Genetic analysis of the function of the plum pox virus CI RNA helicase in virus movement

Virus Research 116 (2006) 136–145

Genetic analysis of the function of the plum pox virus CI RNA helicase in virus movement Marta G´omez de Cedr´on 1,2 , Lourdes Osaba 1,3 , Lissett L´opez 4 , Juan Antonio Garc´ıa ∗ Centro Nacional de Biotecnolog´ıa-CSIC, Campus de la Universidad Aut´onoma de Madrid, 28049 Madrid, Spain Received 11 July 2005; received in revised form 21 September 2005; accepted 21 September 2005 Available online 26 October 2005

Abstract The CI protein forms the cylindrical inclusions typical of potyviral infections and is involved in genome replication and virus movement. In this work, we have analyzed the effect of a series of point mutations at the N-terminal region of the CI protein of Plum pox virus (PPV) on the enzymatic activities and the self-interaction ability of the protein, and on virus replication and movement. DD3,4AA mutation, which had no apparent effects on ATPase and RNA helicase activities in vitro, and on virus replication in protoplasts, drastically impaired cell-to-cell spread of the virus. The effect of KK101,102AA mutation was host-specific. While no signals of virus infection were detected in Chenopodium foetidum inoculated with PPV KK101,102AA, the mutation caused a moderate effect on short distance movement in Nicotiana benthamiana and N. clevelandii, which resulted in a more drastic disturbance of systemic spread. None of the mutations analyzed abolished PPV CI self-interaction in the yeast Two-Hybrid system, but they caused a notable reduction in the binding strength, which appears to positively correlate with their effect on virus movement, suggesting that CI–CI interactions required for RNA replication and virus movement could be rather different. © 2005 Elsevier B.V. All rights reserved. Keywords: Virus movement; Movement protein; RNA helicase; NTPase; Potyvirus; Cylindrical inclusion; Plum pox virus; Sharka

1. Introduction Systemic infection of plants by viruses requires genome replication, cell-to-cell movement of the infectious particles and long-distance spread of them through the vascular tissue (Reichel et al., 1999). Cell-to-cell movement of positive strand RNA viruses typically needs specialized proteins (movement proteins, MP) to facilitate the intracellular and intercellular transport of viruses or ribonucleoprotein complexes (Carrington et al., 1996). Positive strand RNA viruses have developed a large variety of strategies to face the challenge of crossing the cell wall barrier. Some viruses, such as tobacco mosaic virus (TMV)

∗

Corresponding author. Tel.: +34 91 585 4535; fax: +34 91 585 4506. E-mail address: [email protected] (J.A. Garc´ıa). 1 M.G.dC. and L.O. contributed equally to this work. 2 Present address: Centro de Investigaciones Biol´ ogicas-CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain. 3 Present address: PROGENIKA, Edificio 801, Parque Tecnol´ ogico de Zamudio, 48160 Derio, Spain. 4 Present address: INGENASA, Hermanos Garc´ıa Noblejas 39, 28037 Madrid, Spain. 0168-1702/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2005.09.009

encode single dedicated MPs that modify plasmodesmata and facilitate the transport of nucleic acids through the modified channels with no requirement of capsid protein (CP) (Beachy and Heinlein, 2000). Comoviruses and other icosahedral RNA viruses also produce single MPs, but they form tubular structures that pass the cell wall through modified plasmodesmata allowing cell-to-cell movement of the genomic RNA in an encapsidated form (Pouwels et al., 2002). Other groups of viruses use two (Hacker et al., 1992; Marcos et al., 1999), three (Morozov and Solovyev, 2003) and even more (Dolja, 2003) MPs, differing also in the requirement of CP for cell-to-cell spread. Potyviruses are positive-strand RNA viruses that produce at least nine mature proteins from a polyprotein expression strategy (Riechmann et al., 1992). In contrast to most viruses, the potyviruses do not appear to encode a dedicated MP. Rather, the potyvirus movement involves viral proteins that perform additional roles in the life cycle of the virus (Carrington et al., 1996). A unique feature of potyvirus infections is the production of characteristic cylindrical inclusions, formed by the CI protein (Edwardson and Christie, 1996). This protein has RNA helicase activity (Eagles et al., 1994; La´ın et al., 1990) that has been shown to be essential for viral replication (Fern´andez et

