Mutation of Phe50 to Ser50 in the 126/183-kDa proteins of Odontoglossum ringspot virus abolishes virus replication but can be complemented and restored by exact reversion

Hai-He Wang, Hai-Hui Yu and Sek-Man Wong

Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Kent Ridge, Singapore 117543

Correspondence
Sek-Man Wong
dbswsm{at}nus.edu.sg


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Sequence comparison of a non-biologically active full-length cDNA clone of Odontoglossum ringspot virus (ORSV) pOT1 with a biologically active ORSV cDNA clone pOT2 revealed a single nucleotide change of T->C at position 211. This resulted in the change of Phe50 in OT2 to Ser50 in OT1. It was not the nucleotide but the amino acid change of Phe50 that was responsible for the inability of OT1 to replicate. Time-course experiments showed that no minus-strand RNA synthesis was detected in mutants with a Phe50 substitution. Corresponding mutants in Tobacco mosaic virus (TMV) showed identical results, suggesting that Phe50 may play an important role in replication in all tobamoviruses. Complementation of a full-length mutant OT1 was demonstrated in a co-infected local-lesion host, a systemic host and protoplasts by replication-competent mutants tORSV.GFP or tORSV.GFPm, and further confirmed by co-inoculation using tOT1.GFP+tORSV (TTC), suggesting that ORSV contains no RNA sequence inhibitory to replication in trans. Surprisingly, a small number of exact revertants were detected in plants inoculated with tOT1+tORSV.GFPm or tOT1.GFP+tORSV (TTC). No recombination was detected after screening of silent markers in virus progeny extracted from total RNA or viral RNA from inoculated and upper non-inoculated leaves as well as from transfected protoplasts. Exact reversion from TCT (OT1) to TTT (OT2), rather than recombination, restored its replication function in co-inoculated leaves of Nicotiana benthamiana.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Odontoglossum ringspot virus (ORSV) is one of the most prevalent orchid viruses. It is a single-stranded monopartite RNA virus belonging to the tobamovirus group. The complete sequence of a Singapore isolate of ORSV (ORSV-S1) comprises 6609 nt with four open reading frames (Chen et al., 1996). The 126/183-kDa putative RNA-dependent RNA polymerase (RdRp), 30-kDa movement protein (MP) and 18-kDa coat protein (CP) cistrons are respectively located at nucleotides 63–3401/4901, 4879–5718 and 5721–6197 of the genome (Fig. 1a). The 126/183-kDa proteins of tobamoviruses are thought to be involved in virus replication. There is a high degree of similarity between the amino acid sequences of these proteins and that of the putative RdRp of other animal and plant RNA viruses. Three distinct domains are conserved in the proteins (Habili & Symons, 1989; Kamer & Argos, 1984; Rozanov et al., 1992). For ORSV-S1, the three predicted conserved domains are located at aa 72–287, 820–1074 and 1372–1503 (Chng et al., 1996). Mutations in the RdRp domains that disable virus replication have been shown in Brome mosaic virus (Kroner et al., 1989), Potato virus X (Davenport & Baulcombe, 1997; Longstaff et al., 1993) and Tobacco mosaic virus (TMV) (Ogawa et al., 1991). In addition, changes in different regions of the RdRp can alter the synthesis of subgenomic RNA (Watanabe et al., 1987), symptom expression (Bao et al., 1996; Goregaoker et al., 2001; Shintaku et al., 1996), cell-to-cell movement (Hirashima & Watanabe, 2001) and host resistance responses (Hamamoto et al., 1997a, b). Recently, the helicase domain in the TMV RdRp has been shown to induce necrosis in N-gene-carrying tobacco even in the absence of virus replication (Abbink et al., 1998).



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Fig. 1. Replication of ORSV mutants with single- or double-nucleotide mutation at the Phe50 codon. (a) Schematic drawing of the genomic organization of ORSV-S1. The codon for Phe50 is indicated; nucleotide 211 is shown in bold. (b) RNA blots of the replication of positive-strand (+) RNA of tOT1, tORSV.Tyr, tOT2, tORSV.Phe, tORSV.Cys and tORSV.Trp in transfected orchid protoplasts at 24 h p.i. For each lane, 0·5 µg total RNA was loaded. The probes were DIG-UTP-labelled cRNA probes complementary to ORSV CP (nt 6197–5718). (c) Western blot analysis of total proteins from orchid protoplasts transfected with the same constructs, using ORSV CP antiserum. (d) RNA blots of the synthesis of negative-strand (–) RNA in transfected orchid protoplasts. Minus-strand (–) RNA synthesis of tOT1, tORSV.Tyr, tOT2, tORSV.Phe, tORSV.Cys and tORSV.Trp was detected in transfected orchid protoplasts at 36 h p.i. Minus-strand (–) RNA of tOT2 was further detected at the indicated time compared to that of tOT1. For each lane, 3 µg of each total RNA was loaded. DIG-UTP-labelled cRNA probes complementary to ORSV CP gene (nt 5718–6197) was used.

