1 Department of Ornamental Horticulture, Agricultural Research Organization, The Volcani Center, PO Box 6, Bet Dagan 50250, Israel
2 Department of Virology, Agricultural Research Organization, The Volcani Center, PO Box 6, Bet Dagan 50250, Israel
3 Horticulture Institute, Agricultural Research Organization, The Volcani Center, PO Box 6, Bet Dagan 50250, Israel
Correspondence
Tzahi Arazi
tarazi{at}volcani.agri.gov.il
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ABSTRACT |
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Supplementary table presenting CP-Nt net charge of various potyviruses is available in JGV Online.
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INTRODUCTION |
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In contrast to the conserved core, the exposed CP N-terminal domain (CP-Nt) is not conserved in sequence and varies considerably in length (Shukla et al., 1989; Shukla & Ward, 1989
). It has been proposed that this broad variability represents a way for the virus to interact with specific host factors for movement and perhaps other functions in the virus life-cycle (Urcuqui-Inchima et al., 2001
); and it has been demonstrated that CP-Nt assists in aphid transmission via its conserved DAG motif (Atreya et al., 1991
), through interaction with the virus encoded helper component proteinase (Blanc et al., 1997
; Peng et al., 1998
). It was shown that the removal of CP-Nt by limited trypsin proteolysis did not affect particle morphology, suggesting that this domain might not be involved in particle assembly (Jagadish et al., 1993
). Furthermore, recently, it was demonstrated that a deletion of up to 112 aa from the CP N-terminal of Tobacco etch virus (TEV), including its complete CP-Nt, did not abolish its ability to assemble into potyviral-like particles (PVLPs) in bacteria (Voloudakis et al., 2004
). In contrast, a complete deletion of Pepper vein banding virus CP-Nt prevented the truncated recombinant CP to assemble into PVLPs in bacteria suggesting a role for CP-Nt in the initiation of particle assembly (Anindya & Savithri, 2003
). A number of studies have shown that CP-Nt is involved in virus long-distance movement and systemic spread. TEV mutants with deletions in the CP N- or C-terminal domains have produced virions in vivo but the virus was defective in long-distance movement in planta (Dolja et al., 1994
, 1995
). Mutational analysis demonstrated that changes of Ser47 to Pro of the Pea seed-borne mosaic virus CP (Andersen & Johansen, 1998
) can modulate the ability of the virus to move systemically in Chenopodium quinoa. Also, substitution of Asp to Lys in the DAG motif of the Tobacco vein mottling virus (TVMV) and TEV CP-Nts abolished systemic movement (Lopez-Moya & Pirone, 1998
) concluding that charge changes made specifically in the DAG motif alter virus capacity for systemic movement (Lopez-Moya & Pirone, 1998
).
ZYMV CP-Nt is 4345 aa long with its putative trypsin protease motif presumed to be positioned between amino acids Lys42 and Asp43, located in the KDKD motif (Shukla et al., 1988). We have previously demonstrated that foreign peptides of up to 31 aa long can substitute for part or all of ZYMV CP-Nt domain while maintaining systemic infectivity, suggesting that ZYMV CP-Nt per se is not essential for assembly or movement (Arazi et al., 2001a
). Nevertheless, in several cases fusion of positively charged peptides to CP-Nt failed to result in an infectious cDNA clone even though fused peptides were shorter than 31 aa (Arazi et al., unpublished results). These observations were reminiscent of results shown for TMV (Bendahmane et al., 1999
) and Cowpea mosaic virus (CPMV) (Porta et al., 2003
) where fusion of positively charged peptides to exposed parts of their CP affected their systemic movement.
In this study, we investigated the effect of ZYMV CP-Nt charge on virus systemic infection. We generated a series of virus mutants, each harbouring a CP-Nt with modified net charge, and analysed the effect of each charge change on virus infectivity. This analysis revealed a correlation between CP-Nt net charge and virus systemic infectivity, suggesting that maintenance of CP-Nt net charge and not primary sequence is necessary for ZYMV infectivity.