M. G´omez de Cedr´on et al. / Virus Research 116 (2006) 136–145

al., 1997). The most typical inclusions formed by the CI protein are laminate or pinwheel-shaped and accumulate in the cytoplasm of the infected cells (Lesemann, 1988). However, at early times post-infection, CI protein is associated with cone-shaped structures that are anchored at the cell wall or the plasma membrane close to plasmodesmata (Langenberg, 1986; Lawson and Hearon, 1971; Roberts et al., 1998; Rodr´ıguez-Cerezo et al., 1997). In addition to CI protein, these structures were shown to contain CP and viral RNA (Roberts et al., 1998; Rodr´ıguezCerezo et al., 1997). These data led to the suggestion that the CI protein could facilitate cell-to-cell movement of the virus through direct interactions or modifications of plasmodesmata. Furthermore, genetic evidence has demonstrated the involvement of the Tobacco etch virus (TEV) CI protein in viral movement (Carrington et al., 1998). A self-interaction domain at the N-terminal 177 aa of the CI protein of Plum pox virus has been identified by yeast TwoHybrid experiments (L´opez et al., 2001). In this study we analyze the effects of mutations in this domain on the enzymatic activities and self-interaction ability of the protein and assess how these effects influence virus replication and movement. 2. Materials and methods 2.1. Construction of plasmids Point mutations at the N-terminal region of PPV CI were created by the PCR-based mutagenesis method described by Herlitze and Koenen (1990) using as mutator oligodeoxinucleotides (oligos) DD3,4AA (5 -CTTCTATAGCGGCCAAGCTC-3 ), KS91,92AA (5 -TCCAGTTGCTGCGCCCGATC-3 ), KK101,102AA (5 -CGTGGCCCCGCCGCGCTTAAGTG-3 ) and RQ123,125AA (5 -CATTGAATGGTGCACCTGCCAACTGCTTG-3 ), as flanking oligos 3275 (5 -CTCAATGATCAATGAGC-3 ) and 4081 (5 -GTTGATCCAAAGGTGC-3 ) and pGPPV (Riechmann et al., 1990) as template. The products of the two PCR steps were digested with SalI (PPV nt 3628, numbering is according to La´ın et al. (1989)) and NheI (PPV nt 3990) (for DD3,4AA, KS91,92AA and KK101,102AA) or SphI (PPV nt 4056) (for RQ123,125AA), and used to replace the corresponding fragment from pICPPVNK-GFP (Fern´andez-Fern´andez et al., 2001). The resulting plasmids were named pICPPVGFP-DD3,4AA, pICPPVGFPKS91,92AA, pICPPVGFP-KK101,102AA and pICPPVGFPRQ123,125AA. pGPPV-DD3,4AA, pGPPV-KS91,92AA, pGPPVKK101,102AA and pGPPV-RQ123,125AA were obtained by substituting the SalI–BamHI (PPV nt 3628-6931) fragments from pICPPVGFP-DD3,4AA, pICPPVGFP-KS91,92AA, pICPPVGFP-KK101,102AA and pICPPVGFP-RQ123,125AA for the corresponding one from pGPPV (Riechmann et al., 1990). To introduce the CI mutations in plasmids coding for MBPCI fusion proteins, cDNA fragments containing the CI coding sequence from its start until a NheI site were amplified by PCR from pICPPV-NK-GFP, pICPPVGFP-DD3,4AA, pICPPVGFP-KS91,92AA, pICPPVGFP-KK101,102AA and