 
We have obtained a full-length cDNA clone of ORSV-S1 (pOT2) from which in vitro transcribed RNA is infectious (Yu & Wong, 1998). We found that in vitro transcripts from another full-length cDNA clone (pOT1) were not infectious, when tested in local-lesion host Chenopodium quinoa and systemic host Nicotiana benthamiana. We determined that the lack of infectivity of pOT1 was due to a change at nt position 211 which is located outside the three conserved RdRp domains, namely the methyltransferase (MT), helicase and polymerase (H.-H. Yu, unpublished data). We investigated whether nucleotide 211 or its corresponding amino acid Phe50 played an important role in the replication of ORSV, whether the non-infectious virus OT1 could be complemented by replication-competent mutants after co-inoculation and whether reversion or recombination would occur during the replication complementation.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Clones, plasmids, mutants and plants.
The method for constructing pOT1 was described previously (Yu & Wong, 1998). The complete cDNA sequencing of pOT1 was carried out accordingly. Using four primers containing specific point mutations, several mutants (Table 1) were constructed from pOT2 by PCR. These mutants were designated pORSV.Ser (TCT), pORSV.Tyr (TAT) pORSV.Cys (TGT), pORSV.Phe (TTC) [=pORSV (TTC)] and pORSV.Trp (TGG), which referred to their amino acid position 50 changes in the RdRp of ORSV. The resulting mutations were verified by sequencing. The amino acid substitutions were based on consideration for minimum changes to amino acid charge property and hydrophobicity. The first 101 aa residues of the RdRp of 13 tobamoviruses, Chinese rape mosaic virus (Aguilar et al., 1996), Cucumber green mottle mosaic virus-SH strain (Ugaki et al., 1991), Obuda pepper virus (previously known as TMV-Ob strain) (Ikeda et al., 1993), ORSV-Korea isolate (Ryu & Park, 1995), ORSV-Singapore isolate (Chng et al., 1996), Pepper mild mottle virus-S strain (Alonso et al., 1991), Sunn hemp mosaic virus (SHMV) (Silver et al., 1996), Tobacco mild green mosaic virus (Solis & García-Arenal, 1990), Tobacco mosaic virus (TMV)-cruciferae strain (Dorokhov et al., 1994), TMV-vulgare strain (TMV-U1) (Goelet et al., 1982), Tomato mosaic virus (ToMV)-L strain (Ohno et al., 1984), TMV-rakkyo strain (Chen et al., 1996) and Turnip vein-clearing virus (Lartey et al., 1995), were compared for conserved amino acid residues in RdRp.


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Table 1. Infectivity of ORSV RdRp mutants

Mutant nucleotides are shown in bold compared with the wild-type OT2. The correspondingly altered amino acid residue of each mutant is shown in the constructs. +, Infectious; –, non-infectious.

 
To determine the importance of Phe50 in TMV, TMV mutants pTMV.Ser (TCT), pTMV.Phe (TTC) and pTMV.Tyr (TAT) were similarly constructed using the biologically active cDNA clone pU2/U12 (Holt & Beachy, 1991). To investigate whether pOT1 could be complemented by a replication-competent mutant, pORSV.GFP was generated from pOT2 by replacing the CP gene (nt 5721–6197) with a GFP gene. In addition, pOT1.GFP (Fig. 2b) was constructed for complementation tests with pORSV (TTC). The start codon of the CP gene was mutated to ATT (underlined). The next 7 nt, which are essential for the CP RNA subgenomic promoter function in TMV-R (Chen et al., 1996), were inserted upstream of the GFP gene (Fig. 2a). In order to investigate whether recombination or reversion would occur during complementation, a construct pORSV.GFPm containing four silent mutations (Fig. 2c) was made. These four altered nucleotides served as silent markers. It was derived from pORSV.GFP without changing the amino acids Asn49 (AAC->AAT) and Ser51 (TCC->AGT) but changing nucleotides 209 and 213–215 with primers ORSVGFPm (5'-GGGTTAATTTTAGTAAGGTCATTTCTC-3') and GFPORSVm (5'-GAAATGACCTTACTAAAATTAACCC-3') by PCR mutagenesis as described for the construction of ORSV mutants. All mutations were confirmed by sequencing. C. quinoa and N. benthamiana were used as local-lesion and systemic hosts, respectively.



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Fig. 2. Schematic representation of two replication-competent viral constructs and a replication-defective mutant. (a) pORSV.GFP contained TTT at Phe50. Partial sequences of the 3' end of MP gene (in bold) and the 5' end of GFP gene (in italic bold) are shown. ATG was mutated to ATT (underlined) to nullify the initiation codon; the next 7 nt were added as they are essential for the CP RNA subgenomic promoter in TMV-R. (b) pOT1.GFP contained TCT at Ser50. (c) pORSV.GFPm possessed four silent mutations in flanking codons Asn (AAC->AAt) and Ser (TCC->agt) without changing the amino acid residues. Amino acid codons are indicated to show positions of nucleotide change.

