School of Life Sciences and Biotechnology, Korea University, Anam-dong 5-ga, Seongbuk-gu, Seoul 136-701, Korea
Correspondence
Young In Park
yipark{at}korea.ac.kr
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ABSTRACT |
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Present address: Gene Expression Programme, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK.
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INTRODUCTION |
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CMV MP localizes to plasmodesmata and to parietal sieve elements in the phloem (Blackman et al., 1998). The 2a polymerase protein and the 1a protein predominantly co-localize to vacuolar membranes (the tonoplast), and negative-strand CMV RNAs are also associated with the tonoplast (Cillo et al., 2002
). Thus, it appears that the 2a polymerase protein and MP have independent roles and a different subcellular distribution. However, CMV 2a polymerase protein and MP both affect the rate of movement of CMV in zucchini squash, although these two proteins function independently with the host to facilitate virus movement (Choi et al., 2005
).
Studies of other viruses have indicated that the 2a polymerase protein has a similar subcellular distribution to the MP and is involved in movement. In Brome mosaic virus (BMV), a member of the genus Bromovirus in the family Bromoviridae and taxonomically related to CMV, systemic infection in barley plants was inhibited by deletions in 2a proteins that supported strong RNA replication in protoplasts (Traynor et al., 1991). The 1a and 2a proteins co-localized on plant endoplasmic reticulum, the site of nascent BMV RNA synthesis (Restrepo-Hartwig & Ahlquist, 1996
). As in plant systems, the distribution of RNA 3 is associated with the sites of 1a and 2a protein accumulation in yeast cells co-expressing the 1a and 2a proteins and RNA 3 (Restrepo-Hartwig & Ahlquist, 1999
). In Tobacco mosaic virus (TMV), the type member of the genus Tobamovirus in the family Tobamoviridae, a considerably different system from CMV or BMV is found, where the helicase domain of the replicase coding region has been shown to be involved in cell-to-cell movement (Hirashima & Watanabe, 2001
, 2003
).
Since CMV 2a polymerase protein may play roles other than in viral RNA synthesis, we considered that it might regulate other virally encoded proteins, most simply by direct proteinprotein interactions. In this study, we assayed viral protein interactions using yeast two-hybrid and far-Western techniques. The results indicated that CMV MP interacts directly with the 2a polymerase protein and indirectly with the 1a protein via the 2a polymerase protein, suggestive of an additional regulatory mechanism that exploits interactions among viral proteins to promote virus propagation.
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METHODS |
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Far-Western blotting.
Labelling of GST-fused 2a polymerase protein purified from E. coli was done according to Kim et al. (2002). GST fusion protein (100 ng) was incubated in kinase buffer (20 mM Tris/HCl, pH 7·5, 100 mM NaCl, 1 mM dithiothreitol, 12·5 mM MgCl2) including 1 µCi [
-32P]ATP µl1 for 30 min. Unincorporated, free [
-32P]ATP was removed by chromatography through a Sephadex G-25 spin column equilibrated in 20 mM Tris/HCl pH 8·0, 1 mM EDTA, 100 mM NaCl, 0·1 % NP-40 and 1 mM PMSF. SDS-PAGE was performed as described by Schagger & Jagow (1987)
. Proteins in the SDS-polyacrylamide gel were transferred to nitrocellulose membrane in buffer containing 25 mM Tris/195 mM glycine at 250 mA for 1·5 h at 4 °C and the following steps were performed at this temperature. The membrane was rinsed in 100 ml binding buffer (20 mM HEPES/KOH, pH 7·7, 75 mM KCl, 0·1 mM EDTA, 2·5 mM MgCl2, 1 mM DTT, 0·05 % NP-40) for 30 min, soaked in blocking buffer (5 % BSA in binding buffer) for 4 h and hybridized with 32P-labelled (105 c.p.m. ml1) GST2a fusion protein in interaction buffer (1 % BSA in binding buffer) overnight. The membrane was rinsed in interaction buffer for 3 h with three changes of buffer and exposed to X-ray film for 412 h.