137

pICPPVGFP-RQ123,125AA, using as primers oligo 3651 (5 AGCTTGGACGATATAG-3 ) (for wild type sequence and KS91,92AA, KK101,102AA and RQ123,125AA mutations) or oligo 3651dd (5 -AGCTTGGCCGCTATAG-3 ) (for DD3,4AA mutation), and oligo 4081. pMal-CIn, pMal-CInDD3,4AA, pMal-CInKS91,92AA, pMal-CInKK101,102AA and pMalCInRQ123,125AA were obtained by cloning in XmnI/SalIdigested pMal-c2 (New England Biolabs), the PCR products digested with NheI together with a NheI–SalI fragment from pMal-CI (Fern´andez et al., 1995), which supplies the CI coding sequence from the NheI site to its end. To introduce the CI mutations in plasmids coding for fusion products between the CI protein and the AD or DBD domains of protein Gal 4, DNA fragments that code for the first 177 aa of PPV CI with an NcoI site at their 5 end and a stop codon and a BamHI site at their 3 end were amplified by PCR from pICPPV-NK-GFP, pICPPVGFP-DD3,4AA, pICPPVGFP-KS91,92AA, pICPPVGFP-KK101,102AA and pICPPVGFP-RQ123,125AA, using as primers oligo 3651N (5 -CATGCCATGGAGAGCTTGGACGATATAG-3 ) (for wild type sequence and for KS91,92AA, KK101,102AA and RQ123,125AA mutations) or oligo 3651Ndd (5 -CATGCCATGGAGAGCTTGGCCGCTATAG-3 ) (for DD3,4AA mutation), and oligo 4181Bend (5 -CGGGATCCTCAGTGACATTCGTCAAAAATG-3 ). pAS-CI177e, pAS-CI177eDD3, 4AA, pAS-CI177eKS91,92AA, pAS-CI177eKK101,102AA and pAS-CI177eRQ123,125AA, were obtained by cloning between the NcoI and BamHI sites of pAS-CI (L´opez et al., 2001) the PCR products digested with NcoI and BamHI. pACTCI177e, pACT-CI177eDD3,4AA, pACT-CI177eKS91,92AA, pACT-CI177eKK101,102AA and pAS-CI177eRQ123,125AA were obtained by cloning the same NcoI/BamHI-digested PCR products in NcoI/BamHI-digested pACT-CI (L´opez et al., 2001). The accuracy of the constructions was verified by restriction analysis and DNA sequencing of all regions amplified by PCR. 2.2. Purification of MBP-CI recombinant proteins Expression of the recombinant plasmids and partial purification of the corresponding MBP-CI fusion proteins were carried out essentially as previously described (Fern´andez et al., 1995), with minor modifications. After culture at 18 ◦ C in LB medium containing ampicillin (100 ␮g/ml) and induction with 50 ␮M IPTG, Escherichia coli JM109 cells harboring the recombinant plasmids were collected by centrifugation, frozen and thawed, and lysed with lysozyme (75 ␮g/ml) in a buffer containing 10 mM Tris–HCl (pH 7.4), 0.1 M NaCl for 1 h at 4 ◦ C. The crude extract was treated with DNase (10 ␮g/ml) and RNase (10 ␮g/ml) for 30 min at 4 ◦ C, and cell debris were removed by centrifugation. The supernatant was adjusted to Tris–HCl (pH 7.4), 1 M NaCl, 1 mM EDTA and loaded onto an amylose resin column (New England Biolabs) equilibrated in the same buffer. The non-retained proteins were successively washed with the same buffer containing 1, 0.5 and 0.2 M NaCl, and without NaCl. The products specifically retained were eluted with buffer containing 10 mM maltose and no NaCl.

138

M. G´omez de Cedr´on et al. / Virus Research 116 (2006) 136–145

2.3. ATPase activity assay ATPase assays were carried out at 25 ◦ C in 20 ␮l reaction mixtures containing 15 mM HEPES-KOH (pH 7.5), 2.5 mM Mg (CH3 CO2 )2 , 1 mM DTT, 0.1 mM [␥33 P]ATP (∼125 Ci/mol), 0.2 mM poly A and the indicated amount of the partially purified protein fractions. Reactions were stopped in ice after different incubation periods by adding 50 mM EDTA, and samples were analyzed by polyethylenimine cellulose thin-layer chromatography with 0.15 M formic acid-0.15 M LiCl (pH 3) as the liquid phase. Hydrolysis activity was calculated by quantifying the radioactivity of the spots corresponding to ATP and Pi with a Molecular Imager FX System (BioRad). 2.4. RNA helicase activity assay Unwinding reaction mixtures contained 30 mM Tris–HCl (pH 7.5), 1.5 mM MgCl2 , 15 mM DTT, 30 ␮g/ml bovine serum albumin, 0.12 U/␮l RNasin, 2 mM ATP, 32 P-labeled partially dsRNA substrate A (0.15 ␮M), prepared according to La´ın et al. (1990) and the indicated amount of the purified protein fractions. After incubation at 25 ◦ C, the reactions were stopped by adding 3 ␮l of 0.5% SDS/40 mM EDTA. Samples were analysed by electrophoresis in 8% polyacrylamide gels containing 0.1% SDS and 0.5× TBE buffer. Unwinding activity was calculated by quantifying the radioactivity of the ssRNA and the partially dsRNA bands with a Molecular Imager FX System (BioRad).