 
In vitro transcription and mechanical inoculation.
In vitro transcripts of pORSV.Tyr, pORSV.Ser (OT1), pORSV.Cys and pORSV.Phe (TTC), pORSV.Trp, pOT2, pORSV.GFP, pORSV.GFPm and pOT1.GFP were transcribed using T7 RNA polymerase (Ambion) and inoculated onto C. quinoa and N. benthamiana plants, or used to transfect protoplasts from the orchid Dendrobium cultivar Sonia and N. benthamiana. TMV mutants pTMV.Ser, pTMV.Tyr and pTMV.Phe were similarly transcribed and inoculated onto Nicotiana tabacum Xanthi ‘nc’ and N. benthamiana protoplasts. Two independent experiments were carried out, each time with a total of 10 plants as replicates. Transcripts were denoted with the prefix ‘t’ to replace the ‘p’ which represents plasmid constructs.

Isolation, electroporation of Dendrobium cultivar Sonia and N. benthamiana protoplasts and GFP imaging.
Protoplast suspensions (1x106) of Dendrobium cultivar Sonia and N. benthamiana were isolated and electroporated with 10 µg of in vitro transcripts of various ORSV and TMV mutant constructs, according to methods described previously (Hu et al., 1998). Protoplasts of Dendrobium cultivar Sonia, and leaves of C. quinoa and N. benthamiana were harvested and observed for green fluorescence emission under an inverted fluorescence microscope (ZEISS Axiovert 135) using a 366 nm filter at 36 h post-inoculation (p.i.) and at 3, 7, 20 and 30 days p.i.

Northern and Western blot analyses.
At 24 h p.i., protoplasts were pelleted by centrifugation, resuspended in 100 µl sterile water and homogenized in 100 µl RNA extraction buffer (200 mM Tris/HCl pH 8·5, 1 M NaCl, 1 % SDS, 2 mM EDTA). After three phenol/chloroform extractions, total RNA was precipitated with 3 vols ethanol and washed with 70 % ethanol. Plant total RNA was extracted using Trizol reagent (Life Technologies), according to the manufacturer's protocol. Equal amounts of total RNA (0·5 µg for positive-strand RNA, 3·0 µg for negative-strand RNA) were analysed on 1·2 % agarose-formaldehyde denaturing gels. After electrophoresis and blotting to positively charged nylon membranes (Roche), digoxigenin (DIG)-UTP-labelled RNA probes of ORSV CP-specific cRNA (nt 6197–5718 for positive-strand RNA detection; nt 5718–6197 for negative-strand RNA detection) and TMV-specific cRNA (nt 6401–5100) were used.

Total proteins from inoculated leaves, upper non-inoculated leaves with systemic mosaic symptoms and from protoplasts of Dendrobium cultivar Sonia were extracted after grinding samples in liquid nitrogen and resuspended in 500 µl lysis buffer (8 M urea, 10 % SDS, 1 % Triton X-100 and 10 mM Tris/HCl, pH 7·5). Protoplasts were vortexed for 3 min and tissue debris was removed by centrifugation. The supernatant was concentrated and subjected to Western blot analysis. Each sample contained total proteins from 1x106 protoplasts. Protein samples (10 µg) were probed with anti-ORSV CP antiserum and a secondary antibody–AP conjugate after 15 % SDS-PAGE.

RT-PCR and DNA sequencing.
To confirm the sequences from virus particles extracted from upper non-inoculated leaves after co-inoculation of N. benthamiana with tOT1+tORSV.GFP, tOT1+tORSV.GFPm or tOT1.GFP+tORSV (TTC), viral RNA was isolated from the purified virus using phenol/chloroform and precipitated with ethanol. Long-template RT was performed using 0·5 µg viral RNA with an enhanced AMV reverse transcriptase (Sigma) in a final volume of 20 µl using primer A (5'-TAAGCCAGCTTAGACGGG-3', corresponding to nt 5741–5758 of ORSV-S1). PCR was performed using 5 µl of the cDNA synthesized, 40 mM dNTPs, 32 pM each primer and 2·5 U AccuTaq polymerase (Sigma) in a final volume of 50 µl, using primers A and B (5'-CCACCGTTCTAGACGCCC-3', complementary to nt 182–199 of ORSV-S1). For detecting the GFP gene in the inoculated and upper non-inoculated leaves, primers E (5'-GGCAATACAGTCATTATA-3', complementary to nt 4401–4418 of ORSV-S1) and F (5'-CAAGAATTGGGACAACTCC-3', corresponding to nt 28–46 of the GFP gene) were used. The PCR was carried out with an initial denaturation at 98 °C for 30 s, then 2 cycles of 94 °C for 30 s, 48 °C for 1 min and 68 °C for 12 min and 30 cycles of 94 °C for 30 s, 58 °C for 1 min and 68 °C for 12 min.