Yeast plasmids.
The CMV 2a polymerase ORF was amplified by PCR using previously described primers (Kim et al., 2002), digested with SmaI and PstI and ligated to SmaI/PstI-digested pAS2-1, which harbours the GAL4 binding domain (BD) (Clontech). The ORFs for CMV 1a protein (Kim et al., 2002
), 2b protein, MP and CP were also amplified using specific primers as follows. For the 2b ORF: nt 24142421, 5'-GAGACCCGGGGCATATGGAATTGAACGAAGGC-3', and nt 27232746, 5'-GAGAGAGCTCTCAGAAAGCACCTTCCGCCCATTC. For the MP ORF: nt 123140, 5'-GAGACCCGGGGCATATGGCTTTCCAAGGTAC-3', and nt 946963, 5'-GAGAGAGCTCCTAAAGACCGTTGACCAC-3'. For the CP ORF: nt 12611277, 5'-GAGACCCGGGGCATATGGACAAATCTGAATC-3', and nt 18971917, 5'-GAGAGAGCTCTCAAACTGGGAGCACCCCAGA-3'. Restriction sites are underlined and the nucleotide positions of the ORFs after the restriction sites are indicated. The ORF for the Fny strain of CMV (CMV-Fny) MP (GenBank accession no. D10538) was amplified using specific primers (nt 120138, 5'-GAGACCCGGGGATGGCTTTCCAAGGTACCA-3'; nt 941959, 5'-GAGAGAGCTCCTAAAGACCGTTAACCACC-3'). Amplified products were cloned into SmaI/SacI-digested pACT2, which harbours the GAL4 activation domain (AD) (Clontech).
For the yeast three-hybrid assay, the ORFs encoding the 1a protein, 2a polymerase protein and MP were amplified using specific primers sets as described above and cloned into SmaI/PstI-digested pBridge (Clontech). Alternative primer sets were used for ligation to NotI/BglII-digested pAS2-1, which encodes the GAL4 BD as follows. For 1a: nt 95112, 5'-GAGAGCGGCCGCATGGCGACGTCCTCGTTC-3', and nt 30603076, 5'-GAGAAGATCTCTAAGCACGAGCAATAC-3'. For 2a: nt 7996, 5'-GAGAGCGGCCGCATGGCTTTCCCCGCCCCC-3', and nt 26382655, 5'-GAGAAGATCTTCAGACTCGGGTAACTCC-3'. For MP: nt 124141, 5'-GAGAGCGGCCGCATGGCTTTCCAAGGTACC-3', and nt 946963, 5'-GAGAAGATCTCTAAAGACCGTTGACCAC-3'). For prey constructs, PCR products obtained with the above primer sets were digested with SmaI and SacI and cloned into pACT2.
Yeast two-hybrid and three-hybrid analyses and quantification of -galactosidase activity.
Recombinant bait and prey vectors were transformed into Saccharomyces cerevisiae strain Y190, also provided with the MATCHMAKER GAL4 Two-hybrid System 2 (Clontech). Yeast was co-transformed by the lithium acetate method as described previously (Kim et al., 2002). Transformants were plated on solid synthetic dropout minimal complete medium containing 2 % glucose and lacking leucine and tryptophan, or on medium lacking leucine, tryptophan and histidine, and for the yeast three-hybrid assay, on medium lacking methionine as well as these three amino acids. Transformants were spread on selective media and incubated at 30 °C for 46 days. Protein interactions in transformants were detected by assaying colonies on selective media plates and confirmed by assessing
-galactosidase activity with a filter lift assay. Yeast colonies were transferred to Whatman no. 1 filter paper and filters were frozen in liquid nitrogen and allowed to thaw at room temperature. The filters were placed colony-side up on a second filter paper pre-soaked in Z buffer/X-Gal solution comprising 100 ml Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4), 1·67 ml X-Gal stock solution (20 mg 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside in N,N-dimethylformamide ml1) and 0·27 ml
-mercaptoethanol. Filters were incubated at room temperature and interactions were detected by the appearance of blue colonies, generally after 560 min. Colonies isolated from the master plate were assayed quantitatively for
-galactosidase activity as described by O'Reilly et al. (1998)
. Genes encoding the SV40 large T antigen (pSV40) and the human p53 protein were inserted into pAS2-1 and pACT2, respectively, and used as positive interaction controls (Clontech). The human lamin C gene (pLaminC) was cloned downstream of the GAL4 AD in pACT2 and used as a non-interactive control (Clontech). One unit of
-galactosidase was defined as the amount that hydrolysed 1 µmol o-nitrophenyl
-D-galactopyranoside (onpg) to o-nitrophenol and D-galactose min1.