(1 g in 2 ml 5 mM sodium phosphate, pH 7.2) onto three leaves dusted with Carborumdum. Virus infection was assessed by symptom monitoring and by visualizing GFP fluorescence under a Leica MZ FLIII or DM R fluorescence microscope with excitation and barrier filters of 480/40 nm and 510 nm, or 470/40 nm and 525/50 nm, respectively. Pictures were caught with an Olympus DP 70 camera and the software DP Controller and DP manager (Olympus). Immunocapture (Wetzel et al., 1992) followed by RT-PCR using the Titan kit (Roche) and oligos 3597 (5 CTCAATAAACTCAAAGGC-3 , PPV nt 3597–3614) and 4945 (5 -CGAACCAACGCCACTG-3 , complementary to PPV nt 4945–4930) were used as an additional infection criterion. Sequencing of the products of these RT-PCR amplifications confirmed the stability of the PPV mutations. 2.7. Yeast Two-Hybrid assays The yeast Two-Hybrid experiments were carried out using the Saccharomyces cerevisiae strain PJ69/4a (James et al., 1996), following the instructions of the MATCHMAKER System 2 kit (Clontech) as described by L´opez et al. (2001). ␤-Galactosidase activity was determined with the Luminescent ␤-galactosidase detection kit (Clontech). 3. Results 3.1. Design of mutations at the N-terminal region of PPV CI

2.5. Inoculation of plant protoplasts with PPV RNA synthesized in vitro Capped full-length transcripts were synthesized from pGPPV and its derivatives, linearized with PvuII and PstI, using the T7mMESSAGEmMACHINE kit (Ambion). The yield and integrity of the transcripts were analyzed by agarose gel electrophoresis. Protoplasts were isolated from N. clevelandii leaves as described previously (Guo et al., 1998). Samples of 8 × 105 protoplasts resuspended in 0.45 ml of electroporation buffer were exposed together with 20 ␮l of in vitro transcription reaction mixture to an electroporation pulse (110 V, 1000 ␮F, 20–25 ms) in an Electro Cell Manipulator 600 apparatus (BTX). After a 15 min recovery period on ice, the protoplasts were washed, resuspended in 1.2 ml culture medium and incubated under diffuse light at 24 ◦ C for 48 h. To assess virus replication in the protoplasts, PPV RNA was detected by Northern blot analysis as previously described (Fern´andez et al., 1997). 2.6. Analysis of PPV infection in plants The coating of micro gold particles with DNA from pICPPVNK-GFP-derived plasmids and their bombardment on N. clevelandii and N. benthamiana plants with the Helios Gene Gun were performed essentially according to the procedure described by L´opez-Moya and Garc´ıa (2000). For hand inoculation, young N. clevelandii, N. benthamiana or C. foetidum plants were inoculated by rubbing crude extract of previously infected plants

Several mutations in the CI coding region of TEV have been shown to cause different defects in virus spread in tobacco plants, without affecting virus amplification in single cells (Carrington et al., 1998). The fact that most of these mutations lie on the N-terminal region of the protein, the same protein region that is involved in PPV CI–CI interactions (L´opez et al., 2001) raises the possibility of a requirement of formation of cylindrical inclusion structures for the CI function in movement. Thus, in order to determine the way in which the molecular properties of the CI protein (enzymatic activities and self-interaction capacity) are related to its functions in potyvirus infection (replication and movement), we carried out a genetic analysis at the N-terminal region of PPV CI. We decided to analyze the effect of mutations substituting two alanine residues for two aspartic residues at positions 3 and 4 (DD3,4AA), two lysine residues at positions 101 and 102 (KK101,102AA) or one arginine and one glutamine residues at positions 123 and 125 (RQ123,125AA) (Fig. 1). The PPV mutated residues could be aligned with those affected by three TEV CI mutations previously described: DD3,4AA and KR100,101AA, which completely inhibited cell to cell movement, and RE122,124AA, which prevented the systemic spread of the virus but not local infection in the inoculated leaves (Carrington et al., 1998) (Fig. 1). None of these three TEV mutations altered viral RNA replication in tobacco protoplasts (Carrington et al., 1998). We also included in our analysis the mutation KS91,92AA, which affects the helicase motif I, known to be involved in NTP binding (Caruthers and McKay, 2002).