To verify the sequence at nucleotide 211, total RNAs were extracted with Trizol reagent. Two RT-PCR products were obtained from total RNA isolated from the inoculated protoplasts or the upper non-inoculated leaves from plants inoculated with tOT1+tORSV.GFP, tOT1+tORSV.GFPm or tOT1+tORSV (TTC) using the Titan-One RT-PCR kit (Roche), using primers C (5'-ATGGCACACTTCCAACAAAC-3', corresponding to nt 63–82 of ORSV-S1) and D (5'-CGTAATAGAACCAAATGG-3', corresponding to nt 359–376 of ORSV-S1). RT-PCR products were cloned into PGEM-T Easy vector (Promega) and sequenced with primer D.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A single nucleotide change was found between OT1 and OT2
Sequencing of the replication-defective pOT1 and the replication-competent pOT2 revealed a single base change at nucleotide 211 (Fig. 1a). This T->C substitution changed amino acid 50 of the 126/183-kDa proteins from a Phe (TTT) in the replication-competent virus OT2 to a Ser (TCT) in OT1. This amino acid substitution was located 21 aa upstream of the putative MT domain.

Phe50 plays an important role in infectivity of ORSV
Phe50 of the 126/183-kDa proteins is highly conserved among the 13 tobamoviruses compared (data not shown), except for SHMV, which contains Cys50. In order to ascertain whether a change to nucleotide 211 would affect ORSV RdRp activity, different nucleotide residues were introduced into the codon for Phe50 through site-directed mutagenesis. Four mutants tORSV.Ser (TCT), tORSV.Tyr (TAT), tORSV.Cys (TGT) and tORSV.Trp (TGG) did not infect the local-lesion host C. quinoa or systemic host N. benthamiana. Only mutant tORSV.Phe (TTC) could induce local lesions and systemic symptoms identical to those produced by tOT2 (Table 1). Although tORSV.Tyr (TAT) possesses similar charge and hydrophobicity as Phe, it did not infect the test plants. These results suggested that it was the amino acid rather than the nucleotide change that resulted in the loss of infectivity of OT1.

The ability of each mutant to replicate was also studied in protoplasts of the orchid Dendrobium cultivar Sonia. The accumulation of viral plus-strand RNA and CP was detected only for tORSV.Phe (TTC) and not for tORSV.Ser (TCT), tORSV.Tyr (TAT), tORSV.Cys (TGT) or tORSV.Trp (TGG) (Fig. 1b and c). These results were consistent with those observed in whole plants. No minus-strand RNA synthesis was detected at 36 h p.i., except for tOT2 and tORSV.Phe (TTC) (Fig. 1d), demonstrating that mutants with substitution of Phe50 in the 126/183-kDa proteins could not replicate. Moreover, results from time-course experiments confirmed that there was no detectable minus-strand RNA synthesis in the protoplasts transfected with mutant tOT1 (Fig. 1d). Taken together, it is concluded that the mutants tested with a substitution at Phe50 prevented minus-strand RNA synthesis.

Importance of Phe50 in replication of TMV
To investigate the importance of Phe50 in the replication of tobamoviruses, similar mutations were introduced into TMV. Inoculation with in vitro transcripts of three mutants pTMV.Ser (TCT), pTMV.Phe (TTC) and pTMV.Tyr (TAT) (Fig. 3a) produced typical local lesions on inoculated leaves 2–4 days p.i. for wild-type TMV and tTMV.Phe (TTC) on N. tabacum Xanthi ‘nc’. Test plants inoculated with tTMV.Ser (TCT) or tTMV.Tyr (TAT) were not infected (Fig. 3a). In addition, after electroporation of N. benthamiana protoplasts with the transcripts from the mutants, the RNA blot showed that only wild-type tTMV.Phe (TTT) and mutant tTMV.Phe (TTC) were able to replicate, and not tTMV.Ser (TCT) or tTMV.Tyr (TAT) (Fig. 3b) at 24 h p.i. These results showed that Phe50 is probably highly preferred in the replication of tobamoviruses.



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Fig. 3. Analysis and infectivity of wild-type and mutants of TMV in plants and protoplasts. (a) Partial sequence alignment and infectivity of TMV amino acid 50 mutants. (b) Northern blot of TMV and its mutants in transfected N. benthamiana protoplasts at 24 h p.i., probed with DIG-UTP-labelled cRNA complementary to TMV CP gene (nt 6401–5100). Total RNA (2 µg) from each sample was analysed.

 
OT1 can be complemented by a replication-competent mutant ORSV.GFP
To investigate whether tOT1 could be complemented in trans by a replication-competent RdRp, pORSV.GFP was constructed (Fig. 2a). At 7 days p.i., intense green fluorescence was observed along the margins of local lesions 2–3 mm in size on tORSV.GFP-inoculated C. quinoa (Fig. 4a), showing that this mutant was able to replicate.