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METHODS |
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For bacterial expression, the coding region of wild-type CP and mutant CPs with N-terminal fusion of three Lys (CPKKK) or five Asp (CPDDDDD) residues were amplified by PCR with AGII cDNA as a template. Sense and antisense (5'-CCGCTCGAGTTACTGCATTGTATTCACCTAG-3') primers contained a BspHI and a XhoI site (underlined) at their 5' end, respectively, were used. Amplified PCR fragments were digested with BspHI and XhoI and ligated into NcoI/XhoI sites of pET28a(+) expression vector (Novagen) generating pET28a-CP, pET28a-CPKKK and pET28a-CPDDDDD with an additional methionine at the 5'end of each CP to enable expression in bacteria.
For AGII-GFP expression, firstly, GFP S65T coding region (Reichel et al., 1996) was amplified by PCR. Sense and antisense (5'-CGCGTCGACACTGTAATGCTGCAGTCA-3') primers contained a PstI and a SalI (underlined) site at their 5' end, respectively. Amplified PCR fragment was cloned into PstI/SalI sites of AGII cDNA to generate AGII-GFP. Foreign amino acid residues were then inserted upstream of AGII-GFP CP coding sequence using the same strategy as described for AGII mutants except that the sense primer contained SalI instead of PstI.
Plant growth, inoculation and symptom evaluation.
Zucchini squash (Cucurbita pepo L. cv. zucchini) plants were grown in a growth chamber under continuous light at 23 °C. Seedlings were selected for experimental use when their cotyledons were fully expanded. Particle bombardment inoculation of AGII and various mutants was performed with a hand-held device, the handgun (Gal-On et al., 1997). After bombardment inoculation, squash seedlings were grown and examined daily for symptom development, and the first appearance of symptoms on non-inoculated leaves was recorded.
Detection of viral RNA and proteins.
Total RNA (25 µg) was extracted from three leaf disks (one per leaf; 9 mm diameter), collected from non-inoculated leaves of an infected plant, using TRI-reagent (Sigma). RT-PCR of virus progeny was conducted in a one-tube, single-step method with CP-Nt flanking primers 5'-AGCTCCATACATAGCTGAGACA-3' and 5'-TGGTTGAACCAAGAGGCGAA-3' as described by Arazi et al. (2001b). Resulting amplified fragments were sequenced directly.
Total proteins were extracted separately from three squash seedlings. Samples (70 mg; six leaf disks, two of each plant) from non-inoculated leaves were collected. Each sample was ground in 150 µl ESB buffer [75 mM Tris/HCl pH 6·8, 9 M urea, 4·5 % (v/v) SDS, 7·5 % (v/v)
-mercaptoethanol], boiled for 5 min and cooled on ice. Cooled homogenates were centrifuged for 10 min at 10 000 g, and 100 µl supernatant containing total leaf proteins was mixed with 100 µl 2x SDS-PAGE loading buffer. A 1015-µl sample of the mixture was fractionated by SDS-PAGE on a 12·5 % polyacrylamide gel. The fractionated proteins were electroblotted onto nitrocellulose membranes and probed with a monoclonal antiserum specific to ZYMV CP-Nt (AB6; 1 : 500) (Desbiez et al., 1997
) or an anti-CP polyclonal antibody (1 : 2000).
Expression of recombinant CP and evaluation of potyvirus-like particle (PVLP) formation by electron microscopy.
