Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK1
Author for correspondence: John Carr. Fax +44 1223 333953. e-mail jpc1005{at}hermes.cam.ac.uk
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Abstract |
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Host proteins were first implicated in the replication of positive-stranded RNA viruses by the discovery that the replicase of the bacteriophage Q contains four host-encoded proteins: the S1 30S ribosomal protein, a ribosome-associated protein termed HF-1 and two protein synthesis elongation factors, EF-Tu and ET-Ts (Blumenthal & Carmichael, 1979
). Host proteins have also been found that co-purify with the replicase of brome mosaic virus (BMV) (Quadt & Jaspars, 1990
), cowpea mosaic virus (Dorssers et al., 1984
), cucumber mosaic virus (Hayes & Buck, 1990
), red clover necrotic mosaic virus (Bates et al., 1995
), Sindbis and Semliki Forest viruses (Barton et al., 1991
) and turnip yellow mosaic virus (Mouches et al., 1984
). So far, the identities and role(s) in virus replication of most of these host proteins remain elusive. However, there is evidence to suggest that subunits of the eukaryotic translation initiation factor eIF-3 play a role in the replication of BMV (Quadt et al., 1993
) and TMV (Osman & Buck, 1997
).
In the case of TMV, several host proteins have been found to be associated with replicase preparations purified from infected tissue (Osman & Buck, 1997 ; Watanabe et al., 1999
). Osman & Buck (1997)
identified a 56 kDa protein, immunologically related to the GCD10 subunit of yeast eIF-3, in the TMV replicase isolated from infected tomato tissue. The viral 126 and 183 kDa proteins, plus the host-encoded 56 kDa protein, could be purified by using either antibodies against the TMV-L 126 kDa protein or antibodies against yeast GCD10 (Osman & Buck, 1997
). The 56 kDa host protein could not be observed by SDSPAGE analysis of an immuno-purified replicase preparation from tissue infected with TMV-OM because of the large amounts of IgG present (Watanabe et al., 1999
). However, a role for the GCD10-like protein in virus replication is very likely, since the synthesis of TMV genomic ssRNA and dsRNA by the TMV-L replicase was inhibited by antibodies against the GCD10 protein, and this inhibition could be prevented by competition with purified yeast GCD10 protein (Osman & Buck, 1997
).
It is not known whether the plant GCD10-like protein binds the virus-encoded components of the TMV-L replicase complex directly or whether the interaction is via a bridging factor, perhaps another component of the translation initiation apparatus. Since the plant GCD10-like protein has not yet been cloned, we decided to use the yeast two-hybrid system to determine whether the yeast GCD10 protein interacts directly with the TMV-encoded replicase proteins and, if so, which domain(s) of the 126/183 kDa proteins is involved in the interaction.
By using a full-length TMV-L cDNA clone (Ohno et al., 1984 ) as a template, PCR was used to generate cDNA fragments corresponding to the three putative functional domains of the TMV 126/183 kDa protein sequence (Fig. 1
). The methyltransferase-like (designated Dom 1 in Fig. 1
) and helicase-like (Dom 2) domains in this study span amino acids 1484 and 6471116, respectively (Fig. 1
), and correspond to the boundaries determined by primary amino acid comparisons between plant-infecting members of the alphavirus-like superfamily and Sindbis virus (Ahlquist et al., 1985
). The polymerase-like domain (Dom 3) contained the amino acids 11411616 of the 183 kDa readthrough region, which is equivalent to the putative 54 kDa open reading frame (Sulzinski et al., 1985
). The DNA sequences were cloned into the two-hybrid vectors pAS2 and pACTII to generate protein fusions with the GAL4 DNA-binding domain and transcription-activation domain, respectively (Harper et al., 1993
; Li et al., 1994
). All amplified DNA fragments were gel-purified, digested with appropriate restriction enzymes, ligated into pAS2 and pACTII and checked by automated DNA sequencing before use in the yeast two-hybrid system. A DNA clone pMG107 (Garcia-Barrio et al., 1995
) encoding the yeast GCD10 protein sequence was excised with BamHI and XhoI and sub-cloned into pACTII cut with the same restriction enzymes to yield pACTIIGCD10. To sub-clone into pAS2, the GCD10 coding sequence in pMG107 was digested with BamHI and PstI and the fragment was ligated to produce pAS2GCD10.