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Fig. 4. Complementation of tOT1 by tORSV.GFP or tORSV.GFP alone or tOT1.GFP by tORSV (TTC). (a) Margin of a local lesion induced by tORSV.GFP on C. quinoa at 7 days p.i. (bar, 3 mm) showed strong green fluorescence. (b) Orchid Dendrobium cultivar Sonia protoplasts transfected with tOT1+tORSV.GFP. (c) Orchid protoplasts transfected with tORSV.GFP alone at 40 h p.i. (bar, 50 µm). (d) Orchid protoplasts transfected with tOT1.GFP+tORSV (TTC) at 40 h p.i. (bar, 50 µm). (e) Non-infected orchid protoplasts observed under normal bright field at 40 h p.i. (bar, 50 µm). (f) N. benthamiana leaf inoculated with tOT1+tORSV.GFP at 7 days p.i. in the dark field (bar, 10 mm). Fluorescence focus (at 20 days p.i.) on the leaf immediately above the inoculated leaf (inset). (g) N. benthamiana leaf inoculated with tORSV.GFP alone at 7 days p.i. (bar, 5 mm). (h) Non-infected leaf showing no green fluorescence (bar, 10 mm). (i) Larger LL induced by tOT1+ORSV.GFP (arrows) and SL induced by tORSV.GFP alone (arrows with dotted lines) on half-leaves of C. quinoa at 7 days p.i. (bar, 3 mm). Local lesions infected by tOT2 are shown as an inset. (j) Green fluorescence foci observed on N. benthamiana leaf inoculated with tOT1.GFP and tORSV (TTC) at 8 days p.i. (bar, 10 mm).

 
To ascertain whether tOT1 replication could be complemented by the replication-competent tORSV.GFP in protoplasts, orchid protoplasts were transfected with tOT1 alone, tOT2 alone or tOT1+tORSV.GFP. The RNA blot showed that tOT1 was successfully replicated by the complementation of tORSV.GFP at 24 h p.i. (Fig. 5a). Although OT1 was replicated in trans, the replication level was much lower than that of OT2. At 40 h p.i., green fluorescence was detected in protoplasts electroporated with tOT1+tORSV.GFP (Fig. 4b) or tORSV.GFP alone (Fig. 4c). No apparent difference in the green fluorescence intensity was observed between tORSV.GFP alone and tOT1+tORSV.GFP. Virus particles were observed only in the co-inoculated protoplasts when examined under a transmission electron microscope (data not shown). In addition, Western blot analysis confirmed that there was synthesis and accumulation of CP from the replication-defective mutant tOT1 in protoplasts co-inoculated with tORSV.GFP at 48 h p.i. (Fig. 5b). This result showed that tOT1 was complemented by tORSV.GFP in protoplasts. Using RNA extracted from virus particles as templates, long-template RT-PCR did not detect tORSV.GFP. This result implied that ORSV.GFP was not encapsidated, whereas OT1 was assembled (Fig. 5c). To confirm complementation, protoplasts were inoculated with a combination of tOT1.GFP+tORSV (TTC). Green fluorescence was apparent in protoplasts (Fig. 4d) at 40 h p.i., indicating complementation in replication had occurred in protoplasts.



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Fig. 5. Trans-complementation of tOT1 by tORSV.GFP. (a) Northern blot of trans-complementation of OT1 in orchid protoplasts at 24 h p.i. (b) Expression of CP from OT1 virus detected in orchid protoplasts at 48 h p.i. (c) RT-PCR detection of the OT1 and ORSV.GFP in virus particles with primer A (corresponding to nt 182–201 of ORSV RNA) and primer C (complementary to nt 5759–5788 of ORSV RNA) or primer D (corresponding to nt 28–46 of the GFP gene). The amplified 5·6 kb fragment of OT1 is indicated. (d) Northern blot of OT1 in N. benthamiana leaves at 13 days p.i. (e) ORSV CP detected in both inoculated and upper non-inoculated leaves of N. benthamiana. (f) Northern blot showing that only LL contained tOT1 at 7 days p.i. (g) ORSV CP detected in the LL of C. quinoa leaf inoculated with tOT1+tORSV.GFP but not in the SL induced by tORSV.GFP alone. DIG-UTP-labelled RNA probe of ORSV CP gene complementary to nt 6197–5718 was used. Total protein (10 µg) was extracted from leaves infected with tORSV.GFP or tOT1+tORSV.GFP or ORSV virus OT2 and subjected to 15 % SDS-PAGE and Western blot probed with ORSV antiserum. Mock, protoplasts or plants inoculated with buffer only; Inoc. and Upper, inoculated and upper non-inoculated leaves, respectively.