M. G´omez de Cedr´on et al. / Virus Research 116 (2006) 136–145

139

Fig. 1. Localization of the PPV CI mutations analyzed in this work, together with the corresponding ones of TEV CI analyzed by Carrington et al. (1998). The indicated amino acids were replaced by alanines. The position of the CI cistron in the PPV genome is shown at the top. Motifs conserved among RNA helicases are shown as dark areas.

The equivalent KS90,91AA mutation of TEV CI prevented virus replication in single cells (Carrington et al., 1998). The mutations were introduced into: (i) pMal-CIn, producing maltose binding protein (MBP)-CI fusion proteins to be used in the in vitro enzymatic assays; (ii) pAS-CI177e and pACT-CI177e, which express in yeast Two-Hybrid assays PPV N-terminal fragments fused to the Gal 4 activation (AD) or DNA binding (DBD) domains, respectively; (iii) pGPPV, which can be transcribed in vitro, yielding viral RNA to be used in replication analyses in protoplasts; and (iv) pICPPV-NK-GFP, which allows direct virus inoculation onto plants to study the effect of the mutations in local and systemic viral spread. 3.2. Enzymatic activities of MBP-CI proteins harboring mutations at the CI N-terminal region MBP-CI DD3,4AA, MBP-CI KS91,92AA, MBP-CI KK101,102AA and MBP-CI RQ123,125AA fusion proteins were partially purified by amylose affinity chromatography, as previously described (Fern´andez et al., 1997), from E. coli cells transformed with the corresponding mutated pMal-CIn-derived plasmids. As it was expected owing to its mutated NTP binding domain, no ATPase activity could be detected for MBP-CI KS91,92AA (Fig. 2A). In contrast, MBP-CI DD3,4AA, MBPCI KK101,102AA and MBP-CI RQ123,125AA displayed high poly A-stimulated ATPase activity (Fig. 2A), although a rigorous quantitative comparison of the activities was precluded by the rather variable proportion of inactive aggregated products that copurify with the active MBP-CI fusion proteins expressed in E. coli (Fern´andez et al., 1995). The three mutated proteins that had high ATPase activity, MBP-CI DD3,4AA, MBP-CI KK101,102AA and MBP-CI RQ123,125AA, also showed levels of RNA helicase activity comparable to that of wild type MBP-CI protein (Fig. 2B). As expected, the MBP-CI KS91,92AA protein did not show any detectable RNA helicase activity (Fig. 2B). 3.3. Replication in protoplasts of PPV harboring mutations at the CI N-terminal region In order to determine whether mutations at the N-terminal region of PPV CI interfered with virus replication in single cells,

Fig. 2. Enzymatic activities of MBP-CI fusion proteins with mutations at the CI N-terminal region. (A) Basal (250 ng of protein) and poly A-stimulated (100 ng of protein) ATPase activity after 7 min of reaction. The dose of MBP-CI KS91,92AA analyzed was 2 ␮g. (B) RNA helicase activity. Products obtained after 8 min of reaction, were separated by 8% PAGE and detected by autoradiography. Heat denatured substrate (boiled) and native substrate incubated alone in the reaction conditions (native) were used as controls. The RNA substrate employed in the experiment is depicted at the bottom of the figure. The positions of the native partially double stranded substrate (dsRNA) and of the denatured ssRNA are indicated beside the panel.

protoplasts prepared from N. clevelandii leaves were inoculated with transcripts synthesized in vitro from pGPPV-derived plasmids harboring the different mutations (tGPPVx). Northern blot analyses showed bands corresponding to PPV replication of similar intensity in the protoplasts inoculated with tGPPVDD3,4AA, tGPPVKK101,102AA, tGPPVRQ123,125AA and wild type tGPPV, but not in those inoculated with tGPPVKS91,92AA (Fig. 3). 3.4. Infectivity in plants of PPV harboring mutations at the CI N-terminal region In order to investigate the potential effects of the different mutations on virus spread, N. clevelandii plants were inoculated by bombardment with gold particles coated with DNA

