1 Department of Virology and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Vorobiovy Gory Moscow 119899, Russia
2 M. M. Shemyakin and Yu. A. Ovchinnikov Institut of Bioorganic Chemistry, Moscow, Russia
3 Joint Biotechnology Laboratory, Biocity, Turku, Finland
4 University of Helsinki, Institute of Biotechnology, Biocenter, Helsinki, Finland
5 Ludwig Institute of Cancer Research, Biomedical Center, Uppsala, Sweden
Correspondence
Joseph Atabekov
atabekov{at}genebee.msu.su
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ABSTRACT |
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Published ahead of print on 20 December 2002 as DOI 10.1099/vir.0.18972-0.
Present address: Division of Experimental Clinical Chemistry, Lund University, Malmo University Hospital, Malmo, Sweden.
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MAIN TEXT |
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Watanabe et al. (1992) reported that C-terminal residues 234261 are required for TMV MP phosphorylation in tobacco protoplasts, although it is clear that in TMV-infected protoplasts MP can be phosphorylated at multiple internal phosphorylation sites (Haley et al., 1995
). In particular, two distinct domains (residues 61114 and 212231) can be substrates for PK(s) other than the CW-associated PK(s) mentioned above. Similarly, it has been shown that Ser37 and Ser238 of tomato mosaic tobamovirus (ToMV) MP can be phosphorylated in protoplasts. The presence of Ser at position 37 or phosphorylation of Ser37 is important for ToMV MP functionality (Kawakami et al., 1999
). Thus, the results from different groups of workers imply that TMV MP is phosphorylated in vivo, although it is hard to say in what cellular compartments MP phosphorylation occurs. It is important to emphasize that only the C-proximal sites of CW-associated TMV MP are found phosphorylated in planta (Waigmann et al., 2000
), while multiple internal sites of MP could be phosphorylated in infected protoplasts (Haley et al., 1995
; Kawakami et al., 1999
). Despite the apparent contradiction, the various results are not necessarily incompatible. Thus, TMV MP might be transiently phosphorylated when subjected to processes of phosphorylation/dephosphorylation at its internal sites by cytoplasmic PK(s), whereas only the C-proximal sites are selectively phosphorylated by CW-associated PKs.
The TMV genome is accepted widely to be translocated from cell to cell as an MPRNA complex. Moreover, it has been reported that TMV MP is an efficient repressor of in vitro translation and phosphorylation of MP prevents its translation-repressing ability (Karpova et al., 1999). Possible roles of viral MP phosphorylation in regulation of TMV genome expression have been discussed recently by Lee & Lucas (2001)
.
Microsomal fractions from leaves of N. tabacum var. Samsun were isolated by sucrose gradient centrifugation, as described by Mas & Beachy (1999), and analysed for PK activity using preparations of bacterially expressed TMV U1 (His)6-MP as a substrate for labelling in the presence of [
-32P]ATP. Purification of (His)6-MP was carried out as described by Karpova et al. (1997)
. The level of MP phosphorylation activity varied along the sucrose gradient with maximums in fractions 24 and 1215 (Fig. 1
a). The presence of endoplasmic reticulum (ER) lumenal-binding protein (BiP), an ER membrane resident protein (Reichel & Beachy, 1998
), was revealed in fractions 2 and 1113 by Western blotting (Fig. 1b
). These data indicate that MP-specific PK activity was at a maximum in ER-containing fractions of the sucrose density gradient (Fig. 1a, b
). However, there was no BiP in fractions 4 and 15 that phosphorylate the MP. Thus, the PK(s) that phosphorylate the MP may not reside exclusively in the ER.
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To localize the sites phosphorylated by the ER-associated PK activity in TMV MP, (His)6-MP was phosphorylated in vitro in the presence of a microsomal fraction, [-32P]ATP and 1 mM MnCl2. After additional purification of phosphorylated protein on NiNTA resin in the presence of 6 M guanidium/HCl, pH 8·0 (Qiagen), according to the manufacturer's protocol, and immobilization on thiopropylSepharose 6B (Sigma), the MP was digested by sequence-grade trypsin (Sigma) and the resulting peptides were analysed by two different approaches. First, two-dimensional peptide mapping was applied to separate the phosphopeptides (Fig. 2
a). Analyses were performed on HTLE-7002 equipment in accordance with the manufacturer's protocol (CBS Scientific). Several 32P-labelled tryptic peptides could be seen in the phosphopeptide map (Fig. 2a
), which is consistent with TMV MP being phosphorylated by ER-associated PK at multiple sites. The five most prominent spots (Fig. 2a
, numbers 15), corresponding presumably to major 32P-labelled tryptic peptides, were subjected to phospho-amino acid analysis; it was demonstrated that 32P was incorporated into spots corresponding to phosphoserine in peptides 25 and to phosphothreonine in peptide 1 (data not shown). Second, the 32P-labelled phosphopeptides were separated by HPLC and isolated in sufficient amounts for partial amino acid sequencing (first five amino acids were analysed in each peptide). In the present work, we have focused on characterization of peptide 1, which was found to contain Thr104 in the N-terminal sequence ADEAT. Indirect evidence for the importance of Thr104 in TMV MP activity was provided by analyses of functional reversions of Thr104 dysfunctional mutants (Deom & He, 1997
; Boyko et al., 2002
). It is also noteworthy that Thr104 is conserved in MPs encoded by different ToMV (Koonin et al., 1991
).
