John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK1
Author for correspondence: Andy Maule. Fax +44 1603 450045. e-mail andy.maule{at}bbsrc.ac.uk
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Viral MPs fall into several functional classes, of which the best characterized are the tobamovirus-like MPs and the comovirus-like MPs. Tobacco mosaic virus MP is functional as an N-terminal GFP fusion protein and mediates virus movement by directing the translocation of a viral RNAMP complex through plasmodesmata. Comovirus MPs are one group in an increasing list of viral MPs that form tubular structures which displace the plasmodesmal desmotubule to provide a conduit for virus nucleocapsids to pass from cell to cell. Other virus groups present in this list are nepoviruses, bromoviruses, tospoviruses and plant pararetroviruses, including Cauliflower mosaic virus (CaMV).
To study CaMV MP function in isolation from the infection we have exploited the ability of the MP to form tubules at the surface of insect cells after expression in insect cells from baculovirus vectors (Kasteel et al., 1996 ; Thomas & Maule, 1999
). With this expression system, we have shown that the MP tubule-forming domain comprises most (aa 1282) of the MP (Thomas & Maule, 1999
). The non-essential region consists of the C-terminal 45 amino acids (aa 283327). By analogy with structure/function characteristics of the MP of Cowpea mosaic virus (CPMV) (Lekkerkerker et al., 1996
), with which CaMV MP has structural similarity in this region, the C-terminal part of the protein probably interacts with virus particles in the lumen of the tubule during virus translocation from cell to cell. We had proposed previously that the N terminus of the MP could be exposed on the outer surface of the tubule (Thomas & Maule, 1995a
) and also shown that a 6x His addition to the N terminus of the protein did not abolish the movement function in an infection in plants (unpublished data). Using baculovirus expression in insect cells as a model system, we aimed to use GFP fused to the N or C terminus of the relevant domain of the CaMV MP to study the cellular components involved in tubule formation. This model system was selected to: (1) avoid the difficulties of expressing foreign sequences (e.g. GFP) from within the CaMV genome (Fütterer et al., 1990
); (2) analyse tubule formation separate from other associated movement functions of the MP (e.g. RNA binding; Thomas & Maule, 1995b
) or functions associated with its nucleic acid sequence (e.g. splicing; Kiss-László et al., 1995
); and (3) by-pass the inefficiency of tubule formation following transient expression of the CaMV MP in host plant cells (Perbal et al., 1993
).
Translational fusions were made between the gfp gene and CaMV gene I (MP) from CaMV CM1841 (Gardner et al., 1981 ) using PCR and standard cloning techniques. The fusion was then cloned into recombinant baculoviruses (called bvMP) using the Gibco BRL Bac-to-Bac baculovirus expression system. The starting material was a CaMV gene I construct containing the c-myc epitope sequence within the C-proximal spacer region (SPmyc; Thomas & Maule, 1995a
, 1999
). This gene I sequence has been shown to be fully functional in planta when cloned into the CaMV genome (Thomas & Maule, 1995a
).
To construct the MP C-terminal fusion with GFP, a gene I fragment (nt 3641234; Gardner et al., 1981 ) was released from pFastbacSPmyc (Thomas & Maule, 1999
) by digestion with BamHI (immediately 5' to the gene I ATG codon) and HindIII (within the c-myc epitope). This was ligated to gfp, generated by PCR from gfp4 (Haseloff et al., 1997
) with primers containing HindIII (5', but eliminating the ATG initiation codon) and XhoI (3') adaptors. The resulting MPGFP fragment was cloned into a BamHI/XhoI-digested vector (pFastbac). The resulting translational fusion had the junctional sequence CAG AAG CTT AGT (HindIII site in bold type), which inserted an additional two amino acids at the MP/GFP junction. To construct the MP N-terminal fusion with GFP, adaptor PCR primers were similarly used to generate gfp4 with a BamHI site and an EcoRV site outside of the ATG initiation codon and inside the TAA termination codon. The gene I fragment (CaMV nt 364737; Gardner et al., 1981
) was generated using primers that added an EcoRV site outside of the ATG start codon and included the NcoI site at nt 737. Gene I and gfp4 fragments were ligated with BamHI/NcoI-digested pFastbacSPmyc. The translational fusion had the sequence AAA GAT ATC ATG (EcoRV site in bold type) and similarly inserted an additional two amino acids (D, I) at the GFP/MP junction (Fig. 1
). The control vector, pFastbacGFP, containing unfused gfp, was generated by ligating a PCR gfp fragment with primer-adapted sequences (5' BglIIXhoI 3', including translational start and stop sequences) into pFastbac. MPGFP and GFPMP are illustrated in Fig. 1(a)
.