Cultures of E. coli BL21 Rosetta (Novagen) transformed with pET28a-CP, pET28a-CPKKK, pET28a-CPDDDDD and pET28a, were grown overnight at 37 °C. One ml of each culture was diluted 1 : 100 in fresh medium, grown to OD value of 0·5 at 28 °C, induced by the addition of 200 µM IPTG (final concentration) and further incubated at 28 °C overnight. Bacterial cells were then pelleted and resuspended in 5 ml extraction buffer (50 mM Tris/HCl pH 7·6, 2 mM EDTA, 1 mM DTT, 1 mM PMSF). Lysozyme was added to a final concentartion of 200 µg ml1 and the reaction mixture was incubated for 10 min at room temperature followed by 15 min on ice until the bacterial cells lysed. Digestion of bacterial genomic DNA was done by addition of DNase I (Sigma) to a final concentration of 50 µg ml1 together with 3 mM MgCl2 and incubation at room temperature for 10 min. Bacterial extract was then centrifuged for 30 min at 14 000 r.p.m. (Beckman SS34 rotor) and the supernatant fraction was collected. The presence of PVLPs in the bacterial extract was verified by visualization under the electron microscope. Formvar carbon-coated grids were incubated on droplets of supernatants at room temperature for 1 min, washed three times with water and negatively stained with 2 % uranyl acetate. Images were taken on a JEOL JEM-100CXII electron microscope.
Visualization of GFP fluorescence.
AGII, AGII-GFP, AGII-GFP-CPKKK and AGII-GFP-CPDDDDD cDNAs were bombarded into detached cotyledon epidermal cells of squash, under vacuum conditions, using a particle bombardment gun (Gray et al., 1994). Bombarded cotyledons were placed on 1 % agarose in closed Petri-dish and maintained in a growth chamber under 16 h light at 26 °C. Images were acquired using a confocal laser-scanning microscope system (Olympus 1X81) equipped with an argon laser. The GFP images were obtained at an excitation wavelength of 488 nm and a 515525 nm emission filter. Transmitted light images were acquired using Nomarski differential interference contrast.
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RESULTS |
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A CP-Nt net positive charge larger than +1 prevents virus systemic infectivity
To validate our hypothesis, we constructed a series of mutant AGII cDNA clones designed to express versions of full-length CP-Nt with increased net positive charge, by N-terminal fusion of either Lys or Arg residues (Fig. 2a and b), and tested the systemic infectivity of the mutant cDNAs. Squash seedlings were inoculated by particle bombardment with various mutant cDNA clones. Symptom appearance on non-inoculated leaves, indicative of systemic spread, was recorded (Table 1
) and the presence of virions was verified under the electron microscope after partial purification (data not shown). RT-PCR of virus progeny and direct sequencing of the amplified product was done in each case to confirm the authenticity and genetic stability of each mutant (Table 1
). Virus accumulation of genetically stable mutants was estimated by Western blot analysis 14 days p.i. with anti-CP AB6 monoclonal antibody (Desbiez et al., 1997
). Modification of CP-Nt by the addition of one positively charged Lys (AGII-CPK) or Arg (AGII-CPR) residue did not compromise virus systemic infectivity and CP accumulation level (Table 1
and Fig. 2c
). However, modification of CP-Nt by addition of two Lys (AGII-CPKK) or Arg (AGII-CPRR) residues (Fig. 2b
) resulted in non-infectious clones that sometimes caused a delayed systemic infection (Table 1
). RT-PCR and sequencing analysis of progeny viruses from infected plants did not recover the original clone sequence. Instead, a change of one of the added Lys to an acidic Glu or one of the added Arg to a weakly charged amino acid residue (Cys, His or Gln) was found (Table 1
). All the changes occurred by means of a single nucleotide change in planta. These changes, which spontaneously eliminated one positively charged residue, suggest that a CP-Nt net charge of +1 is tolerable by the virus, as was the case with infectious AGII-CPK and AGII-CPR (Table 1
). Fusion of three Lys residues to CP-Nt (AGII-CPKKK) resulted in a non-infectious clone and late systemic infection was not recorded (Table 1
). In addition, no accumulation of CPKKK could be detected by Western blot analysis (Fig. 2c
). To test whether AGII-CPKKK lack of infectivity is directly correlated to the specific fusion of three basic residues to CP-Nt, we fused three neutral Ala residues at a similar location to generate AGII-CPAAA, which harbours a CP-Nt net charge of 0 like the wild-type CP-Nt. In contrast to AGII-CPKKK, AGII-CPAAA was systemically infectious and accumulated to a similar level as parental AGII (Table 1
and Fig. 2c
). To confirm that CP-Nt net positive charge added in AGII-CPKKK is the cause for non-infectivity, we generated mutants where added positive charge was partially neutralized by the addition of two negatively charged Asp residues either upstream (AGII-CPDDKKK) or downstream (AGII-CPKKKDD) of the KKK residues (Fig. 2b
). These additions changed the CP-Nt net charge from +3 to +1, which is predicted to support systemic infectivity as was demonstrated with AGII-CPK and AGII-CPR (Table 1
). Indeed, AGII-CPDDKKK and AGII-CPKKKDD were systemically infectious 7 days p.i., accumulated to a similar level as parental AGII (Fig. 2c
) and were genetically stable (Table 1
). A control clone (AGII-CPKKKAA) harbouring two neutral Ala residues instead of Asp upstream of the KKK residues was not infectious (Table 1
).