Co-immunoprecipitation of interacting proteins in yeast.
Antibodies and co-immunoprecipitation analysis were carried out as described previously (Kim et al., 2002). Immune complexes were detected by Western blotting (Sambrook et al., 1989
), using an anti-MP rabbit antibody and an anti-2a rabbit antibody. Briefly, yeast cells were resuspended with glass beads in lysis buffer (2 % Triton X-100, 1 % SDS, 100 mM NaCl, 10 mM Tris/HCl, pH 8·0, 1 mM EDTA) and vortexed five times for 5 s each at 1 min intervals. After centrifugation, the lysates were incubated with anti-2a or anti-MP antibody for 2 h at 4 °C. Immune complexes were collected and precipitated with 20 µl 50 % slurry of protein ASepharose beads (Bio-Rad). The beads were washed three times with PBS and the immunoprecipitates were separated by 8 % SDS-PAGE and transferred to a nitrocellulose membrane. Proteins were detected with anti-MP or anti-2a antibody and an alkaline phosphatase-conjugated secondary antibody (Bio-Rad).
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RESULTS |
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In the filter-lift assays, yeast colonies containing the 2a polymerase and MP genes developed a blue colour within 30 min, whereas cells carrying p53 and pSV40 as positive controls took at least 2 h for colour to develop. Yeast cells harbouring the pLaminC and pSV40 constructs as negative controls did not undergo colour changes, even after overnight incubation. As shown in Fig. 3, a strong interaction between the 2a polymerase and MP was evident, compared with the control interaction between p53 and pSV40.
-Galactosidase activity was assessed by the ONPG method and transformants carrying the interacting 2a polymerase protein and MP constructs exhibited a 20- to 50-fold higher degree of activity than control cells carrying the 2a polymerase protein bait and a vector with the AD alone. Similar results were observed in baitprey swapping experiments (data not shown). More than ten different colony variants assayed exhibited an activity approximately 20- to 50-fold higher than that of the control in three independent tests, suggesting that the 2a polymerase protein and the MP interact strongly in the yeast system.
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Co-immunoprecipitation analysis confirming 2a polymeraseMP interaction
To confirm the interaction between the 2a polymerase and MP observed by the yeast two-hybrid analysis, total yeast cell lysates were immunoprecipitated with anti-2a and anti-MP antibodies. The immune complexes were fractionated by SDS-PAGE and subjected to immunoblot analysis with anti-MP and anti-2a antibodies, respectively. Specific interactions were also identified in this experiment (Fig. 4). Total lysate from cells containing the vectors pAS2-1 and pACT2 as negative controls did not exhibit reactivity. As shown in Fig. 4
(lane 2), yeast cell lysates containing pAS2-1/2a and pACT2 were not recognized by specific antibodies. Yeast lysates containing pACT2 and pAS2-1/2a were also unreactive (Fig. 4
, lane 3). Proteins in yeast cell lysates picked from three independent colonies containing pAS2-1/2a and pACT2/MP were detected by immunoblotting with the anti-2a antibody and anti-MP antibody, respectively, confirming a specific interaction between the 2a polymerase and the MP (Fig. 4
, lanes 46).