 
When N. benthamiana plants were co-inoculated with tOT1+tORSV.GFP, highly intense green fluorescent foci around 2–3 mm in size were observed on the co-inoculated leaves at 7 days p.i. (Fig. 4f). There seemed no apparent difference in fluorescent intensity from cells inoculated with tORSV.GFP alone (Fig. 4g). No green fluorescence was observed on upper non-inoculated leaves. Five of 30 co-inoculated plants showed a 6–7 day delay in symptom appearance in upper non-inoculated leaves and the other 25 plants showed no symptoms. Systemic symptoms in plants infected with tOT2 appeared at approximately 12 days p.i. In addition, tOT1 was replicated in the same five plants. ORSV accumulation in one of the systemic plants is shown in Fig. 5(d). Accumulation of CP derived from tOT1 was detected in both the inoculated and upper non-inoculated leaves from the same five systemically infected plants (Fig. 5e) at 20 days p.i. Numerous green fluorescent foci were observed in the epidermal cells of leaves immediately above the inoculated leaf at 20 days p.i. (Fig. 4f inset shows one such focus). No green fluorescence was found in the upper non-inoculated leaves of the same five systemically infected plants up to 30 days p.i., suggesting that tORSV.GFP may be unable to move long distances. This result also showed that the replication-defective tOT1 was complemented in trans by the replication-competent ORSV.GFP.

At 7 days p.i., on C. quinoa inoculated with tORSV.GFP and tOT1+tORSV.GFP on opposite halves of the same leaf, necrotic lesions of two different sizes appeared. Small lesions (SL) were found on both half-leaves, whereas large lesions (LL) were only found on the half-leaf inoculated with tOT1+tORSV.GFP, which resembled similar sized lesions from those infected with OT2 alone (Fig. 4i). Only tOT1 was detected from the LL in the Northern blot, probed with a CP gene of ORSV (Fig. 5f). Western blot further confirmed the presence of ORSV CP from the LL but not from the SL (Fig. 5g). This showed that replication of tOT1 was complemented by tORSV.GFP.

Complementation of tOT1.GFP+tORSV (TTC)
To confirm the complementation event further, N. benthamiana plants were co-inoculated with tOT1.GFP+tORSV (TTC). ORSV (TTC) was a mutant construct of OT2 (TTT), with its Phe50 encoded by a different codon. Green fluorescent foci on the inoculated leaves at 8 days p.i. (Fig. 4j) were found in 4 of 20 plants, and the codon (TCT) derived from tOT1 was also detected using RT-PCR and sequencing. Systemic symptoms appeared in 19 of the 20 co-inoculated plants at 12 days p.i. None of the four plants showing green fluorescence on the inoculated leaves showed any green fluorescence on the upper non-inoculated leaves (Table 2). This result verified that tOT1 was complemented by a replication-competent mutant on the inoculated leaves and that long-distance movement of tOT1.GFP was not supported by the CP derived from tORSV (TTC).


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Table 2. Exact reversion of replication-defective virus OT1 to wild-type OT2 after complementation with replication-competent mutants on N. benthamiana

cDNA sequences corresponding to nt 63–376 of ORSV genome were determined after RT-PCR. Independent experiments were carried out twice with 10 plants each as replicates. NA, Not applicable.

 
OT1 was not detected in either inoculated or upper non-inoculated leaves of N. benthamiana when complemented by ORSV.GFP at 20 days p.i.
To determine whether tOT1 after complementation by tORSV.GFP was retained in the five systemically infected N. benthamiana plants, total RNA from the inoculated and upper non-inoculated leaves as well as viral RNA of ORSV were extracted at 20 days p.i. Using RT-PCR, ORSV cDNA fragments encompassing nt 182–5758 were amplified from viral RNA extracted from the upper non-inoculated leaves from each of the five plants. RT-PCR fragments corresponding to nt 182–376 of ORSV were amplified from total RNA of the inoculated and upper non-inoculated leaves. Sequencing of 10 cloned RT-PCR fragments from each of the inoculated and upper non-inoculated leaves showed that TCT (tOT1) was not detected at 20 days p.i. Similarly, the wild-type sequence TTT (tOT2) was detected in both inoculated and upper non-inoculated leaves. The sequence encompassing the MP : GFP in tORSV.GFP (nt 4401–5720 of ORSV and nt 1–46 of GFP) (Fig. 2a) was detected only in the inoculated leaf and the leaf immediately above in the five systemically infected plants. No RT-PCR fragment was detected from tOT1-inoculated leaves at 3 days p.i. This ruled out the possibility of detecting residual tOT1 inoculum in the complementation experiment with tOT1+tORSV.GFP. Each experiment was carried out twice and the same results were obtained.

Reversion rather than recombination occurred in systemic leaves of N. benthamiana
To study whether recombination had occurred and, if so, whether it had led tOT1 to restore its replication function, a construct designated pORSV.GFPm was made (Fig. 2c). As shown in Table 2, at 3 days p.i., both tOT1 and tORSV.GFPm were detected in the co-inoculated leaves of all 20 plants, whereas no tOT1 was detected in leaves inoculated by tOT1 alone at 3 days p.i., as determined by RT-PCR and sequencing. The number of plants showing green fluorescent foci on inoculated leaves was recorded. At 18 days p.i., mosaic symptoms appeared on three infected plants. Long-template RT-PCR and sequencing using viral RNA extracted from virus particles purified from the upper non-inoculated leaves revealed that silent marker sequences from tORSV.GFPm (AAtTTTagt) were not detected, whereas the wild-type TTT sequence was found. The sequences of tOT1 (TCT) and tORSV.GFPm were not detected in the total RNA extracted from the upper non-inoculated leaves up to 20 days p.i. In addition, results from the complementation experiment of tOT1.GFP+tORSV (TTC) showed that no recombination but rather reversion occurred in two of the 20 co-inoculated plants.