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When baculovirus bvMPGFP or bvGFPMP was used to infect Sf21 cells grown on glass slides (Thomas & Maule, 1999 ) aggregates of fluorescing material were visible without immunostaining (Fig. 1b
, c
) and corresponded to the pattern of distribution of the fusion protein detected using the anti-MP serum (not shown). In each case, the distribution differed from bvGFP expressing free GFP (Fig. 1d
). It also differed from bvSPmyc, which showed the formation of surface tubules by immuno-staining (Fig. 1e
), as seen previously (Thomas & Maule, 1999
). This inability of the fusion protein to form tubules was contrary to results for similar experiments with MPs of Alfalfa mosaic virus (Zheng et al., 1997
) and Olive latent virus 2 (Grieco et al., 1999
) that were fused to GFP, which formed tubules at the surface of protoplasts. Until we know more about the three-dimensional structure of these tubule-forming MPs, we can only speculate that this contradiction represents differences in the positioning of the C and N termini relative to the core of the MP. For CaMV MP specifically, the failure to form tubules must be due to an inhibition of MP function attributable to incorrect folding of the MP when fused to GFP or to the interference of GFP in the MP aggregation into tubules. Analysis of the protein content of CPMV MP tubules (Kasteel et al., 1997
) indicated that there were no other proteins involved in tubule formation.
To test whether GFP fusion proteins could result in steric hindrance at the N or C termini to the ordered aggregation of the MP into a tubule, bvMPGFP or bvGFPMP was co-expressed with bvSPmyc in insect cells in an m.o.i. ratio of 1:4 and 1:1 (Thomas & Maule, 1999 ). Immunoblot analysis of total insect cell proteins extracted (Thomas & Maule, 1999
) at 48 h p.i., with anti-MP serum (Fig. 2a
) and anti-GFP (Clontech) (Fig. 2b
), showed that the GFP-tagged and untagged MP were both expressed in the insect cells and approximately in proportion to their relative m.o.i. In each case, when the co-infected cells were examined for GFP fluorescence, long thread-like structures, interpreted previously as tubules (Kasteel et al., 1996
; Thomas & Maule, 1999
), were seen extending from the cell surface (Fig. 2c
, d
). These structures were more abundant and more fluorescent with an m.o.i. ratio of 1:4 than with a ratio of 1:1. These structures were not seen when bvSPmyc was absent (i.e. no unfused MP) or when bvGFP was co-infected with bvSPmyc (i.e. no MPGFP or GFPMP fusion present), although in the latter case, tubules could be detected by immuno-staining with anti-MP serum (data not shown).
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Construct pFastbacGFP2AMP was made by using PCR to add 72 nt of FMDV 2A sequence (Ryan et al., 1991 ) (with a BglII adaptor) and a StuI site to the 3' and 5' ends, respectively, of gfp (Crameri et al., 1996
). This was ligated with a CaMV gene I fragment [isolated from pFastbacSPmyc digested with BamHI (5') and XhoI (3')], into StuI/XhoI-digested pFastbac. This resulted in the insertion of 24 amino acids of FMDV with a GFP2AMP junction sequence of CCCAAGATCCCTATG (BglII/BamHI fusion in bold type) where auto-proteolysis would leave four amino acids (PKIP) at the N terminus of the MP (Fig. 1a
). Immunoblot analysis of proteins extracted 48 h p.i., from insect cells infected with bvGFP2AMP, showed that the fusion protein (Mr 66 kDa) cleavage by 2A was about 80% efficient, generating free MP (Mr 46 kDa; Fig. 3a
) and GFP with a C-terminal addition of 23 amino acids (Mr 29 kDa; Fig. 3b
). When insect cells were examined for GFP fluorescence, long tubules were seen (Fig. 3c
). A similar number of tubules was seen when the insect cells were immunostained for MP distribution (Fig. 3d
).