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DISCUSSION |
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Our data suggest that ZYMV is less affected by negatively charged residues added to CP-Nt. We have demonstrated that up to four negatively charged residues can be fused to CP-Nt without abolishing systemic infection (AGII-CPDDDD). Nevertheless, this fusion resulted in a delayed systemic infection (Table 2) and reduced virus accumulation (Fig. 3b
), indicating that it was not optimal for virus life-cycle. In addition, AGII mutants with CP-Nt net charge of 3 (AGII-CPDDD, AGII-CPEDD and AGII-CPDDDDDKK) were, in general, genetically unstable (Table 2
). This result is enigmatic because of the infectivity and genetic stability of AGII-CPDDDD (4) and AGII-Myc-CP
33 (3) clones. One possible explanation is that in these clones some of CP-Nt negative charge was masked, resulting in an actual higher CP-Nt net charge, which allowed systemic infection. Indeed, addition of less than three acidic residues to CP-Nt (net charge>3) does not affect virus systemic infectivity. This is also supported by progeny viruses of unstable clones, which mutated CP-Nt to reduce its net charge to either 1 or 2 (Table 2
). Moreover, fusion of basic Lys residues, which partially neutralizes CP-Nt net charge [AGII-CPKDDD (2), AGII-CPDDDDKK (2) and AGII-CPDDDDDKK (3)], considerably improved virus infectivity and accumulation.
Currently, we cannot determine the exact reason for lack of infectivity upon change of CP-Nt net charge. Formation of ZYMV PVLPs in bacteria following expression of recombinant CPDDDDD protein suggest that the change of CP-Nt net charge to negative does not affect particle assembly. However, we cannot exclude the possibility that a net positive charge of CP-Nt disturbs virus assembly in planta. Nevertheless, in TEV and TVMV, substitutions that involved positively charged amino acids and increased CP-Nt net charge above +1 affected virus movement and not assembly or replication (Lopez-Moya & Pirone, 1998). Indeed, AGII-GFP loses its ability to move efficiently from cell-to-cell upon fusion of charged residues to its CP-Nt (AGII-GFP-CPKKK and AGII-GFP-CPDDDDD; Fig. 5
), suggesting that a neutral CP-Nt net charge may be required to enable efficient potyviral cell-to-cell movement. Interestingly, the deleterious effect of high CP pI on virus infectivity was already demonstrated in TMV and CPMV where expression of very basic immunogenic peptides (pI
10·81 and
12·00) as fusions with their CPs reduced chimaeric virus infectivity by promoting cell death in TMV (Bendahmane et al., 1999
) and restricting long distance movement in CPMV (Porta et al., 2003
). Like in ZYMV, in TMV and CPMV basic peptides were expressed in CP regions known to be highly exposed on the virus surface. Thus, an exposed positive charge abolished systemic infectivity in three distinct virus families. This charge might disturb an interaction with an unknown host component, which is essential for virus cell-to-cell or systemic movement.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Andersen, K. & Johansen, I. E. (1998). A single conserved amino acid in the coat protein gene of pea seed-borne mosaic potyvirus modulates the ability of the virus to move systemically in Chenopodium quinoa. Virology 241, 304311.[CrossRef][Medline]
Anindya, R. & Savithri, H. S. (2003). Surface-exposed amino- and carboxy-terminal residues are crucial for the initiation of assembly in Pepper vein banding virus: a flexuous rod-shaped virus. Virology 316, 325336.[CrossRef][Medline]
Arazi, T., Shiboleth, Y. M. & Gal-On, A. (2001a). A nonviral peptide can replace the entire N terminus of zucchini yellow mosaic potyvirus coat protein and permits viral systemic infection. J Virol 75, 63296336.