To investigate further whether reversion could occur rapidly in the newly infected plant cells, protoplasts of N. benthamiana were co-inoculated with tOT1+tORSV.GFPm. Only tOT1 or tORSV.GFPm sequences were detected from 50 cDNA clones of RT-PCR products amplified from the region flanking nt 182–376 of ORSV. No wild-type virus TTT sequence was detected. Therefore, reversion was not detected in the co-inoculated protoplasts by 48 h p.i.


   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
RdRp functions as the catalytic subunit of the viral replicase required for the replication of all RNA viruses. To verify the importance and conservation of Phe50 in RdRp of tobamoviruses, the first 101 aa of the RdRp of 13 tobamoviruses were compared. Interestingly, the Phe residue is highly conserved among all tobamoviruses, except for a Cys50 in SHMV (Silver et al., 1996). In this study, tORSV.Cys (TGT) was not infectious (Table 1; Fig. 1b–d). Phylogenetically the SHMV RdRp is the most distantly related to other tobamoviruses (results not shown). Our results confirmed that it was Phe50 rather than the nucleotide(s) that plays an important role in the replication of ORSV and TMV (Figs 2 and 3), although Phe50 is not located in any of the putative conserved domains of 126/183-kDa proteins. Our results showed that no nascent minus-strand RNA was detected in protoplasts up to 36 h p.i. (Fig. 1d), indicating that none of the mutant replicases with alteration in Phe50 could support minus-strand RNA synthesis, and that another domain(s) may also be affected. This result showed that alteration of an important amino acid could render a replicase non-functional.

The 110 aa region downstream of the core MT in the 126/183-kDa proteins of ToMV was shown to bind to its 3' UTR (Osman & Buck, 2003). A similar binding domain might also be present in ORSV. Aromatic amino acids, including Phe and Tyr, are known to cross-link with nucleic acids (Golden et al., 1999). However, alteration of Ser50 to Tyr did not restore the replication. Therefore, neither Tyr50 nor Phe50 may participate in direct binding to RNA. In the Ras superfamily, a group of small GTPases that function as molecular switches or timers to regulate cell fate, Phe156 is conserved and mutation of Phe results in disruption of secondary structure (Quilliam et al., 1995). Similarly, it is possible that substitution of Phe50 may result in a conformational change that renders it non-functional. Similar results obtained from the TMV mutants further suggest that Phe50 may play an important role in replication among all tobamoviruses. The exact effects of altering Phe50 could be studied if the complete 126/183-kDa proteins could be expressed and crystallized. However, it is still a challenge to express and purify large membrane-bound RdRp proteins of TMV from E. coli (Osman & Buck, 2003) and only limited success has been achieved with partially purified RdRp of Bamboo mosaic virus (Li et al., 1998). Since OT1 and OT2 share the same known individual domains with only one amino acid change outside of those domains, studies using individual domains are unable to distinguish the differences between OT1 and OT2.

Trans-complementation is a common phenomenon among animal and plant viruses (Atabekov et al., 1999; Garcia-Arenal et al., 2001; Heldsinger & Sharmeen, 2001; Lough et al., 2001; Osbourn et al., 1990; Saphire et al., 2002; van Kuppeveld et al., 2002). A full-length replication-defective mutant of the coxsackie B3 virus 2B protein can be complemented by its wild-type virus (van Kuppeveld et al., 2002). Replicase components produced by a replication-competent mutant of TMV are able to complement replication-defective mutants with various deletions in trans (Knapp et al., 2001; Lewandowski & Dawson, 1998; Ogawa et al., 1991, 1992). However, efficient trans-complementation of a full-length replication-deficient genome is uncommon among tobamoviruses (Ogawa et al., 1991). Full-length defective RNA encoding only the 126-kDa protein could not be complemented in trans by a construct encoding the functional 126/183-kDa proteins (Lewandowski & Dawson, 1998; Ogawa et al., 1992), as it contains sequences that are inhibitory to replication (Lewandowski & Dawson, 1998). Efficient replication requires the 183-kDa protein to form a heterodimer with the 126-kDa protein which has already bound to the target RNA (Lewandowski & Dawson, 2000). Recent evidence shows that the helicase domain interacts with itself to form hexamer-like oligomers (Goregaoker et al., 2001). Our results showed that full-length tOT1 was trans-complemented by various replication-competent mutants in both protoplasts and host plants (Fig. 5 and Table 2). The results thus confirmed that a full-length replication-defective mutant could be complemented by a replication-competent ORSV, indicating that there is no inhibitory RNA sequence in ORSV.