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To examine the phosphorylation patterns of mutated MPs, the preparations of bacterially expressed recombinant T104A and T104D proteins were phosphorylated as described above and analysed by two-dimensional peptide mapping. Fig. 2(b, c) show that substitution of Thr104 by either of the amino acids Ala or Asp led to the disappearance of one major and one minor spot (Fig. 2a
, labelled 1 and A) from the phosphopeptide map. The major spots, 25 (Fig. 2
), were still present in the phosphopeptide maps of the mutant MPs. These results indicate that (i) Thr104 can be phosphorylated in vitro by ER-associated PK(s) in wild-type MP and (ii) the level of phosphorylation of phosphopeptides 25 was not decreased by replacement of Thr104 with either of the amino acids. It is noteworthy that two minor spots (Fig. 2a, b
, labelled B and C) observed in phosphopeptide maps of wild-type and T104A MP were missing from phosphorylated T104D MP (Fig. 2c
). One can speculate that substitution of Thr104 by Asp may change the MP conformation so that these two sites are not exposed to phosphorylation. Finally, the ER-associated PKs responsible for T104A, T104D and wild-type MP phosphorylation were examined by gel PK assay. The number and apparent molecular masses of PKs revealed were similar in the experiments when wild-type and mutant MPs were used as substrate (data not shown). Therefore, no particular ER-associated PK was responsible for the Thr104 phosphorylation only.
To elucidate the functional importance of Thr104 for TMV cell-to-cell movement, mutations were introduced into the MP gene of a full-length TMV U1 cDNA copy to substitute Thr104 by Ala or Asp in modified MP. The mutant viruses referred to as TMV T104A and TMV T104D, respectively, were compared by inoculation of indicator plants reacting to TMV infection by production of local lesions (N. tabacum cv. Xanthi nc.) or systemic symptoms (N. tabacum var. Samsun, N. benthamiana). Opposite halves of the same leaf were inoculated and mean values for at least 10 inoculated leaves were compared. The specific infectivity levels (number of the local lesions produced by 1·5 µg RNA on Xanthi nc. leaves) of wild-type and T104A transcripts were very similar, as was the size of lesions produced by T104A and wild-type RNA (Fig. 3a). In contrast, the specific infectivity of T104D RNA transcripts dramatically decreased (13±5 and 86±17 lesions per half-leaf were induced by T104D and wild-type TMV, respectively). It should be emphasized that only tiny local lesions were produced by T104D mutant (Fig. 3a
), suggesting that the Thr to Asp substitution at position 104 strongly inhibited virus cell-to-cell movement. However, our results do not rule out that the MP produced by mutant T104D is less stable than wild-type and T104A MPs.
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In conclusion, our in vivo experiments have shown that: (i) replacement of Thr104 in TMV MP with neutral Ala did not cause significant changes in cell-to-cell movement of TMV, indicating that phosphorylation of Thr104 was not essential for MP functions; (ii) substitution of Thr104 by a negatively charged Asp residue led to a strong inhibition of the local lesion development in Xanthi nc. tobacco. This inhibition could be eliminated in Xanthi nc. plants transgenic for MP gene. Presuming that this substitution functionally mimics phosphorylation, we suggest that Thr104 phosphorylation renders TMV MP dysfunctional. If this is the case, it seems logical to hypothesize that Thr104 phosphorylation in vivo represents a defence mechanism that protects the plant from virus infections. It should be mentioned that inactivation of the MP by the Asp104 mutation may not be directly due to mimicry of phosphorylation but due to the change of the MP conformation. It is evident that our data do not provide direct evidence that MP is in fact phosphorylated at Thr104 during infection. Alternatively, it may be phosphorylated only transiently in vivo. Experiments on examination of in vivo Thr104 phosphorylation are in progress.
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ACKNOWLEDGEMENTS |
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Received 12 November 2002;
accepted 10 December 2002.