Arazi, T., Slutsky, S. G., Shiboleth, Y. M., Wang, Y., Rubinstein, M., Barak, S., Yang, J. & Gal-On, A. (2001b). Engineering zucchini yellow mosaic potyvirus as a non-pathogenic vector for expression of heterologous proteins in cucurbits. J Biotechnol 87, 6782.[CrossRef][Medline]
Atreya, P. L., Atreya, C. D. & Pirone, T. P. (1991). Amino acid substitutions in the coat protein result in loss of insect transmissibility of a plant virus. Proc Natl Acad Sci U S A 88, 78877891.[Abstract]
Baratova, L. A., Efimov, A. V., Dobrov, E. N., Fedorova, N. V., Hunt, R., Badun, G. A., Ksenofontov, A. L., Torrance, L. & Jarvekulg, L. (2001). In situ spatial organization of potato virus A coat protein subunits as assessed by tritium bombardment. J Virol 75, 96969702.
Bendahmane, M., Koo, M., Karrer, E. & Beachy, R. N. (1999). Display of epitopes on the surface of tobacco mosaic virus: impact of charge and isoelectric point of the epitope on virus-host interactions. J Mol Biol 290, 920.[CrossRef][Medline]
Blanc, S., Lopez-Moya, J. J., Wang, R., Garcia-Lampasona, S., Thornbury, D. W. & Pirone, T. P. (1997). A specific interaction between coat protein and helper component correlates with aphid transmission of a potyvirus. Virology 231, 141147.[CrossRef][Medline]
Desbiez, C., Gal-On, A., Raccah, B. & Lecoq, H. (1997). Characterization of epitopes on zucchini yellow mosaic potyvirus coat protein permits studies on the interactions between strains. J Gen Virol 78, 20732076.[Abstract]
Dolja, V. V., Haldeman, R., Robertson, N. L., Dougherty, W. G. & Carrington, J. C. (1994). Distinct functions of capsid protein in assembly and movement of tobacco etch potyvirus in plants. EMBO J 13, 14821491.[Abstract]
Dolja, V. V., Haldeman-Cahill, R., Montgomery, A. E., Vandenbosch, K. A. & Carrington, J. C. (1995). Capsid protein determinants involved in cell-to-cell and long distance movement of tobacco etch potyvirus. Virology 206, 10071016.[CrossRef][Medline]
Gal-On, A., Meiri, E., Elman, C., Gray, D. J. & Gaba, V. (1997). Simple hand-held devices for the efficient infection of plants with viral-encoding constructs by particle bombardment. J Virol Methods 64, 103110.[CrossRef][Medline]
Gray, D. J., Hiebert, E., Lin, C. M., Compton, M. E., McColley, D. W., Harrison, R. J. & Gaba, V. P. (1994). Simplified construction and performance of a device for particle bombardment. Plant Cell Tissue Organ Cult 37, 179184.
