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Coat proteins of two filamentous plant viruses display NTPase activity in vitro
FEBS 29884
FEBS Letters 579 (2005) 4955–4960
Coat proteins of two filamentous plant viruses display NTPase activity in vitro Daria V. Rakitinaa,*, Omar L. Kantidzea, Anna D. Leshchinera, Andrey G. Solovyeva, Viktor K. Novikovb, Sergey Yu. Morozova, Natalia O. Kalininaa a
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russia b Department of Virology, Moscow State University, Moscow 119992, Russia Received 5 April 2005; revised 22 June 2005; accepted 19 July 2005 Available online 11 August 2005 Edited by Julian Schroeder
Abstract Coat proteins (CPs) of plant viruses are involved in different stages of the viral life cycle such as virion assembly, replication, movement, vector transmission, and regulation of host defense responses. Here, we report that the CPs of two filamentous RNA viruses, potato virus X (PVX, Potexvirus) and potato virus A (PVA, Potyvirus) exhibit an enzyme activity. The CP isolated from PVX virions possesses ATP-binding and ATPase activities. Recombinant PVX and PVA CPs produced in Escherichia coli show Mg2+-dependent ATPase and UTPase activities inhibited by antibodies against virus particles. Deletion of the C-terminal regions of these proteins diminishes their ATPase activity. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Plant RNA viruses; Coat protein; ATPase; UTPase
1. Introduction The primary function of the coat (capsid) proteins (CPs) of plant viruses is encapsidation of viral genomic nucleic acids. In most plant RNA viruses, CPs also play an essential role in viral spread throughout the infected plants, i.e., in long-distance transport through the vasculature and cell-to-cell movement (for review, see [1,2]). Moreover, CP may participate in other stages of the viral life cycle such as replication, vector transmission, and silencing suppression (for review, see [1,3,4]). Although the viral genome structure and expression differ considerably in families Flexiviridae (includes Potexvirus) and Potyviridae (includes Potyvirus), their CPs show weak amino acid sequence similarity [5,6]. Furthermore, the CPs encoded by viruses of these two families are required for virus movement. Accordingly, the CPs and virions have been found in plasmodesmata, specialized cell wall-traversing channels recruited by viruses for transport between plant cells [7–14]. Other components of transport systems are dissimilar in Flexiviridae and Potyviridae. In addition to the CP, three movement proteins (MPs) referred to as TGBp1, TGBp2, and * Corresponding author. Fax: +7 95 939 3181. E-mail address: [email protected] (D.V. Rakitina).
Abbreviations: CP, coat protein; PVX, potato virus X; PVA, potato virus A; MP, movement protein; TGB, triple gene block; IgG, immunoglobulins
TGBp3 are required for cell-to-cell movement of potexviruses [15], whereas the viral transport requires the helper component-proteinase (HC-Pro) and the cylindrical inclusion protein (CI) in potyviruses [13,14,16,17]. Interestingly, both virus genera encode viral NTPases/helicases involved in virus cell-to-cell movement [15,18]. The proposed role of virus particles as a transport-competent form of the filamentous virus genomes has not been proven yet [8,18]. On the other hand, analyses of CP mutants show that the virion formation is not sufficient for effective functioning of cell-to-cell and long-distance transport systems, suggesting additional movement-related activities of the CP molecules [10–12,19,20]. Importantly, transient complementation studies showed that the CPs of potex- and potyviruses share some (yet unknown) function(s) necessary for the transport process [20,21]. In this paper, we studied the in vitro biochemical activities of the CPs of members of the genera Potexvirus (potato virus X, PVX) and Potyvirus (potato virus A, PVA). The PVX and PVA CPs were shown to possess Mg2+-dependent NTPase activity.
2. Materials and methods 2.1. Isolation and purification of PVX virions from infected plants Leaves of infected Datura stramonium plants (25–30 days after inoculation) were frozen at 20 C, homogenized in a Waring blender in buffer containing 0.3 M glycine-KOH and 1% Na2SO3, pH 7.5, were strained through two layers of cheese cloth, and centrifuged at 12 000 rpm for 30 min. The supernatant was filtered through a Miracloth filter and, after addition of Triton X-100 to a final concentration of 1%, allowed to stand for 20 min. Then polyethylene glycol (Mr 6000) and NaCl were added to final concentrations of 5% and 2%, respectively. The solution was incubated at 4 C for 2 h or overnight, centrifuged at 10 000 · g for 30 min, and the virus particles were extracted twice from the pellet with buffer A (25 mM Tris/HCl, pH 8.0) by stirring for 1–2 h. The extract was centrifuged at 10 000 · g for 20 min and the supernatant was ultracentrifuged at 35 000 rpm for 90 min (Ti-60 rotor, Beckman L5-50). The virus pellet was resuspended with a glass homogenizer in buffer A until completely free of lumps and loaded onto 2 ml of 30% sucrose cushion in tubes for Ti-50 rotor and centrifuged at 40 000 rpm for 2.5 h (Beckman L5-50). The virus pellet was dissolved in buffer A. All manipulations were carried out at 4 C. To obtain PVX CP, a suspension of virus particles was supplemented with 2 M LiCl, left at 20 C for two days, and centrifuged at 10 000 · g for 15 min. The CP-containing supernatant was recentrifuged and dialyzed against 10 mM Tris/HCl (pH 7.5). The isolation and purification of TMV virions from infected plants was performed as described in [22]. Briefly, wild type (U1) TMV was propagated on N. tabacum var. Samsun at 24 C. The virus was
0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.07.083
4956 extracted from plant sap with 10 mM phosphate buffer (pH 7.0) with 2 mM EDTA and purified by chloroform–butanol extraction, polyethylene glycol (Mr 6000) precipitation, and several cycles of differential centrifugation (90 min at 32 000 rpm in a SW50.1 rotor, Beckman L5-50, and 20 min at 12 000 rpm in a JA20 rotor, Beckman J2-20). Anti-PVX, anti-PVA, and anti-TMV antibodies were obtained by standard rabbit immunization with purified virion preparations and the fraction of immunoglobulins (IgG) was purified as described previously [23]. 2.2. Construction of recombinant clones For expression of the recombinant PVX CP, the PVX CP gene was amplified by PCR from a full-length PVX cDNA clone. The PCR product was digested with NcoI and BamHI and cloned into similarly digested pQE30 plasmid (QIAGEN). A frameshift mutant DCfsCPPVX (with 18 C-terminal amino acids replaced with a non-viral amino acid sequence), previously characterized in vivo [20], was generated by partial digestion of the PVX CP gene with XhoI, filling in by Klenow enzyme, and religation. To obtain deletion mutant of PVX CP – DC18CPPVX, expression clone PQE-cpPVX, carrying the PVX CP gene and coding for a protein with 6-His-tag on the N-terminus, was digested with EcoRI and XhoI, and the resulting fragment was introduced into similarly digested pQE30 plasmid. The plasmid pQE-cpPVA containing an N-terminal 6-His-tagged PVA CP gene was kindly provided by Dr. A. Merits (Institute of Biotechnology, Viikki Biocenter, University of Helsinki, Finland). A mutant DC49CPPVA (with 49 C-terminal amino acids deleted) was generated by cloning of an EcoRI/PstI fragment of expression vector pQE-cpPVA into similarly digested pQE30 plasmid. 2.3. Expression and purification of histidine-tagged CPs and their mutant forms E. coli strain M15 transformed with the recombinant vectors was grown at 37 C in liquid medium until OD600 of 0.8–0.9 was reached. Expression of the proteins was induced with 1 mM IPTG followed by growth for 2–4 h at 37 C. The purification of recombinant proteins from cultures was performed following the general procedure described by the manufacturerÕs instructions (QIAGEN) for denaturing Ni-NTA chromatography. 2.4. Cross-linking of ATP to PVX CP [a-32P] ATP (6000 Ci/mmol; 1 lCi per probe) was incubated on ice with 1–1.5 lg of the PVX CP for 20 min in reaction buffer containing 10 mM Tris/HCl, pH 8.0, 10% glycerol, 1 mM DTT, and 5 mM MgCl2. Samples were UV-irradiated in a Stratalinker 1800 unit as described elsewhere [24], heated with an equal volume of 2· Laemmli sample buffer, and separated by SDS–PAGE. Proteins were visualized by autoradiography of the dried gels. 2.5. NTP hydrolysis assay The reaction mixture contained 10 mM Tris/HCl, pH 8.0, 10% glycerol, 1 mM DTT, 5 mM MgCl2, 1 lCi [c-32P]ATP (6000 Ci/mmol) or 1 lCi [c-32P]UTP (3000 Ci/mmol), and 20–500 ng of protein in a final volume of 10 ll. When indicated, various NTPs, IgG, or chlorides of divalent cations (instead of MgCl2, at the same concentration) were added to the reaction mixture. Samples were incubated for 1 h at 37 C and the reaction was stopped by the addition of EDTA to a final concentration of 20 mM. To estimate NTPase activity, unreacted NTP was precipitated by addition of 200 ll of 7.5% activated charcoal in 50 mM HCl and 5 mM H3PO4; the mixtures were vortexed and allowed to stand for 5 min, charcoal was removed by centrifugation for 10 min, and a half of the supernatant was analyzed by Cherenkov counting of free 32Pi released by hydrolysis of labeled NTP [24]. The sample incubated without protein was used as a negative control. The total sample activity without incubation and precipitation procedure (minus a negative control sample) was taken as 100%. NTPase activity of proteins was expressed as relative activity (% hydrolysis of 32 P-labeled substrate). Kinetic parameters of ATP hydrolysis were measured in the linear phase of the reaction as described in [25,26]. The reaction mixture, which contained 10 mM Tris/HCl, pH 8.0, 10% glycerol, 1 mM DTT, and 5 mM MgCl2, was supplemented with 200 ng of the PVX CP or 400 ng of the PVA CP, ATP (from 1 to 65 lM), and 1 lCi
D.V. Rakitina et al. / FEBS Letters 579 (2005) 4955–4960 (8 nM) [c-32P]ATP. After 30 min incubation, ATP hydrolysis was analyzed using the activated charcoal method as described above. The experiment was repeated four times. Assuming that cold ATP and radiolabeled ATP are hydrolyzed at the same rate, the degree of [c-32P]ATP hydrolysis was taken as the degree of unlabeled ATP hydrolysis. Multiplying this degree by the amount of ATP in each sample (the amount of radiolabeled ATP was neglected), we achieved the amount of ATP hydrolyzed in each sample. Dividing the amount of the ATP hydrolyzed in each sample by the time of reaction, we achieved v (reaction velocity) at each substrate concentration. v was plotted versus ATP concentration. The Lineweaver–Burke doublereciprocal plots were then used to calculate Km and Vmax of ATP hydrolysis.