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Our results indicate that the GCD10 protein interacts with the methyltransferase-like domain of the TMV-L 126/183 kDa proteins (Table 1). The interaction was found to be orientation-specific. That is to say, the interaction between GCD10 and the TMV 126/183 kDa methyltransferase domain was only observed with the two-hybrid vector combination pAS2GCD10pACTIIDom1 (Table 1
; Fig. 2
). This result was confirmed spectrophotometrically by using extracts of transformed yeast. In three separate experiments, we found that the level of
-galactosidase activity in cells transformed with the vector combination pAS2GCD10pACTIIDom1 was a minimum of fivefold greater in all cases than that in cells transformed with the vector combination pAS2Dom1pACTIIGCD10 or in untransformed yeast (data not shown). It was also found that Y190 cells containing the vector combination pAS2GCD10pACTIIDom1 grew well on medium lacking histidine, tryptophan and leucine but containing 25 mM 3-aminotriazole (Yocum et al., 1984
) (data not shown). This indicated that the second reporter gene his3 was induced in these cells and further confirmed the interaction.
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We also carried out experiments to determine whether the cloned domains (Fig. 1) of the 126 and 183 kDa proteins were able to interact with each other to form homo- or heterodimers. However, we found no evidence for dimerization (data not shown). This is consistent with work with BMV, in which the 1a helicase-like domain interacted with the 2a polymerase-like protein (Smirnyagina et al., 1996
) but did so via an interaction with a region of 115 amino acids that lies upstream of the conserved polymerase-like domain (OReilly et al., 1998
) and not with the polymerase-like domain itself (Kao & Ahlquist, 1992
; OReilly et al., 1995
). The 115 amino acid region, necessary and sufficient for the 1a2a interaction in BMV, is absent from the TMV 126/183 kDa protein sequence. This is not surprising, since the BMV 1a2a complex is the functional equivalent of the TMV 183 kDa protein, where the methyltransferase-, helicase- and polymerase-like domains are found within a single polypeptide chain. However, we cannot exclude the possibility that our results are due to the misfolding of the domains when fused with the GAL4 activation or DNA-binding moieties.
Recently, Watanabe et al. (1999) used antibodies specific for either the RNA polymerase (anti-P) or the methyltransferase (anti-M) domains of the TMV 126/183 kDa proteins to carry out co-immunoprecipitation experiments on a replicase preparation from tobacco infected with TMV-OM. They found that anti-M precipitated both the 126 and 183 kDa proteins, with the 126 kDa protein being present in excess of the 183 kDa protein, while anti-P precipitated similar amounts of the 126 and 183 kDa proteins (Watanabe et al., 1999
). This led them to suggest that the 183 kDa protein occurs in the viral replicase complex predominantly as a heterodimer with the 126 kDa protein. However, we have seen no interaction in the yeast two-hybrid system between either the Dom 1 (methyltransferase) or Dom 2 (helicase) constructs with the Dom 3 (polymerase) construct (data not shown). This suggests that dimerization between the 126 and 183 kDa proteins requires either a bridging factor, such as a host factor, or involves an interaction between the polymerase domain of the 183 kDa protein and the region of the 126 kDa protein lying between amino acid residues 484 and 647 (Fig. 1
).
In summary, we have used the yeast two-hybrid system to show that the yeast GCD10 protein appears to interact with the TMV 126/183 kDa proteins via the methyltransferase-like domain. Whether this represents a direct interaction between the two polypeptides or an indirect interaction via a third (bridging) factor present in yeast and plant cells awaits further studies in vitro. However, our work confirms and extends that of Osman & Buck (1997) , who showed that a TMV replicase preparation purified from tomato contained a 56 kDa host protein related to the GCD10 subunit of yeast eIF-3. The interaction between a plant GCD10-like protein and the methyltransferase-like domain of the 126/183 kDa protein may constitute a link between the viral replicase and the host cell translation machinery. One possible function may be to facilitate transfer of nascent, capped (+)-strand RNA to the translation machinery of the host cell. Alternatively, the GCD10-like protein may have an as yet unidentified role in (-)-strand and sub-genomic RNA synthesis. Our results suggest that synthesis of viral RNA may be closely co-ordinated with viral protein synthesis.
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References |
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Received 14 December 1999;
accepted 1 March 2000.