In this study, we confirmed that ORSV.GFPm could not move long distances and was not assembled, as shown by RT-PCR using appropriate primers, whereas OT1 was assembled in N. benthamiana protoplasts (Fig. 5c). Another result showed that OT1.GFP, which has CP substituted with GFP, was not detected in systemic leaves (Table 2). It indicates that CP is required for efficient long-distance movement. Our observation of two sizes of necrotic lesions on C. quinoa may indicate differences in efficiency of cell-to-cell movement among the mutants tOT1+tORSV.GFP, tORSV.GFP and tOT2.

Since tOT1 alone is unable to replicate and tORSV.GFP is unable to move long-distances without CP, neither virus mutant could move into the upper non-inoculated leaves independently. Only virus mutants that acquire a functional RdRp gene and CP gene either through recombination or reversion will be able to move systemically and induce symptoms. Therefore, it is reasonable to speculate that reversion occurs in the inoculated leaves, as recombination has not been detected. Revertants were not detected in the inoculated leaves; this was probably due to the lack of detection sensitivity at early stages of infection.

If recombination between OT1 and ORSV.GFPm occurred during trans-complementation to restore the replication of OT1, the marker nucleotides in the silent mutations of ORSV.GFPm (AAtTTTagt) would appear in the recombinant viruses present in the upper non-inoculated leaves. Only the revertant sequence TTT was detected in the extracted virions. None of the silent marker sequences from ORSV.GFPm were detected in the total RNA or virions extracted from the upper non-inoculated leaves (Table 2), indicating that the tOT1 TCT sequence reverted without recombination. The reversion of a mutant Y1867F back to wild-type sequence after undergoing systemic spread has been reported in Tobacco vein mottling virus (Murphy et al., 1996). Another example comes from mutant BW5.123 of Beet western yellow virus which reverted to its original wild-type sequence without complementation and/or recombination (Brault et al., 2000).

Reversion of mutants could occur in vivo. In general, restoration of function by second or other site mutations have been reported. For example, in ToMV, restoration of the MP function occurred, resulting from a Leu to Ile mutation, rather than reversion (Kawakami et al., 2003). Another example is a mutation at the amino acid level in the HEL region of the TMV RdRp which reverted to the wild-type in nucleotide sequence (Goregaoker et al., 2001). We observed exact reversion, but not second-site mutation, that resulted in restoration of replication in OT1. A mutant containing GUA in the 3' non-coding region of RNA3 of Brome mosaic virus reverted back to the conserved AUA wild-type sequence without any other mutation occurring (Rao & Hall, 1993). A similar report showed that an infectious Rubella virus variant with a motif substitution of GDD with ADD in the RdRp region reverted back during infection (Wang & Gillam, 2001). In these cases, it seems that reversion is likely to be the only way to restore function. In another example, a single nucleotide substitution in the amber codon of 130/180-kDa proteins of ToMV resulted in a mutant without expression of the 130-kDa protein. This mutant was unstable and the mutated codon rapidly reverted back to an amber codon, resulting in the expression of both 130-kDa and 180-kDa proteins (Ishikawa et al., 1986). Here reversion occurred and resulted in the restoration of replication for OT1.

Reversion to Phe50 restored the replication function of OT1 (TCT). Since there are only two possible codons for Phe (TTT or TTC), mutation from TCT to TTC can also restore the replication function. However, this would require two mutation events. Reversion from TCT to TTT is more straightforward, as it involves a change in only one nucleotide to regain its function. Therefore, this mutation is more likely to occur, resulting in an exact reversion. During the replication of tOT1 complemented by the functional RdRp of tORSV.GFP, the host might also be involved in the selection of the revertant for replication in vivo. In Potato virus X, restoration of a stem–loop structure required for accumulation indicated host selection (Miller et al., 1999).

Two lines of evidence have shown exact reversion of Ser50 to Phe50 in vivo [Table 2; tOT1+tORSV.GFP and tOT1.GFP+tORSV (TTC)], this may have resulted from virus mutation, virus adaptation, host selection (Domingo et al., 2002; Rozanov et al., 1992) or any of the above combinations. Failure to detect the reversion in protoplasts could be due to insufficient cycles, small population size, lack of host selection or that reversion was such a rare event that it escaped detection. The mechanism of exact reversion is still unclear. However, the error-prone RdRp and mutation may contribute to exact reversion during virus replication. Further understanding and investigation into the biochemistry of each component of the functional RdRp complex may provide new insights into the mechanism of exact reversion.


   ACKNOWLEDGEMENTS
 
We thank Professor Peter Palukaitis of the Scottish Crop Research Institute, Scotland for suggestions, helpful discussions and critical review of this manuscript. This research was supported by National University of Singapore through research grant R-154-000-186-112. H.-H. W. was a recipient of a post-doctoral fellowship from the former National Science and Technology Board, Republic of Singapore.


   REFERENCES
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ABSTRACT
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METHODS
RESULTS
DISCUSSION
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Received 28 February 2004; accepted 7 April 2004.