3. Results 3.1. PVX CP isolated from virions demonstrates ATP binding and ATPase activities The role of PVX CP in virus cell-to-cell movement involves protein function(s) other than encapsidation; hence, the CP may have some functions of viral MPs [15,21]. MPs of different plant viruses are known to have rNTP-binding activity [27–32]. Therefore, we studied whether the PVX CP is able to bind rNTPs. For these experiments, the CP was isolated from PVX virions. Typically, such PVX CP preparations contain two protein forms, the full-length Ps form and the Pf form which lacks 24 N-terminal amino acids as a result of natural proteolysis during isolation and storage [33]. The UV-crosslinking assay revealed the ability of both Ps and Pf forms of the PVX CP to effectively bind [a-32P]ATP (Fig. 1A). Since ATP-binding proteins often have ATPase activity too, the PVX CP was tested for the ability to hydrolyze rATP. After incubation of the protein with [c-32P]ATP, the percentage of hydrolyzed ATP was determined using activated charcoal. This experiment revealed ATPase activity of the PVX CP (Fig. 1B). Moreover, we found that PVX virion preparations also exhibited ATPase activity (Fig. 1B). No detectable ATPase activity was found for preparations of tobacco mosaic virus (TMV) virions purified from plant extracts either by the method routinely employed for TMV isolation (Fig. 1B) or by the PVX isolation procedure used in this paper (data not shown). These data could indicate that plant cellular ATPases were likely absent from the PVX virion preparations. PVX-specific IgG (but not TMV-specific IgG) substantially inhibited this virion activity (Fig. 1C). As reported previously, PVX TGBp1, the PVX MP with NTPase/helicase activity [34,35], is associated with one end of PVX filamentous virions in vitro [36]. Accordingly, Western blot analysis with a TGBp1-specific antibodies demonstrated the presence of a minor amount of the TGBp1 in preparations of PVX virions (our unpublished data). To determine whether the observed ATPase activity of PVX virion preparation was due to the found enzymatic function of the CP or to the activity of the TGBp1, anti-PVX virion or anti-TGBp1 IgG were added to the ATPase assay reaction. It was found that the ATPase activity of virions was only partially suppressed by TGBp1-specific IgG, while IgG against PVX virions severely inhibited the virion activity (Fig. 1C). In the control, TGBp1-specific IgG completely suppressed ATPase activity of recombinant TBGp1 (data not shown). Importantly, the virion-specific IgG had no cross-reactivity with TBGp1 and healthy plant extract proteins, and the TGBp1-specific IgG had no cross-reactivity with PVX CP
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Fig. 1. ATP binding and ATPase activity of the wild-type PVX CP and PVX virions. (A) UV-crosslinked binding of [a-32P]ATP to the PVX CP. Samples containing 1 lg of the PVX CP isolated from the virion were incubated on ice with [a-32P]ATP for 20 min. [a-32P]ATP was UV crosslinked to the CP. The samples were analyzed by SDS–PAGE, gels were dried, and proteins were visualized by autoradiography. Lanes: 1, PVX CP isolated from virus particles (Coomassie blue staining); the mobilities of two forms of PVX CP, the full-length Ps form and the truncated Pf form, are indicated; 2–3, autoradiograph of the PVX CP incubated with [a-32P]ATP after or without UV-crosslinking. (B) Comparison of ATPase activities of the virion-derived PVX CP as well as PVX and TMV virions. Samples containing 1–4 lg of protein were incubated for 1 h at 37 C and analyzed as described in Section 2. The reaction product, free radioactive Pi, was measured by Cherenkov counting after unreacted [c-32P]ATP was precipitated by the addition of activated charcoal in the presence of excess unlabeled Pi. Relative NTPase activity of proteins is presented (% hydrolysis of 32Plabeled substrate). (C) Inhibition of ATPase activity of the PVX virion by immunoglobulins (IgG). Anti-PVX virion IgG, anti-TGBp1 IgG, or antiTMV virion IgG were added to the ATPase reaction samples containing PVX virions (2 lg of protein).
and proteins of healthy plant extract (data not shown). Thus, the detected PVX virion ATPase activity can be at least in part attributed to the viral CP, which therefore retains its enzymatic activity when included into virus particles. 3.2. Mg2+-dependent ATPase activity of recombinant PVX CP To further investigate the PVX CP ATPase activity, we used the recombinant 6-His-tagged protein overexpressed in E. coli and purified by affinity chromatography on Ni-NTA agarose (Fig. 2A). The recombinant CP exhibited significant ATPase activity. Fig. 2B shows the percentage of [c-32P]ATP hydrolyzed by the CP plotted against the CP concentration. Up to 80–90% of [c-32P]ATP can be hydrolyzed by 150 ng of the protein within 1 h. ATPase activity of recombinant PVX CP was drastically suppressed by IgG against PVX virions but not by IgG against TMV virions (Fig. 2C). These results indicated that the observed ATPase activity was an intrinsic property of the PVX CP, and was not due to contaminating bacterial proteins. Further experiments examined the influence of different divalent cations on ATPase activity of the PVX CP. CaCl2, MnCl2, ZnCl2, or CuCl2 were added to the reaction mixture instead of MgCl2 (used in the standard reaction). No ATPase activity was detected in the presence of EDTA. All tested cations stimulated ATPase activity of the CP, and Mg2+ had the most pronounced effect (Fig. 2D). The kinetic parameters of the PVX CP for ATP hydrolysis were determined as described in Section 2. Km of ATP hydrolysis, calculated from the Lineweaver–Burke double-reciprocal plots, was 7 lM, with an apparent Vmax of 5 nmol min 1 mg 1 of the PVX CP. The C-terminally truncated PVX CP with a frame-shift mutation to encode 219 (out of the 237) amino acids plus 10 non-viral amino acids proved unable to support virus cell-to-
cell movement [20]. To test whether this mutation also affected the ATPase activity, the truncated mutant (referred to as DCfsCPPVX) was expressed in E. coli and purified by affinity chromatography on Ni-NTA agarose (Fig. 2A). The ATPase assay revealed that DCfsCPPVX hydrolyzed less than 10% of ATP (Fig. 2B). Additionally, we constructed the CP mutant DC18CPPVX with a stop codon that truncated the C-terminus of the protein by 18 amino acids. The ATPase assay carried out with the purified DC18CPPVX (Fig. 2A) demonstrated that this mutant hydrolyzed up to 30% of ATP (Fig. 2B). These data further support that the observed ATPase activity was due to the CP but not to contaminating cellular proteins. 3.3. The recombinant PVA CP exhibits Mg+2-dependent ATPase activity The CPs of two potyviruses, potato viruses A and Y, have been shown to complement the cell-to-cell movement of the PVX CP mutant lacking 18 C-terminal amino acids [20,21]. The recombinant 6-His-tagged PVA CP, overexpressed in E. coli and purified by Ni-NTA chromatography, was used to study whether potyviral CPs, which are distantly related in amino acid sequence to potexviral CPs [5,6], also have ATPase activity (Fig. 3A). Under standard reaction conditions, the PVA CP demonstrated ATPase activity (Fig. 3B). ATPase activity of the PVA CP was inhibited by specific (anti-PVA virion) but not by non-specific (anti-TMV virion) antibodies (Fig. 3C). The PVA CP activity was lower than that of the PVX CP: within 1 h of incubation, 150 ng of the PVA and PVX CP hydrolyzed up to 40–45% and 80–90% of total [c-32P]ATP, respectively. This observation was confirmed by the difference of apparent Vmax values: 1.5 and 5 nmol min 1 mg 1, respectively. At the same time, the Km of ATP hydrolysis was 7 lM for both CPs, which indicated their similar affinity to ATP. The influence of different divalent
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Fig. 2. Characterization of ATPase activity of the recombinant PVX CP. (A) SDS–PAGE analysis of the wild-type PVX CP isolated from virus particles (two forms), the recombinant PVX CP and its mutant (frame-shift, DCfsCPPVX, and deletion, DC18CPPVX) variants purified from E. coli. The sizes of molecular weight markers are shown at the left side. (B) Comparison of ATPase activities of the full-length PVX CP and its mutants, DCfsCPPVX and DC18CPPVX. (C) Inhibition of ATPase activity of the PVX CP by specific immunoglobulins (IgG). Anti-PVX virion IgG and antiTMV virion IgG were added at indicated concentrations to the reaction mixtures containing 60 ng of the CP. (D) Effect of various divalent cations on ATPase activity of PVX CP. 80 ng of the CP were incubated in standard reaction mixtures containing 5 mM of the indicated divalent cations as chlorides.