140

M. G´omez de Cedr´on et al. / Virus Research 116 (2006) 136–145

Fig. 3. Viral RNA accumulation, assessed by Northern blot analysis, in N. clevelandii protoplasts inoculated with transcripts synthesized in vitro from pGPPV-derived plasmids harboring different mutations at the coding sequence of the CI N-terminal region. The arrow indicates the position of full-length PPV RNA. Two independent experiments are shown in panels A and B.

of the corresponding pICPPV-NK-GFP-derived plasmids. Viral spread was assessed by symptom inspection and by monitoring expression of the GFP gene included in the viral genome. Plants inoculated either with pICPPVGFP-RQ123,125AA or with non-mutated pICPPV-NK-GFP were indistinguishable both in time of appearance of the systemic symptoms (10–11 dpi) and in their severity (not shown). Large GFP expression foci (more than 100 cells) were detected in the inoculated leaves by 6 dpi (Fig. 4) and in the systemically infected leaves by 10 dpi (not shown). Sequencing of a PPV cDNA fragment amplified by IC-RT-PCR from systemically infected leaves demonstrated the stability of the RQ123,125AA mutation. Plants inoculated with either pICPPVGFP-DD3,4AA or pICPPVGFP-KS91,92AA failed to elicit symptoms within the time-length of the study (until 41 dpi), and GFP expression was restricted to isolated cells at the inoculated leaves (Fig. 4), although the number of cells expressing GFP was larger in the case of the DD3,4AA mutant. Since the DD3,4AA mutation did not affect viral RNA replication in protoplasts (Fig. 3), the confinement of GFP expression to single cells in the leaves inoculated with pICPPVGFP-DD3,4AA suggests that this mutation completely disrupts the activity of PPV CI in cell-to-cell movement. Besides, given the drastic effect of the KS91,92AA mutation in RNA replication in protoplasts (Fig. 3), the GFP expression detected in some single cells of the leaves inocu-

lated with pICPPVGFP-KS91,92AA would probably be due to transient nuclear expression from the bombarded plasmid. Plants inoculated with pICPPVGFP-KK101,102AA showed delayed (18–21 dpi) and weaker symptoms in comparison with those induced by inoculation with pICPPVGFP-RQ123,125AA or non-mutated pICPPV-NK-GFP (not shown). GFP expression foci in the leaves inoculated with pICPPVGFP-KK101,102AA were usually smaller (30–50 cells at 6 dpi) than those of plants bombarded with pICPPVGFP-RQ123,125AA or non-mutated pICPPV-NK-GFP, suggesting that the KK101,102AA mutation causes a partial deficiency in virus cell-to-cell-spread (Fig. 4). In agreement with the attenuation and the delay in the appearance of symptoms, at 10 dpi systemic GFP expression in pICPPVGFPKK101,102AA-inoculated plants was limited to small foci, mainly restricted to the proximity of major veins, in contrast with the large systemic GFP foci of pICPPV-NK-GFP- or pICPPVGFP-RQ123,125AA-inoculated plants, which at 10 dpi covered almost the whole leaf lamina (data not shown). In order to characterize in more detail the movement defect caused by the KK101,102AA mutation, N. clevelandii and N. benthamiana plants were inoculated by hand-rubbing with viral progeny from plants infected by biolistic inoculation with pICPPVGFP-KK101,102AA. The mutation appeared to affect more drastically PPV infection in N. clevelandii than in N. benthamiana: in a total of nine different experiments, only some

Fig. 4. GFP expression foci in leaves of N. clevelandii plants inoculated by particle bombardment with pICPPV-NK-GFP-derived plasmids harboring different mutations at the coding sequence of the CI N-terminal region. Photographs were taken at 6 dpi under a Leica DM R fluorescence microscope. The scale bars correspond to 40 ␮m.