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DISCUSSION |
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A previous study with CMV has implied a role for the polymerase protein in virus movement. It was shown that two nucleotide changes in the polymerase gene affected the elicitation of a hypersensitive response and restriction of CMV to a local lesion in cowpea (Kim & Palukaitis, 1997). Previous studies have demonstrated that the CMV 2a polymerase protein can be detected as early as 3 h post-infection in squash and tobacco protoplasts (Gal-On et al., 1994
; Kim & Palukaitis, 1997
), although phosphorylated 2a polymerase protein was undetectable prior to 48 h post-infection in the tobacco protoplast system (Kim et al., 2002
). Phosphorylated 2a polymerase protein then continued to accumulate up to 72 h post-infection. Phosphorylated 2a polymerase protein did not interact with the 1a protein, but the continued accumulation of the 2a polymerase protein suggested that it has other functions. Thus, it is conceivable that the 2a polymerase is involved in viral movement. In addition to this, some data from previous studies with TMV have shown the possibility of cooperation between the replicase and MP. It was shown that the failure of cell-to-cell movement was not caused by the MP. It is also possible that the replicase and MP associate with viral RNA and/or with a host factor(s) and collaborate indirectly (Hirashima & Watanabe, 2001
, 2003
).
Although the temporal order of the interactions in vivo is not yet known, it is suspected that the replicase and MP cooperate in regulating the intercellular movement of progeny viral RNA by an unknown mechanism. CMV transfers its genomes across plasmodesmata in the form of a ribonucleoprotein complex. Although co-localization of the MP and the replicase complex in CMV has not been detected, it is assumed that the MP can locate adjacent to the site of RNA synthesis. This would result in a more coordinated recognition of newly synthesized RNA by the MP and more rapid transfer of viral RNA to and through the plasmodesmata. During infection by TMV, despite the system being quite different from CMV, virus replication complexes containing membrane-associated replicase, genomic RNAs and MP move to adjacent cells through plasmodesmata as large bodies (Asurmendi et al., 2004; Kawakami et al., 2004
). It has been proposed that these complexes contain all the necessary components that initiate rapid spread of infection (Kawakami et al., 2004
).
Interaction of CMV-Fny 2a polymerase protein with CMV-As MP in our yeast two-hybrid system suggests that subgroup IA CMV-Fny MP, which exhibits 94·3 % amino acid sequence identity with subgroup IB CMV-As MP, may have a similar conformation to that of subgroup IB MPs. The 2a polymerase proteins of CMV-Fny and CMV-As also share 93·8 % amino acid sequence identity.
Co-expression of CMV MP and CP complements defects in the movement of Tomato mosaic virus and Potato virus X, but this effect is not seen with either protein alone (Tamai et al., 2003). CMV MP requires its cognate CP in order to transfer viral genomes through the plasmodesmata (Canto et al., 1997
; Nagano et al., 2001
). Truncated CMV MP lacking the 33 C-terminal amino acids supports the cell-to-cell movement of CMV as well as the chimeric BMV (Nagano et al., 1997
). It is suggested that it is the C-terminal 33 amino acids of CMV MP that are involved in conferring specificity for the viral genome. A recent report showed that the C-terminal 33 amino acids of CMV MP affected virus movement, RNA binding and inhibition of infection and translation (Kim et al., 2004
). Deletion of the C-terminal 33 amino acids resulted in CP-independent cell-to-cell movement, although CP was still required for long-distance movement. In addition, the truncated MP had a greater affinity for the viral RNA than the wild-type MP. The biological differences may be a consequence of differences in the architecture of binding of the two MPs (Andreev et al., 2004
). It has been suggested that the CP may alter the conformation of the MP available for complex formation (Kim et al., 2004
). The CP also may not interact directly with the MP, but via a host protein(s), which may explain the failure to detect direct interactions between the MP and CP as shown here.
In conclusion, our study constitutes the first report showing that, among CMV virally encoded proteins, the MP interacts specifically with the 2a polymerase protein. Further studies should allow us to identify the function of the interaction between the 2a polymerase and MP.
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ACKNOWLEDGEMENTS |
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Received 27 April 2005;
accepted 18 August 2005.
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