Fig. 3. ATPase activity of the recombinant PVA CP. (A) Analysis of the recombinant PVA CP and its mutant lacking 49 C-terminal amino acids (DC49CPPVA) purified from E. coli by SDS–PAGE. The sizes of molecular weight markers are shown at the left side. The position of the PVA CP trimer is indicated. (B) Comparison of ATPase activities of the full-length PVA CP and mutant DC49CPPVA. The ATPase assay was performed under standard conditions. (C) Inhibition of ATPase activity of the PVA CP by specific IgG. Anti-PVA virion IgG and anti-TMV virion IgG were added at indicated concentrations to the reaction mixtures containing 150 ng of protein. (D) Effect of various divalent cations on ATPase activity of PVA CP. 150 ng of the CP were incubated in the reaction mixtures containing 5 mM of the indicated divalent cations as chlorides.
cations on ATPase activity was similar in the PVA and PVX CPs, except that the PVA CP activity was only slightly stimulated by Ca2+ (Fig. 3D).
To analyze the role of the C-terminal region of PVA CP in ATPase activity, a mutant which lacked the 49 C-terminal residues (DC49CPPVA) was constructed, expressed in E. coli, and
D.V. Rakitina et al. / FEBS Letters 579 (2005) 4955–4960
Fig. 4. Inhibition of ATPase activity of the PVX and PVA CPs by various NTPs and UTPase activity of the recombinant CPs. (A) Inhibition of ATPase activity of the PVX CP by rNTPs and dNTPs added to the reaction mixtures containing 40 ng of the CP. (B) Inhibition of ATPase activity of the PVA CP by rNTPs and dNTPs added to the reaction mixtures containing 150 ng of the protein. (C) UTPase activity of the PVX and PVA CPs.
purified (Fig. 3A). In the ATPase assay, DC49CPPVA showed a reduced ATPase activity in comparison with the full-length PVA CP (Fig. 3B). 3.4. The PVX and PVA CPs are NTPases To examine the influence of different rNTPs and dNTPs on the PVX CP ATPase activity, increasing concentrations of each unlabeled NTP were added to the ATPase reaction mixtures. All rNTPs inhibited the hydrolysis of [c-32P]ATP with a similar efficiency, while the effect of dNTPs was small (Fig. 4A). Similar results were obtained for the PVA CP: almost no inhibition of the [c-32P]ATP hydrolysis was observed in the presence of unlabeled dNTPs, whereas rNTPs inhibited the hydrolysis (Fig. 4B). The competition experiments suggest that the PVX and PVA CPs are able to hydrolyze rNTPs but not dNTPs. Therefore, we studied their UTPase activity in the standard NTPase assay. Both recombinant CPs were able to hydrolyze [c-32P]UTP with the efficiency similar to that of ATP hydrolysis (Fig. 4C). Thus, the CPs can hydrolyze both the purine triphosphate ATP and the pyrimidine triphosphate UTP. Taken together, these results indicate that the PVX and PVA CPs possess rNTPase activity.
4. Discussion Coat proteins (CPs) of plant RNA viruses are involved in several stages of the viral life cycle such as virion assembly, replication, movement, vector transmission, and regulation of host defense responses [1,3,4]. In this paper, we demonstrate
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that CPs of plant RNA viruses can have an enzyme activity. We found that the CPs encoded by two genera of viruses with flexuous filamentous particles, PVX (Potexvirus) and PVA (Potyvirus), exhibited Mg2+-dependent ATPase and UTPase activities. The Km values of the PVX and PVA CPs calculated for ATP hydrolysis were similar (7 lM). In addition, PVX virus particles and PVX CP isolated from these particles could bind and hydrolyze ATP although with a lower efficiency than the recombinant PVX CP. While the location of the enzyme active site could not be predicted based on the amino acid sequences of the CPs of filamentous viruses, the data obtained demonstrate that the C-terminal region of the PVX and PVA CPs may play an important role in the maintenance of the protein enzymatic core. The deletions of 18 C-terminal amino acids of the PVX CP (DC18CPPVX) and of 49 C-terminal residues of the PVA CP (DC49CPPVA) likely destabilized the overall CP structure and considerably decreased the ATPase activity (Figs. 2B, 3B). We suggest that the CP C-terminal sequence is not a major part of the NTPase active site, but can affect its proper conformation. This conclusion is supported by a much lower ATPase activity of DCfsCPPVX, with the 18 C-terminal amino acids replaced by a heterologous sequence, as compared to DC18CPPVX (Fig. 2B). The functional role(s) of ATPase activity of PVX and PVA CPs in viral infection is obscure. As suggested previously, the ATP-binding and ATP hydrolysis could be involved in cell-to-cell movement of viral genomes either at the stage of intracellular transport to plasmodesmata or at the stage of translocation of the viral genome-containing complex through plasmodesmal microchannels, which can be accompanied by energy-dependent complex disassembly or rearrangement [15]. ATPase activity of free CP molecules can also be required at other stages of the virus life cycle. Further studies of the biological role of the ATPase activity described for the CPs of filamentous plant viruses will require mapping of the ATPase active site and analysis of ATPase-deficient CP mutants. A pair of positively and negatively charged residues, conserved in the CPs of filamentous viruses and previously proposed to form a salt bridge important for the CP structure stabilization [5], can be considered as possible structural elements of the active site (E.V. Koonin, personal communication). Acknowledgments: We thank Dr. A.K. Bobkova for preparation of antibodies against PVX, PVA, and TMV virus particles and Dr. A. Merits for the PVA CP expression clone. We are grateful to Dr. A.V. Efimov for helpful discussion of the PVX and PVA CP structures and possible functions of different protein regions. We thank Dr. E.V. Koonin for valuable discussions. This work was supported by the Grant No. 04-04-49356 of the Russian Foundation for Basic Research and in part by the Grant No. 1291.2003.4 of the Leading Scientific School Program of the Russian Ministry of Education and Science.
References [1] Callaway, A., Giesman-Cookmeyer, D., Gillock, E.T., Sit, T.L. and Lommel, S.A. (2001) The multifunctional capsid proteins of plant RNA viruses. Annu. Rev. Phytopathol. 39, 419–460. [2] Carrington, J.C., Kasschau, K.D., Mahajan, S.K. and Schaad, M.C. (1996) Cell-to-cell and long-distance transport of viruses in plants. Plant Cell 10, 1669–1681. [3] Thomas, C.L., Leh, V., Lederer, C. and Maule, A.J. (2003) Turnip crinkle virus coat protein mediates suppression of RNA silencing in Nicotiana benthamiana. Virology 306, 33–41.