M. G´omez de Cedr´on et al. / Virus Research 116 (2006) 136–145

141

Fig. 5. Patterns of GFP expression in N. benthamiana plants infected with wild type PPVGFP or PPVGFP-KK101,102AA: (A) Systemically infected leaves at 14 dpi. (B) Inoculated leaves at 3 and 5 dpi. Photographs were taken under a Leica MZ FLIII fluorescence microscope. The scale bars correspond to 1 mm.

leaves of six N. clevelandii plants out of 26 inoculated ones were systemically infected, while 21 out of 22 N. benthamiana plants inoculated became systemically infected, always homogenously. Moreover, when systemic symptoms of the KK101,102AA mutant were appreciable in N. clevelandii, they were milder and appeared with some delay compared with N. benthamiana infection. Under the fluorescence microscope, it was observed that, as it was also the case for plants inoculated with plasmid DNA, GFP expression in systemically infected leaves of plants inoculated with PPVGFP-KK101,102AA progeny virus remained localized to the proximity of the vascular system, at a post-inoculation time at which leaves systemically infected with wild type PPVGFP showed widespread GFP expression (Fig. 5A). At later infection times, GFP expression spread through the whole leaf also in the KK101,102AA infected plants (data not shown). The restricted distribution at early times post inoculation of PPVGFP-KK101,102AA could be due to a delayed exit of the virus from the inoculated leaves, to an inefficient movement through the vascular tissue, to a low rate of spread of the virus in the newly invaded leaf or to a combination of these possibilities. To evaluate the contribution of the defect in cell-to-cell spread of PPVGFP-KK101,102AA in the delay of its systemic propagation, the number of infection foci and their expansion rate were analyzed in N. benthamiana leaves inoculated by hand rubbing with crude extracts of plants infected with either PPVGFP or PPVGFP-KK101,102AA (Table 1). Moreover, we evaluated the time needed by the virus to leave the inoculated leaf, by removing the inoculated leaves at different times post inoculation (Table 2). The number of infection foci was very variable among the different inoculated leaves, although it appeared to be lower in

the leaves inoculated with PPVGFP-KK101,102AA, probably due to a lower virus concentration in the extracts used as inocula. More interestingly, the size of the infection foci, both at 3 dpi and at 5 dpi, was noticeably lower in the leaves inoculated with the mutant than in those inoculated with the wild type virus, clearly confirming the effect of the KK101,K102AA mutation on local virus movement (Fig. 5B and Table 1). The inoculated leaf depletion assay appeared to indicate that the reduction in the speed of cell-to-cell spread of the KK101,102AA mutant significantly affected the efficiency of its systemic movement. Whereas wild type PPVGFP was able to infect systemically most of the plants depleted of inoculated leaves at 3 dpi, PPVGFP-KK101,102AA was able to infect only 50% of the plants that conserved the inoculated leaves until 5 dpi (Table 2). Unexpectedly, in spite of the inefficiency of PPVGFPKK101,102AA infection in N. benthamiana and N. clevelandii, serial passages of the mutant in these plants (9 passages in N. benthamiana, 2 passages in N. benthamiana plus 3 in N. clevelandii, or 2 passages in N. benthamiana plus 2 in N. clevelandii plus 5 again in N. benthamiana) did not give rise to the emergence of more efficient virus variants, and neither reversions of the mutation nor compensatory second mutations were detected. PPV causes in Chenopodium foetidum local lesions that tend to necrotize in their central region, but is unable to establish a systemic infection in this host. PPVGFP produces similar local lesions, which display GFP fluorescence in the non-necrotic ring area. Neither GFP foci nor local lesions were observed in leaves of C. foetidum inoculated with PPVGFP-KK101,102AA (Table 1). We detected a few single cells that showed some green fluorescence (not shown), but we could not verify whether or not

M. G´omez de Cedr´on et al. / Virus Research 116 (2006) 136–145

142

Table 1 Number and size of local infection foci in plants inoculated with PPVGFP or PPVGFP-KK101,102AA Nicotiana benthamiana

Experiment 1 PPVGFP Extract 1 Extract 2 Extract 2a PPVGFP-KK101,102AA Extract 1 Extract 2 Experiment 2 PPVGFP Extract 1 Extract 2 PPVGFP-KK101,102AA Extract 1 Extract 2 a b c d e f g h i j k l m n o p q

Chenopodium foetidum

Number of foci

Area (3 dpi)

Area (5 dpi)

