based on the ORF VI present in both the pregenomic and monocistronic RNAs (shown in Fig. 1), the poly(A) site of both mRNAs would be located at the same position. Attempts to determine the 5'-end of the monocistronic mRNA by 5' RACE PCR were not successful, as the PCR products had various 5'-ends scattered between nucleotide positions 4420 and 4579 of the viral DNA and no TATA or TATA-like box was located upstream of these 5'-ends.
The pregenomic RNA of plant pararetroviruses carries a long leader sequence which contains several short ORFs (sORFs) and has the potential to form a large stemloop structure; both features are known to be inhibitory for downstream translation (Pooggin et al., 1999 ). To bypass these scanning-inhibitory regions, a ribosome shunt mechanism has been shown to be used in the case of CaMV (Fütterer et al., 1990
, 1993
) and Rice tungro bacilliform virus (Fütterer et al., 1996
). Also, in other plant pararetroviruses, such a ribosome shunt mechanism has been predicted to exist because of the conservation of the consensus cis-elements (Pooggin et al., 1999
). However, our results showed that the SbCMV pregenomic RNA leader sequence was 293 nt in length, containing only one sORF overlapping ORF VII. Moreover, no extensive secondary structure was predicted to exist in this leader sequence, as mentioned by Pooggin et al. (1999)
. This indicated that the ribosome shunt mechanism is not likely to be used in the case of SbCMV.
We have previously reported that a DNA fragment with a sequence corresponding to a region within ORF III of SbCMV, called promoter IV, could promote the expression of a downstream connected GUS gene in tobacco mesophyll protoplasts (Hasegawa et al., 1989 ; Conci et al., 1993
). However, no transcript for ORF IV and its downstream ORFs could be detected in analysis of SbCMV-infected P. vulgaris leaves. No internal transcription units for ORFs I to V have been identified in other caulimoviruses, although a subgenomic RNA for ORF V of CaMV has been postulated (Plant et al., 1985
; Hohn et al., 1990
). Therefore, SbCMV promoter IV might not be functional in situ, although the excised fragment has promoter activity in vitro.
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Acknowledgments |
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References |
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Received 23 November 2000;
accepted 12 February 2001.