4960 [4] Qiu, W. and Scholthof, K.B. (2004) Satellite panicum mosaic virus capsid protein elicits symptoms on a nonhost plant and interferes with a suppressor of virus-induced gene silencing. Mol. Plant Microbe Interact. 17, 263–271. [5] Dolja, V.V., Boyko, V.P., Agranovsky, A.A. and Koonin, E.V. (1991) Phylogeny of capsid proteins of rod-shaped and filamentous RNA plant viruses: Two families with distinct patterns of sequence and probably structure conservation. Virology 184, 79– 86. [6] Koonin, E.V. and Dolja, V.V. (1993) Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences. Crit. Rev. Biochem. Mol. Biol. 28, 375–430. [7] Oparka, K.J., Roberts, A.G., Roberts, I.M., Prior, D.A.M. and Santa Cruz, S. (1996) Viral coat protein is targeted to, but does not gate, plasmodesmata during cell-to-cell movement of potato virus X. Plant J. 10, 805–813. [8] Santa Cruz, S., Roberts, A.G., Prior, D.A.M., Chapman, S. and Oparka, K.J. (1998) Cell-to-cell and phloem-mediated transport of potato virus X: the role of virions. Plant Cell 10, 495–510. [9] Chapman, S., Hills, G., Watts, J. and Baulcombe, D. (1992) Mutational analysis of the coat protein gene of potato virus X: effects on virion morphology and viral pathogenicity. Virology 191, 223–230. [10] Forster, R.L.S., Beck, D.l., Guilford, P.G.J., Voot, D.M., van Dolleweerd, C.J. and Andersen, M.T. (1992) The coat protein of white clover mosaic potexvirus has a role in facilitating cell-to-cell transport in plants. Virology 191, 480–484. [11] Dolja, V.V., Haldeman, R., Robertson, N.L., Dougherty, W.G. and Carrington, J.C. (1994) Distinct functions of capsid protein in assembly and movement of tobacco etch potyvirus in plants. EMBO J. 13, 1482–1491. [12] Dolja, V.V., Haldeman-Cahill, R., Montgomery, A.E., Vandenbosch, K.A. and Carrington, J.C. (1995) Capsid protein determinants involved in cell-to-cell and long distance movement of tobacco etch potyvirus. Virology 206, 1007–1016. [13] Rojas, M.R., Zerbini, F.M., Allison, R.F., Gilbertson, R.L. and Lucas, W.J. (1997) Capsid protein and helper componentproteinase function as potyvirus cell-to-cell movement proteins. Virology 237, 283–295. [14] Rodrıguez-Cerezo, E., Findlay, K., Shaw, J.G., Lomonossoff, G.P., Qiu, S.G., Linstead, P., Shanks, M. and Risco, C. (1997) The coat and cylindrical inclusion proteins of a potyvirus are associated with connections between plant cells. Virology 236, 296–306. [15] Morozov, S.Yu. and Solovyev, A.G. (2003) Triple gene block: modular design of a multifunctional machine for plant virus movement. J. Gen. Virol. 84, 1351–1366. [16] Carrington, J.C., Jensen, P.E. and Schaad, M.C. (1998) Genetic evidence for an essential role for potyvirus CI protein in cell-tocell movement. Plant J. 14, 393–400. [17] Roberts, I.M., Wang, D., Findlay, K. and Maule, A.J. (1998) Ultrastructural and temporal observations of the potyvirus cylindrical inclusions (CIs) show that the CI protein acts transiently in aiding virus movement. Virology 245, 173–181. [18] Revers, F., Le Gall, O., Candresse, T. and Maule, A.J. (1999) New advances in understanding the molecular biology of plant/ potyvirus interactions. Mol. Plant Microbe Interact. 12, 367–376. [19] Lough, T.J., Netzler, N.E., Emerson, S.J., Sutherland, P., Carr, F., Beck, D.L., Lucas, W.J. and Forster, R.L.S. (2000) Cell-to-cell movement of potexviruses: evidence for a ribonucleoprotein complex involving the coat protein and first triple gene block protein. Mol. Plant Microbe Interact. 13, 962–974. [20] Fedorkin, O.N., Merits, A., Luccesi, J., Solovyev, A.G., Saarma, M., Morozov, S.Yu. and Makinen, K. (2000) Complementation
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[21]
[22]
[23]
[24] [25]
[26]
[27] [28]
[29]
[30] [31]
[32]
[33] [34]
[35]
[36]
of the movement-deficient mutations in potato virus X: potyvirus coat protein mediates cell-to-cell trafficking of C-terminal truncation but not deletion mutant of potexvirus coat protein. Virology 270, 31–42. Fedorkin, O.N., Solovyev, A.G., Yelina, N.E., Zamyatnin Jr, A.A., Zinovkin, R.A., Makinen, K., Schiemann, J. and Morozov, S.Yu. (2001) Cell-to-cell movement of potato virus X involves distinct functions of the coat protein. J. Gen. Virol. 82, 449–458. Dobrov, E.N., Abu-Eid, M.M., Solovyev, A.G., Kust, S.V. and Novikov, V.K. (1997) Properties of the coat protein of a new tobacco mosaic virus coat protein ts-mutant. J. Protein Chem. 16, 27–36. Dobrov, E.N., Bobkova, A.F., Goldshtein, M.I., Timofeeva, E.K. and Atabekov, J.G. (1991) Comparative study of antigenic properties of potato virus X and its coat protein in solution and after adsorbsion to solid support. J. Virol. Methods 32, 317–326. Rikkonen, M., Peranen, J. and Kaariainen, L. (1994) ATPase and GTPase activities associated with semliki forest virus nonstructural protein nsP2. J. Virol. 68, 5804–5810. Makino, Y., Yamano, K., Kanemaki, M., Morikawa, K., Kishimoto, T., Shimbara, N., Tanaka, K. and Tamura, T. (1997) SUG1, a component of the 26S proteasome, is an ATPase stimulated by specific RNAs. J. Biol. Chem. 272, 23201–23205. Hagman, W., Nies, A.T., Kıınig, J., Frey, M., Zentgraf, H. and Keppler, D. (1999) Purification of the human apical conjugate export pump MRP2. Reconstruction and functional characterization as substrate-stimulated ATPase. Eur. J. Biochem. 265, 281–289. Rouleau, M., Smith, R.J., Bancroft, J.B. and Mackie, G.A. (1994) Purification, properties, and subcellular localization of foxtail mosaic potexvirus 26-kDa protein. Virology 204, 254–265. Li, Q. and Palukaitis, P. (1996) Comparison of the nucleic acidand NTP-binding properties of the movement protein of cucumber mosaic cucumovirus and tobacco mosaic tobamovirus. Virology 216, 71–79. Bleykasten, C., Gilmer, D., Guilley, H., Richards, K.E. and Jonard, G. (1996) Beet necrotic yellow vein virus 42 kDa triple gene block protein binds nucleic acid in vitro. J. Gen. Virol. 77, 889–897. Donald, R.G., Lawrence, D.M. and Jackson, A.O. (1997) The barley stripe mosaic virus 58-kilodalton beta(b) protein is a multifunctional RNA binding protein. J. Virol. 71, 1538–1546. Kalinina, N.O., Samuilova, O.V., Fedorkin, O.N., Zelenina, D.A. and Morozov, S.Yu. (1998) Biochemical characterization and subcellular localization of a 25K movement protein of potato virus X. Mol. Biol. (Mosk) 4, 686–691, In Russia. Carvalho, C.M., Pouwels, J., van Lent, J.W.M., Bisseling, T., Goldbach, R.W. and Wellink, J. (2004) The movement protein of cowpea mosaic virus binds GTP and single-stranded nucleic acid in vitro. J. Virol. 78, 1591–1594. Koenig, R., Tremaine, J.H. and Shepard, J.F. (1978) In situ degradation of the protein chain of potato virus X at the N- and C-termini. J. Gen. Virol. 38., 329–337. Kalinina, N.O., Fedorkin, O.N., Samuilova, O.V., Maiss, E., Korpela, T., Morozov, S.Yu. and Atabekov, J.G. (1996) Expression and biochemical analyses of the recombinant potato virus X 25K movement protein. FEBS Lett. 397, 75–78. Kalinina, N.O., Rakitina, D.V., Solovyev, A.G., Schiemann, J. and Morozov, S.Yu. (2002) RNA helicase activity of the plant virus movement proteins encoded by the first gene of the triple gene block. Virology 296, 321–329. Atabekov, J.G., Rodionova, N.P., Karpova, O.V., Kozlovsky, S.V. and Poljakov, V.Yu. (2000) The movement protein-triggered in situ conversion of potato virus X virion RNA from nontraslatable into a translatable form. Virology 271, 259–263.