Hellens, R. P., Edwards, E. A., Leyland, N. R., Bean, S. & Mullineaux, P. M. (2000). pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42, 819832.[CrossRef][Medline]
Jagadish, M. N., Ward, C. W., Gough, K. H., Tulloch, P. A., Whittaker, L. A. & Shukla, D. D. (1991). Expression of potyvirus coat protein in Escherichia coli and yeast and its assembly into virus-like particles. J Gen Virol 72, 15431550.[Abstract]
Jagadish, M. N., Huang, D. & Ward, C. W. (1993). Site-directed mutagenesis of a potyvirus coat protein and its assembly in Escherichia coli. J Gen Virol 74, 893896.[Abstract]
Lopez-Moya, J. J. & Pirone, T. P. (1998). Charge changes near the N terminus of the coat protein of two potyviruses affect virus movement. J Gen Virol 79, 161165.[Abstract]
McLachlan, A. D., Bloomer, A. C. & Butler, P. J. (1980). Structural repeats and evolution of tobacco mosaic virus coat protein and RNA. J Mol Biol 136, 203224.[Medline]
Peng, Y. H., Kadoury, D., Gal-On, A., Huet, H., Wang, Y. & Raccah, B. (1998). Mutations in the HC-Pro gene of zucchini yellow mosaic potyvirus: effects on aphid transmission and binding to purified virions. J Gen Virol 79, 897904.[Abstract]
Porta, C., Spall, V. E., Findlay, K. C., Gergerich, R. C., Farrance, C. E. & Lomonossoff, G. P. (2003). Cowpea mosaic virus-based chimaeras. Effects of inserted peptides on the phenotype, host range, and transmissibility of the modified viruses. Virology 310, 5063.[CrossRef][Medline]
Reichel, C., Mathur, J., Eckes, P., Langenkemper, K., Koncz, C., Schell, J., Reiss, B. & Maas, C. (1996). Enhanced green fluorescence by the expression of an Aequorea victoria green fluorescent protein mutant in mono- and dicotyledonous plant cells. Proc Natl Acad Sci U S A 93, 58885893.
Rojas, M. R., Zerbini, F. M., Allison, R. F., Gilbertson, R. L. & Lucas, W. J. (1997). Capsid protein and helper component-proteinase function as potyvirus cell-to-cell movement proteins. Virology 237, 283295.[CrossRef][Medline]
Sawyer, l., Tollin, P. & Wilson, R. H. (1987). A comparison between the predicted secondary structures of potato virus X and papaya mosaic virus coat proteins. J Gen Virol 68, 12291232.
Shukla, D. D. & Ward, C. W. (1989). Identification and classification of potyviruses on the basis of coat protein sequence data and serology. Brief review. Arch Virol 106, 171200.[Medline]
Shukla, D. D., Strike, P. M., Tracy, S. L., Gough, K. H. & Ward, C. W. (1988). The N and C termini of the coat proteins of potyviruses are surface-located and the N terminus contains the major virus-specific epitopes. J Gen Virol 69, 14971508.
Shukla, D. D., Tribbick, G., Mason, T. J., Hewish, D. R., Geysen, H. M. & Ward, C. W. (1989). Localization of virus-specific and group-specific epitopes of plant potyviruses by systematic immunochemical analysis of overlapping peptide fragments. Proc Natl Acad Sci U S A 86, 81928196.[Abstract]
Shukla, D. D., Ward, C. W. & Brunt, A. A. (1994). The Potyviridae. Wallingford: CAB International.
Urcuqui-Inchima, S., Haenni, A. L. & Bernardi, F. (2001). Potyvirus proteins: a wealth of functions. Virus Res 74, 157175.[CrossRef][Medline]
Varrelmann, M. & Maiss, E. (2000). Mutations in the coat protein gene of Plum pox virus suppress particle assembly, heterologous encapsidation and complementation in transgenic plants of Nicotiana benthamiana. J Gen Virol 81, 567576.
Voloudakis, A. E., Malpica, C. A., Aleman-Verdaguer, M. E., Stark, D. M., Fauquet, C. M. & Beachy, R. N. (2004). Structural characterization of Tobacco etch virus coat protein mutants. Arch Virol 149, 699712.[CrossRef][Medline]
Received 4 July 2004;
accepted 5 August 2004.
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