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Acknowledgments |
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References |
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Fütterer, J., Bonneville, J. M. & Hohn, T. (1990). Cauliflower mosaic virus as a gene expression vector for plants. Physiologia Plantarum 79, 154-157.
Gardner, R. C., Howarth, A. J., Hahn, P., Brown-Luedi, M., Shepherd, R. J. & Messing, J. (1981). The complete nucleotide sequence of an infectious clone of cauliflower mosaic virus by M13mp7 shotgun sequencing. Nucleic Acids Research 9, 2871-2888.[Abstract]
Grieco, F., Castellano, M. A., Di Sansebastiano, G. P., Maggipinto, G., Neuhaus, J.-M. & Martelli, G. P. (1999). Subcellular localization and in vivo identification of the putative movement protein of olive latent virus 2. Journal of General Virology 80, 1103-1109.[Abstract]
Harker, C. L., Mullineaux, P. M., Bryant, J. A. & Maule, A. J. (1987). Detection of CaMV gene 1 and gene VI protein products in vivo using antisera raised to COOH-terminal -galactosidase proteins. Plant Molecular Biology 8, 275-287.
Haseloff, J., Siemering, K. R., Prasher, D. C. & Hodge, S. (1997). Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proceedings of National Academy of Sciences, USA 94, 2122-2127.
Kasteel, D. T. J., Perbal, M.-C., Boyer, J.-C., Wellink, J., Goldbach, R. W., Maule, A. J. & van Lent, J. W. M. (1996). The movement proteins of cowpea mosaic virus and cauliflower mosaic virus induce tubular structures in plant and insect cells. Journal of General Virology 77, 2857-2864.[Abstract]
Kasteel, D. T. J., Wellink, J., Goldbach, R. W. & van Lent, J. W. M. (1997). Isolation and characterisation of tubular structures of cowpea mosaic virus. Journal of General Virology 78, 3167-3170.[Abstract]
King, L. A. & Possee, R. D. (1992). The Baculovirus Expression System. A Laboratory Guide. London: Chapman & Hall.
Kiss-László, Z., Blanc, S. & Hohn, T. (1995). Splicing of cauliflower mosaic virus 35S RNA is essential for viral infectivity. EMBO Journal 14, 3552-3562.[Abstract]
Lekkerkerker, A., Wellink, J., Yuan, P., van Lent, J., Goldbach, R. W. & van Kammen, A. (1996). Distinct functional domains in the cowpea mosaic virus movement protein. Journal of Virology 70, 5658-5661.[Abstract]
Maule, A. J., Usmany, M., Wilson, I. G., Boudazin, G. & Vlak, J. M. (1992). Biophysical and biochemical properties of baculovirus-expressed CaMV P1 protein.Virus Genes 6, 5-18.[Medline]
Oparka, K. J., Boevink, P. & Santa Cruz, S. (1996). Studying the movement of plant viruses using green fluorescent protein. Trends in Plant Science 1, 412-418.
Perbal, M.-C., Thomas, C. L. & Maule, A. J. (1993). Cauliflower mosaic virus gene 1 product (P1) forms tubular structures which extend from the surface of infected protoplasts. Virology 195, 281-285.[Medline]
Ryan, M. D. & Drew, J. (1994). Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein. EMBO Journal 13, 928-933.[Abstract]
Ryan, M. D., King, A. M. Q. & Thomas, G. P. (1991). Cleavage of foot-and-mouth disease virus polyprotein is mediated by residues located within a 19 amino acid sequence.Journal of General Virology 72, 2727-2732.[Abstract]
Thomas, C. L. & Maule, A. J. (1995a). Identification of structural domains within the cauliflower mosaic virus movement protein by scanning deletion mutagenesis and epitope tagging. Plant Cell 7, 561-572.
Thomas, C. L. & Maule, A. J. (1995b). Identification of the cauliflower mosaic virus movement protein RNA binding domain. Virology 206, 1145-1149.[Medline]
Thomas, C. L. & Maule, A. J. (1999). Identification of inhibitory mutants of cauliflower mosaic virus movement protein function after expression in insect cells. Journal of Virology 73, 7886-7890.
Zheng, H., Wang, G. & Zhang, L. (1997). Alfalfa mosaic virus movement protein induces tubules in plant protoplasts. Molecular PlantMicrobe Interactions 10, 1010-1014.
Received 10 January 2000;
accepted 14 March 2000.