FEBS Letters 579 (2005) 4955–4960
Coat proteins of two filamentous plant viruses display NTPase activity in vitro Daria V. Rakitinaa,*, Omar L. Kantidzea, Anna D. Leshchinera, Andrey G. Solovyeva, Viktor K. Novikovb, Sergey Yu. Morozova, Natalia O. Kalininaa a
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russia b Department of Virology, Moscow State University, Moscow 119992, Russia Received 5 April 2005; revised 22 June 2005; accepted 19 July 2005 Available online 11 August 2005 Edited by Julian Schroeder
Abstract Coat proteins (CPs) of plant viruses are involved in different stages of the viral life cycle such as virion assembly, replication, movement, vector transmission, and regulation of host defense responses. Here, we report that the CPs of two filamentous RNA viruses, potato virus X (PVX, Potexvirus) and potato virus A (PVA, Potyvirus) exhibit an enzyme activity. The CP isolated from PVX virions possesses ATP-binding and ATPase activities. Recombinant PVX and PVA CPs produced in Escherichia coli show Mg2+-dependent ATPase and UTPase activities inhibited by antibodies against virus particles. Deletion of the C-terminal regions of these proteins diminishes their ATPase activity. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Plant RNA viruses; Coat protein; ATPase; UTPase
1. Introduction The primary function of the coat (capsid) proteins (CPs) of plant viruses is encapsidation of viral genomic nucleic acids. In most plant RNA viruses, CPs also play an essential role in viral spread throughout the infected plants, i.e., in long-distance transport through the vasculature and cell-to-cell movement (for review, see [1,2]). Moreover, CP may participate in other stages of the viral life cycle such as replication, vector transmission, and silencing suppression (for review, see [1,3,4]). Although the viral genome structure and expression differ considerably in families Flexiviridae (includes Potexvirus) and Potyviridae (includes Potyvirus), their CPs show weak amino acid sequence similarity [5,6]. Furthermore, the CPs encoded by viruses of these two families are required for virus movement. Accordingly, the CPs and virions have been found in plasmodesmata, specialized cell wall-traversing channels recruited by viruses for transport between plant cells [7–14]. Other components of transport systems are dissimilar in Flexiviridae and Potyviridae. In addition to the CP, three movement proteins (MPs) referred to as TGBp1, TGBp2, and * Corresponding author. Fax: +7 95 939 3181. E-mail address: [email protected] (D.V. Rakitina).
Abbreviations: CP, coat protein; PVX, potato virus X; PVA, potato virus A; MP, movement protein; TGB, triple gene block; IgG, immunoglobulins
TGBp3 are required for cell-to-cell movement of potexviruses [15], whereas the viral transport requires the helper component-proteinase (HC-Pro) and the cylindrical inclusion protein (CI) in potyviruses [13,14,16,17]. Interestingly, both virus genera encode viral NTPases/helicases involved in virus cell-to-cell movement [15,18]. The proposed role of virus particles as a transport-competent form of the filamentous virus genomes has not been proven yet [8,18]. On the other hand, analyses of CP mutants show that the virion formation is not sufficient for effective functioning of cell-to-cell and long-distance transport systems, suggesting additional movement-related activities of the CP molecules [10–12,19,20]. Importantly, transient complementation studies showed that the CPs of potex- and potyviruses share some (yet unknown) function(s) necessary for the transport process [20,21]. In this paper, we studied the in vitro biochemical activities of the CPs of members of the genera Potexvirus (potato virus X, PVX) and Potyvirus (potato virus A, PVA). The PVX and PVA CPs were shown to possess Mg2+-dependent NTPase activity.
2. Materials and methods 2.1. Isolation and purification of PVX virions from infected plants Leaves of infected Datura stramonium plants (25–30 days after inoculation) were frozen at 20 C, homogenized in a Waring blender in buffer containing 0.3 M glycine-KOH and 1% Na2SO3, pH 7.5, were strained through two layers of cheese cloth, and centrifuged at 12 000 rpm for 30 min. The supernatant was filtered through a Miracloth filter and, after addition of Triton X-100 to a final concentration of 1%, allowed to stand for 20 min. Then polyethylene glycol (Mr 6000) and NaCl were added to final concentrations of 5% and 2%, respectively. The solution was incubated at 4 C for 2 h or overnight, centrifuged at 10 000 · g for 30 min, and the virus particles were extracted twice from the pellet with buffer A (25 mM Tris/HCl, pH 8.0) by stirring for 1–2 h. The extract was centrifuged at 10 000 · g for 20 min and the supernatant was ultracentrifuged at 35 000 rpm for 90 min (Ti-60 rotor, Beckman L5-50). The virus pellet was resuspended with a glass homogenizer in buffer A until completely free of lumps and loaded onto 2 ml of 30% sucrose cushion in tubes for Ti-50 rotor and centrifuged at 40 000 rpm for 2.5 h (Beckman L5-50). The virus pellet was dissolved in buffer A. All manipulations were carried out at 4 C. To obtain PVX CP, a suspension of virus particles was supplemented with 2 M LiCl, left at 20 C for two days, and centrifuged at 10 000 · g for 15 min. The CP-containing supernatant was recentrifuged and dialyzed against 10 mM Tris/HCl (pH 7.5). The isolation and purification of TMV virions from infected plants was performed as described in [22]. Briefly, wild type (U1) TMV was propagated on N. tabacum var. Samsun at 24 C. The virus was
0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.07.083
4956 extracted from plant sap with 10 mM phosphate buffer (pH 7.0) with 2 mM EDTA and purified by chloroform–butanol extraction, polyethylene glycol (Mr 6000) precipitation, and several cycles of differential centrifugation (90 min at 32 000 rpm in a SW50.1 rotor, Beckman L5-50, and 20 min at 12 000 rpm in a JA20 rotor, Beckman J2-20). Anti-PVX, anti-PVA, and anti-TMV antibodies were obtained by standard rabbit immunization with purified virion preparations and the fraction of immunoglobulins (IgG) was purified as described previously [23]. 2.2. Construction of recombinant clones For expression of the recombinant PVX CP, the PVX CP gene was amplified by PCR from a full-length PVX cDNA clone. The PCR product was digested with NcoI and BamHI and cloned into similarly digested pQE30 plasmid (QIAGEN). A frameshift mutant DCfsCPPVX (with 18 C-terminal amino acids replaced with a non-viral amino acid sequence), previously characterized in vivo [20], was generated by partial digestion of the PVX CP gene with XhoI, filling in by Klenow enzyme, and religation. To obtain deletion mutant of PVX CP – DC18CPPVX, expression clone PQE-cpPVX, carrying the PVX CP gene and coding for a protein with 6-His-tag on the N-terminus, was digested with EcoRI and XhoI, and the resulting fragment was introduced into similarly digested pQE30 plasmid. The plasmid pQE-cpPVA containing an N-terminal 6-His-tagged PVA CP gene was kindly provided by Dr. A. Merits (Institute of Biotechnology, Viikki Biocenter, University of Helsinki, Finland). A mutant DC49CPPVA (with 49 C-terminal amino acids deleted) was generated by cloning of an EcoRI/PstI fragment of expression vector pQE-cpPVA into similarly digested pQE30 plasmid. 2.3. Expression and purification of histidine-tagged CPs and their mutant forms E. coli strain M15 transformed with the recombinant vectors was grown at 37 C in liquid medium until OD600 of 0.8–0.9 was reached. Expression of the proteins was induced with 1 mM IPTG followed by growth for 2–4 h at 37 C. The purification of recombinant proteins from cultures was performed following the general procedure described by the manufacturerÕs instructions (QIAGEN) for denaturing Ni-NTA chromatography. 2.4. Cross-linking of ATP to PVX CP [a-32P] ATP (6000 Ci/mmol; 1 lCi per probe) was incubated on ice with 1–1.5 lg of the PVX CP for 20 min in reaction buffer containing 10 mM Tris/HCl, pH 8.0, 10% glycerol, 1 mM DTT, and 5 mM MgCl2. Samples were UV-irradiated in a Stratalinker 1800 unit as described elsewhere [24], heated with an equal volume of 2· Laemmli sample buffer, and separated by SDS–PAGE. Proteins were visualized by autoradiography of the dried gels. 2.5. NTP hydrolysis assay The reaction mixture contained 10 mM Tris/HCl, pH 8.0, 10% glycerol, 1 mM DTT, 5 mM MgCl2, 1 lCi [c-32P]ATP (6000 Ci/mmol) or 1 lCi [c-32P]UTP (3000 Ci/mmol), and 20–500 ng of protein in a final volume of 10 ll. When indicated, various NTPs, IgG, or chlorides of divalent cations (instead of MgCl2, at the same concentration) were added to the reaction mixture. Samples were incubated for 1 h at 37 C and the reaction was stopped by the addition of EDTA to a final concentration of 20 mM. To estimate NTPase activity, unreacted NTP was precipitated by addition of 200 ll of 7.5% activated charcoal in 50 mM HCl and 5 mM H3PO4; the mixtures were vortexed and allowed to stand for 5 min, charcoal was removed by centrifugation for 10 min, and a half of the supernatant was analyzed by Cherenkov counting of free 32Pi released by hydrolysis of labeled NTP [24]. The sample incubated without protein was used as a negative control. The total sample activity without incubation and precipitation procedure (minus a negative control sample) was taken as 100%. NTPase activity of proteins was expressed as relative activity (% hydrolysis of 32 P-labeled substrate). Kinetic parameters of ATP hydrolysis were measured in the linear phase of the reaction as described in [25,26]. The reaction mixture, which contained 10 mM Tris/HCl, pH 8.0, 10% glycerol, 1 mM DTT, and 5 mM MgCl2, was supplemented with 200 ng of the PVX CP or 400 ng of the PVA CP, ATP (from 1 to 65 lM), and 1 lCi
D.V. Rakitina et al. / FEBS Letters 579 (2005) 4955–4960 (8 nM) [c-32P]ATP. After 30 min incubation, ATP hydrolysis was analyzed using the activated charcoal method as described above. The experiment was repeated four times. Assuming that cold ATP and radiolabeled ATP are hydrolyzed at the same rate, the degree of [c-32P]ATP hydrolysis was taken as the degree of unlabeled ATP hydrolysis. Multiplying this degree by the amount of ATP in each sample (the amount of radiolabeled ATP was neglected), we achieved the amount of ATP hydrolyzed in each sample. Dividing the amount of the ATP hydrolyzed in each sample by the time of reaction, we achieved v (reaction velocity) at each substrate concentration. v was plotted versus ATP concentration. The Lineweaver–Burke doublereciprocal plots were then used to calculate Km and Vmax of ATP hydrolysis.
3. Results 3.1. PVX CP isolated from virions demonstrates ATP binding and ATPase activities The role of PVX CP in virus cell-to-cell movement involves protein function(s) other than encapsidation; hence, the CP may have some functions of viral MPs [15,21]. MPs of different plant viruses are known to have rNTP-binding activity [27–32]. Therefore, we studied whether the PVX CP is able to bind rNTPs. For these experiments, the CP was isolated from PVX virions. Typically, such PVX CP preparations contain two protein forms, the full-length Ps form and the Pf form which lacks 24 N-terminal amino acids as a result of natural proteolysis during isolation and storage [33]. The UV-crosslinking assay revealed the ability of both Ps and Pf forms of the PVX CP to effectively bind [a-32P]ATP (Fig. 1A). Since ATP-binding proteins often have ATPase activity too, the PVX CP was tested for the ability to hydrolyze rATP. After incubation of the protein with [c-32P]ATP, the percentage of hydrolyzed ATP was determined using activated charcoal. This experiment revealed ATPase activity of the PVX CP (Fig. 1B). Moreover, we found that PVX virion preparations also exhibited ATPase activity (Fig. 1B). No detectable ATPase activity was found for preparations of tobacco mosaic virus (TMV) virions purified from plant extracts either by the method routinely employed for TMV isolation (Fig. 1B) or by the PVX isolation procedure used in this paper (data not shown). These data could indicate that plant cellular ATPases were likely absent from the PVX virion preparations. PVX-specific IgG (but not TMV-specific IgG) substantially inhibited this virion activity (Fig. 1C). As reported previously, PVX TGBp1, the PVX MP with NTPase/helicase activity [34,35], is associated with one end of PVX filamentous virions in vitro [36]. Accordingly, Western blot analysis with a TGBp1-specific antibodies demonstrated the presence of a minor amount of the TGBp1 in preparations of PVX virions (our unpublished data). To determine whether the observed ATPase activity of PVX virion preparation was due to the found enzymatic function of the CP or to the activity of the TGBp1, anti-PVX virion or anti-TGBp1 IgG were added to the ATPase assay reaction. It was found that the ATPase activity of virions was only partially suppressed by TGBp1-specific IgG, while IgG against PVX virions severely inhibited the virion activity (Fig. 1C). In the control, TGBp1-specific IgG completely suppressed ATPase activity of recombinant TBGp1 (data not shown). Importantly, the virion-specific IgG had no cross-reactivity with TBGp1 and healthy plant extract proteins, and the TGBp1-specific IgG had no cross-reactivity with PVX CP
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Fig. 1. ATP binding and ATPase activity of the wild-type PVX CP and PVX virions. (A) UV-crosslinked binding of [a-32P]ATP to the PVX CP. Samples containing 1 lg of the PVX CP isolated from the virion were incubated on ice with [a-32P]ATP for 20 min. [a-32P]ATP was UV crosslinked to the CP. The samples were analyzed by SDS–PAGE, gels were dried, and proteins were visualized by autoradiography. Lanes: 1, PVX CP isolated from virus particles (Coomassie blue staining); the mobilities of two forms of PVX CP, the full-length Ps form and the truncated Pf form, are indicated; 2–3, autoradiograph of the PVX CP incubated with [a-32P]ATP after or without UV-crosslinking. (B) Comparison of ATPase activities of the virion-derived PVX CP as well as PVX and TMV virions. Samples containing 1–4 lg of protein were incubated for 1 h at 37 C and analyzed as described in Section 2. The reaction product, free radioactive Pi, was measured by Cherenkov counting after unreacted [c-32P]ATP was precipitated by the addition of activated charcoal in the presence of excess unlabeled Pi. Relative NTPase activity of proteins is presented (% hydrolysis of 32Plabeled substrate). (C) Inhibition of ATPase activity of the PVX virion by immunoglobulins (IgG). Anti-PVX virion IgG, anti-TGBp1 IgG, or antiTMV virion IgG were added to the ATPase reaction samples containing PVX virions (2 lg of protein).
and proteins of healthy plant extract (data not shown). Thus, the detected PVX virion ATPase activity can be at least in part attributed to the viral CP, which therefore retains its enzymatic activity when included into virus particles. 3.2. Mg2+-dependent ATPase activity of recombinant PVX CP To further investigate the PVX CP ATPase activity, we used the recombinant 6-His-tagged protein overexpressed in E. coli and purified by affinity chromatography on Ni-NTA agarose (Fig. 2A). The recombinant CP exhibited significant ATPase activity. Fig. 2B shows the percentage of [c-32P]ATP hydrolyzed by the CP plotted against the CP concentration. Up to 80–90% of [c-32P]ATP can be hydrolyzed by 150 ng of the protein within 1 h. ATPase activity of recombinant PVX CP was drastically suppressed by IgG against PVX virions but not by IgG against TMV virions (Fig. 2C). These results indicated that the observed ATPase activity was an intrinsic property of the PVX CP, and was not due to contaminating bacterial proteins. Further experiments examined the influence of different divalent cations on ATPase activity of the PVX CP. CaCl2, MnCl2, ZnCl2, or CuCl2 were added to the reaction mixture instead of MgCl2 (used in the standard reaction). No ATPase activity was detected in the presence of EDTA. All tested cations stimulated ATPase activity of the CP, and Mg2+ had the most pronounced effect (Fig. 2D). The kinetic parameters of the PVX CP for ATP hydrolysis were determined as described in Section 2. Km of ATP hydrolysis, calculated from the Lineweaver–Burke double-reciprocal plots, was 7 lM, with an apparent Vmax of 5 nmol min 1 mg 1 of the PVX CP. The C-terminally truncated PVX CP with a frame-shift mutation to encode 219 (out of the 237) amino acids plus 10 non-viral amino acids proved unable to support virus cell-to-
cell movement [20]. To test whether this mutation also affected the ATPase activity, the truncated mutant (referred to as DCfsCPPVX) was expressed in E. coli and purified by affinity chromatography on Ni-NTA agarose (Fig. 2A). The ATPase assay revealed that DCfsCPPVX hydrolyzed less than 10% of ATP (Fig. 2B). Additionally, we constructed the CP mutant DC18CPPVX with a stop codon that truncated the C-terminus of the protein by 18 amino acids. The ATPase assay carried out with the purified DC18CPPVX (Fig. 2A) demonstrated that this mutant hydrolyzed up to 30% of ATP (Fig. 2B). These data further support that the observed ATPase activity was due to the CP but not to contaminating cellular proteins. 3.3. The recombinant PVA CP exhibits Mg+2-dependent ATPase activity The CPs of two potyviruses, potato viruses A and Y, have been shown to complement the cell-to-cell movement of the PVX CP mutant lacking 18 C-terminal amino acids [20,21]. The recombinant 6-His-tagged PVA CP, overexpressed in E. coli and purified by Ni-NTA chromatography, was used to study whether potyviral CPs, which are distantly related in amino acid sequence to potexviral CPs [5,6], also have ATPase activity (Fig. 3A). Under standard reaction conditions, the PVA CP demonstrated ATPase activity (Fig. 3B). ATPase activity of the PVA CP was inhibited by specific (anti-PVA virion) but not by non-specific (anti-TMV virion) antibodies (Fig. 3C). The PVA CP activity was lower than that of the PVX CP: within 1 h of incubation, 150 ng of the PVA and PVX CP hydrolyzed up to 40–45% and 80–90% of total [c-32P]ATP, respectively. This observation was confirmed by the difference of apparent Vmax values: 1.5 and 5 nmol min 1 mg 1, respectively. At the same time, the Km of ATP hydrolysis was 7 lM for both CPs, which indicated their similar affinity to ATP. The influence of different divalent
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Fig. 2. Characterization of ATPase activity of the recombinant PVX CP. (A) SDS–PAGE analysis of the wild-type PVX CP isolated from virus particles (two forms), the recombinant PVX CP and its mutant (frame-shift, DCfsCPPVX, and deletion, DC18CPPVX) variants purified from E. coli. The sizes of molecular weight markers are shown at the left side. (B) Comparison of ATPase activities of the full-length PVX CP and its mutants, DCfsCPPVX and DC18CPPVX. (C) Inhibition of ATPase activity of the PVX CP by specific immunoglobulins (IgG). Anti-PVX virion IgG and antiTMV virion IgG were added at indicated concentrations to the reaction mixtures containing 60 ng of the CP. (D) Effect of various divalent cations on ATPase activity of PVX CP. 80 ng of the CP were incubated in standard reaction mixtures containing 5 mM of the indicated divalent cations as chlorides.
Fig. 3. ATPase activity of the recombinant PVA CP. (A) Analysis of the recombinant PVA CP and its mutant lacking 49 C-terminal amino acids (DC49CPPVA) purified from E. coli by SDS–PAGE. The sizes of molecular weight markers are shown at the left side. The position of the PVA CP trimer is indicated. (B) Comparison of ATPase activities of the full-length PVA CP and mutant DC49CPPVA. The ATPase assay was performed under standard conditions. (C) Inhibition of ATPase activity of the PVA CP by specific IgG. Anti-PVA virion IgG and anti-TMV virion IgG were added at indicated concentrations to the reaction mixtures containing 150 ng of protein. (D) Effect of various divalent cations on ATPase activity of PVA CP. 150 ng of the CP were incubated in the reaction mixtures containing 5 mM of the indicated divalent cations as chlorides.
cations on ATPase activity was similar in the PVA and PVX CPs, except that the PVA CP activity was only slightly stimulated by Ca2+ (Fig. 3D).
To analyze the role of the C-terminal region of PVA CP in ATPase activity, a mutant which lacked the 49 C-terminal residues (DC49CPPVA) was constructed, expressed in E. coli, and
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Fig. 4. Inhibition of ATPase activity of the PVX and PVA CPs by various NTPs and UTPase activity of the recombinant CPs. (A) Inhibition of ATPase activity of the PVX CP by rNTPs and dNTPs added to the reaction mixtures containing 40 ng of the CP. (B) Inhibition of ATPase activity of the PVA CP by rNTPs and dNTPs added to the reaction mixtures containing 150 ng of the protein. (C) UTPase activity of the PVX and PVA CPs.
purified (Fig. 3A). In the ATPase assay, DC49CPPVA showed a reduced ATPase activity in comparison with the full-length PVA CP (Fig. 3B). 3.4. The PVX and PVA CPs are NTPases To examine the influence of different rNTPs and dNTPs on the PVX CP ATPase activity, increasing concentrations of each unlabeled NTP were added to the ATPase reaction mixtures. All rNTPs inhibited the hydrolysis of [c-32P]ATP with a similar efficiency, while the effect of dNTPs was small (Fig. 4A). Similar results were obtained for the PVA CP: almost no inhibition of the [c-32P]ATP hydrolysis was observed in the presence of unlabeled dNTPs, whereas rNTPs inhibited the hydrolysis (Fig. 4B). The competition experiments suggest that the PVX and PVA CPs are able to hydrolyze rNTPs but not dNTPs. Therefore, we studied their UTPase activity in the standard NTPase assay. Both recombinant CPs were able to hydrolyze [c-32P]UTP with the efficiency similar to that of ATP hydrolysis (Fig. 4C). Thus, the CPs can hydrolyze both the purine triphosphate ATP and the pyrimidine triphosphate UTP. Taken together, these results indicate that the PVX and PVA CPs possess rNTPase activity.
4. Discussion Coat proteins (CPs) of plant RNA viruses are involved in several stages of the viral life cycle such as virion assembly, replication, movement, vector transmission, and regulation of host defense responses [1,3,4]. In this paper, we demonstrate
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that CPs of plant RNA viruses can have an enzyme activity. We found that the CPs encoded by two genera of viruses with flexuous filamentous particles, PVX (Potexvirus) and PVA (Potyvirus), exhibited Mg2+-dependent ATPase and UTPase activities. The Km values of the PVX and PVA CPs calculated for ATP hydrolysis were similar (7 lM). In addition, PVX virus particles and PVX CP isolated from these particles could bind and hydrolyze ATP although with a lower efficiency than the recombinant PVX CP. While the location of the enzyme active site could not be predicted based on the amino acid sequences of the CPs of filamentous viruses, the data obtained demonstrate that the C-terminal region of the PVX and PVA CPs may play an important role in the maintenance of the protein enzymatic core. The deletions of 18 C-terminal amino acids of the PVX CP (DC18CPPVX) and of 49 C-terminal residues of the PVA CP (DC49CPPVA) likely destabilized the overall CP structure and considerably decreased the ATPase activity (Figs. 2B, 3B). We suggest that the CP C-terminal sequence is not a major part of the NTPase active site, but can affect its proper conformation. This conclusion is supported by a much lower ATPase activity of DCfsCPPVX, with the 18 C-terminal amino acids replaced by a heterologous sequence, as compared to DC18CPPVX (Fig. 2B). The functional role(s) of ATPase activity of PVX and PVA CPs in viral infection is obscure. As suggested previously, the ATP-binding and ATP hydrolysis could be involved in cell-to-cell movement of viral genomes either at the stage of intracellular transport to plasmodesmata or at the stage of translocation of the viral genome-containing complex through plasmodesmal microchannels, which can be accompanied by energy-dependent complex disassembly or rearrangement [15]. ATPase activity of free CP molecules can also be required at other stages of the virus life cycle. Further studies of the biological role of the ATPase activity described for the CPs of filamentous plant viruses will require mapping of the ATPase active site and analysis of ATPase-deficient CP mutants. A pair of positively and negatively charged residues, conserved in the CPs of filamentous viruses and previously proposed to form a salt bridge important for the CP structure stabilization [5], can be considered as possible structural elements of the active site (E.V. Koonin, personal communication). Acknowledgments: We thank Dr. A.K. Bobkova for preparation of antibodies against PVX, PVA, and TMV virus particles and Dr. A. Merits for the PVA CP expression clone. We are grateful to Dr. A.V. Efimov for helpful discussion of the PVX and PVA CP structures and possible functions of different protein regions. We thank Dr. E.V. Koonin for valuable discussions. This work was supported by the Grant No. 04-04-49356 of the Russian Foundation for Basic Research and in part by the Grant No. 1291.2003.4 of the Leading Scientific School Program of the Russian Ministry of Education and Science.
References [1] Callaway, A., Giesman-Cookmeyer, D., Gillock, E.T., Sit, T.L. and Lommel, S.A. (2001) The multifunctional capsid proteins of plant RNA viruses. Annu. Rev. Phytopathol. 39, 419–460. [2] Carrington, J.C., Kasschau, K.D., Mahajan, S.K. and Schaad, M.C. (1996) Cell-to-cell and long-distance transport of viruses in plants. Plant Cell 10, 1669–1681. [3] Thomas, C.L., Leh, V., Lederer, C. and Maule, A.J. (2003) Turnip crinkle virus coat protein mediates suppression of RNA silencing in Nicotiana benthamiana. Virology 306, 33–41.
4960 [4] Qiu, W. and Scholthof, K.B. (2004) Satellite panicum mosaic virus capsid protein elicits symptoms on a nonhost plant and interferes with a suppressor of virus-induced gene silencing. Mol. Plant Microbe Interact. 17, 263–271. [5] Dolja, V.V., Boyko, V.P., Agranovsky, A.A. and Koonin, E.V. (1991) Phylogeny of capsid proteins of rod-shaped and filamentous RNA plant viruses: Two families with distinct patterns of sequence and probably structure conservation. Virology 184, 79– 86. [6] Koonin, E.V. and Dolja, V.V. (1993) Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences. Crit. Rev. Biochem. Mol. Biol. 28, 375–430. [7] Oparka, K.J., Roberts, A.G., Roberts, I.M., Prior, D.A.M. and Santa Cruz, S. (1996) Viral coat protein is targeted to, but does not gate, plasmodesmata during cell-to-cell movement of potato virus X. Plant J. 10, 805–813. [8] Santa Cruz, S., Roberts, A.G., Prior, D.A.M., Chapman, S. and Oparka, K.J. (1998) Cell-to-cell and phloem-mediated transport of potato virus X: the role of virions. Plant Cell 10, 495–510. [9] Chapman, S., Hills, G., Watts, J. and Baulcombe, D. (1992) Mutational analysis of the coat protein gene of potato virus X: effects on virion morphology and viral pathogenicity. Virology 191, 223–230. [10] Forster, R.L.S., Beck, D.l., Guilford, P.G.J., Voot, D.M., van Dolleweerd, C.J. and Andersen, M.T. (1992) The coat protein of white clover mosaic potexvirus has a role in facilitating cell-to-cell transport in plants. Virology 191, 480–484. [11] Dolja, V.V., Haldeman, R., Robertson, N.L., Dougherty, W.G. and Carrington, J.C. (1994) Distinct functions of capsid protein in assembly and movement of tobacco etch potyvirus in plants. EMBO J. 13, 1482–1491. [12] Dolja, V.V., Haldeman-Cahill, R., Montgomery, A.E., Vandenbosch, K.A. and Carrington, J.C. (1995) Capsid protein determinants involved in cell-to-cell and long distance movement of tobacco etch potyvirus. Virology 206, 1007–1016. [13] Rojas, M.R., Zerbini, F.M., Allison, R.F., Gilbertson, R.L. and Lucas, W.J. (1997) Capsid protein and helper componentproteinase function as potyvirus cell-to-cell movement proteins. Virology 237, 283–295. [14] Rodrıguez-Cerezo, E., Findlay, K., Shaw, J.G., Lomonossoff, G.P., Qiu, S.G., Linstead, P., Shanks, M. and Risco, C. (1997) The coat and cylindrical inclusion proteins of a potyvirus are associated with connections between plant cells. Virology 236, 296–306. [15] Morozov, S.Yu. and Solovyev, A.G. (2003) Triple gene block: modular design of a multifunctional machine for plant virus movement. J. Gen. Virol. 84, 1351–1366. [16] Carrington, J.C., Jensen, P.E. and Schaad, M.C. (1998) Genetic evidence for an essential role for potyvirus CI protein in cell-tocell movement. Plant J. 14, 393–400. [17] Roberts, I.M., Wang, D., Findlay, K. and Maule, A.J. (1998) Ultrastructural and temporal observations of the potyvirus cylindrical inclusions (CIs) show that the CI protein acts transiently in aiding virus movement. Virology 245, 173–181. [18] Revers, F., Le Gall, O., Candresse, T. and Maule, A.J. (1999) New advances in understanding the molecular biology of plant/ potyvirus interactions. Mol. Plant Microbe Interact. 12, 367–376. [19] Lough, T.J., Netzler, N.E., Emerson, S.J., Sutherland, P., Carr, F., Beck, D.L., Lucas, W.J. and Forster, R.L.S. (2000) Cell-to-cell movement of potexviruses: evidence for a ribonucleoprotein complex involving the coat protein and first triple gene block protein. Mol. Plant Microbe Interact. 13, 962–974. [20] Fedorkin, O.N., Merits, A., Luccesi, J., Solovyev, A.G., Saarma, M., Morozov, S.Yu. and Makinen, K. (2000) Complementation
D.V. Rakitina et al. / FEBS Letters 579 (2005) 4955–4960
[21]
[22]
[23]
[24] [25]
[26]
[27] [28]
[29]
[30] [31]
[32]
[33] [34]
[35]
[36]
of the movement-deficient mutations in potato virus X: potyvirus coat protein mediates cell-to-cell trafficking of C-terminal truncation but not deletion mutant of potexvirus coat protein. Virology 270, 31–42. Fedorkin, O.N., Solovyev, A.G., Yelina, N.E., Zamyatnin Jr, A.A., Zinovkin, R.A., Makinen, K., Schiemann, J. and Morozov, S.Yu. (2001) Cell-to-cell movement of potato virus X involves distinct functions of the coat protein. J. Gen. Virol. 82, 449–458. Dobrov, E.N., Abu-Eid, M.M., Solovyev, A.G., Kust, S.V. and Novikov, V.K. (1997) Properties of the coat protein of a new tobacco mosaic virus coat protein ts-mutant. J. Protein Chem. 16, 27–36. Dobrov, E.N., Bobkova, A.F., Goldshtein, M.I., Timofeeva, E.K. and Atabekov, J.G. (1991) Comparative study of antigenic properties of potato virus X and its coat protein in solution and after adsorbsion to solid support. J. Virol. Methods 32, 317–326. Rikkonen, M., Peranen, J. and Kaariainen, L. (1994) ATPase and GTPase activities associated with semliki forest virus nonstructural protein nsP2. J. Virol. 68, 5804–5810. Makino, Y., Yamano, K., Kanemaki, M., Morikawa, K., Kishimoto, T., Shimbara, N., Tanaka, K. and Tamura, T. (1997) SUG1, a component of the 26S proteasome, is an ATPase stimulated by specific RNAs. J. Biol. Chem. 272, 23201–23205. Hagman, W., Nies, A.T., Kıınig, J., Frey, M., Zentgraf, H. and Keppler, D. (1999) Purification of the human apical conjugate export pump MRP2. Reconstruction and functional characterization as substrate-stimulated ATPase. Eur. J. Biochem. 265, 281–289. Rouleau, M., Smith, R.J., Bancroft, J.B. and Mackie, G.A. (1994) Purification, properties, and subcellular localization of foxtail mosaic potexvirus 26-kDa protein. Virology 204, 254–265. Li, Q. and Palukaitis, P. (1996) Comparison of the nucleic acidand NTP-binding properties of the movement protein of cucumber mosaic cucumovirus and tobacco mosaic tobamovirus. Virology 216, 71–79. Bleykasten, C., Gilmer, D., Guilley, H., Richards, K.E. and Jonard, G. (1996) Beet necrotic yellow vein virus 42 kDa triple gene block protein binds nucleic acid in vitro. J. Gen. Virol. 77, 889–897. Donald, R.G., Lawrence, D.M. and Jackson, A.O. (1997) The barley stripe mosaic virus 58-kilodalton beta(b) protein is a multifunctional RNA binding protein. J. Virol. 71, 1538–1546. Kalinina, N.O., Samuilova, O.V., Fedorkin, O.N., Zelenina, D.A. and Morozov, S.Yu. (1998) Biochemical characterization and subcellular localization of a 25K movement protein of potato virus X. Mol. Biol. (Mosk) 4, 686–691, In Russia. Carvalho, C.M., Pouwels, J., van Lent, J.W.M., Bisseling, T., Goldbach, R.W. and Wellink, J. (2004) The movement protein of cowpea mosaic virus binds GTP and single-stranded nucleic acid in vitro. J. Virol. 78, 1591–1594. Koenig, R., Tremaine, J.H. and Shepard, J.F. (1978) In situ degradation of the protein chain of potato virus X at the N- and C-termini. J. Gen. Virol. 38., 329–337. Kalinina, N.O., Fedorkin, O.N., Samuilova, O.V., Maiss, E., Korpela, T., Morozov, S.Yu. and Atabekov, J.G. (1996) Expression and biochemical analyses of the recombinant potato virus X 25K movement protein. FEBS Lett. 397, 75–78. Kalinina, N.O., Rakitina, D.V., Solovyev, A.G., Schiemann, J. and Morozov, S.Yu. (2002) RNA helicase activity of the plant virus movement proteins encoded by the first gene of the triple gene block. Virology 296, 321–329. Atabekov, J.G., Rodionova, N.P., Karpova, O.V., Kozlovsky, S.V. and Poljakov, V.Yu. (2000) The movement protein-triggered in situ conversion of potato virus X virion RNA from nontraslatable into a translatable form. Virology 271, 259–263.