Number of foci

24.5 ± 32.1b 10.8 ± 9.5b 4.2 ± 5.8b

0.6 ± 0.28c

3.23 ± 1.45d

10.7 ± 3.8e 24.3 ± 3.9e 9.7 ± 1.7e

6.9 ± 8.5f 15.7 ± 22f

0.3 ± 0.11g

1.29 ± 0.5h

0i 0i

28.3 ± 8.4j 27.1 ± 18.6m

ND

2.12 ± 0.74k

50.2 ± 31l 56.8 ± 27.8n

12.8 ± 6.9o 23 ± 23m

ND

0.64 ± 0.24p

0q 0q

Dilution 1:2 of the extract 2. Average of six leaves from two different plants analyzed at 5 dpi. Average of the area of eight lesions (mm2 ). Average of the area of 39 lesions (mm2 ). Average of three leaves from three different plants analyzed at 9 dpi. Average of six leaves from three different plants analyzed at 5 dpi. Average of the area of 14 lesions (mm2 ). Average of the area of 37 lesions (mm2 ). Average of eight leaves from two different plants analyzed at 23 dpi. Average of seven leaves from four different plants analyzed at 5 and 9 dpi. Average of the area of 30 lesions (mm2 ). Average of four leaves from two different plants analyzed at 5 and 9 dpi. Average of eight leaves from four different plants analyzed at 5 and 9 dpi. Average of five leaves from two different plants analyzed at 5 and 9 dpi. Average of four leaves from two different plants analyzed at 5 and 9 dpi. Average of the area of 23 lesions (mm2 ). Average of eight leaves from two different plants analyzed at 9 dpi.

this corresponded to GFP expression associated to viral replication. Thus, in contrast with its effect in N. clevelandii and N. benthamiana, the KK101,102AA mutation appears to cause a complete blockade of PPV movement in C. foetidum. 3.5. Effects of mutations at the CI N-terminal region in self-interaction Yeast Two-Hybrid and pull-down experiments have mapped a self-interaction domain to the N-terminal 177 aa of the CI protein (L´opez et al., 2001). In order to assess whether Table 2 Effect of detachment of the inoculated leaves on the establishment of a systemic infection by PPVGFP and PPVGFP-KK101,102AA Time of detachment

PPVGFP PPVGFP-KK101,102AA a

3 dpi

5 dpi

No cut

5/6a

6/6 3/6

6/6 6/6

0/6

Number of plants systemically infected at 18 dpi/number of inoculated plants. All plants showed GFP expression foci in the inoculated leaves.

point mutations at this domain could affect CI–CI interactions, DD3,4AA, KS91,92AA, KK101,102AA and RQ123,125AA mutations were introduced in plasmids derived from pACTCI177e and pAS-CI177e, which encode the domains of transcription activation and DNA binding, respectively, of protein Gal4 fused to the N-terminal 177 aa of PPV CI (L´opez et al., 2001). Couples of the pACT-CI177e- and pAS-CI177e-derived mutated plasmids were transferred to the yeast strain PJ69/4a. In all cases, the transformed yeasts were able to grow in restrictive conditions, indicating that none of the mutations disturbed the CI–CI interaction completely. The ␤-galactosidase gene is activated in yeast PJ69/4a by the joint action of the Gal4 transcription activation and DNA binding domains. In order to assess whether the mutations that we were analyzing affected the strength of the CI–CI interaction, ␤-galactosidase assays were carried out with extracts of yeast colonies cultured in restrictive conditions. All the mutations, even RQ123,125AA, which appears not to affect PPV infectivity, caused a notable reduction in ␤-galactosidase activity, suggesting weaker CI–CI interactions (Table 3). However, whereas the RQ123,125AA mutation only caused a decrease in ␤-galactosidase activity of approximately 50%, the mutation

M. G´omez de Cedr´on et al. / Virus Research 116 (2006) 136–145

143

Table 3 Effect of point mutations at the N-terminal region of PPV CI on CI–CI interactions in the yeast Two-Hybrid system Coding sequence of CI fused to: DBDa

(pAS-)

CI177e CI177eDD3,4AA CI177eKS91,92AA CI177eKK101,102AA CI177eRQ123,125AA

ADa

(pACT-)

CI177e CI177eDD3,4AA CI177eKS91,92AA CI177eKK101,102AA CI177eRQ123,125AA

Growth in the absence of histidine and adenine

␤-Galactosidase activityb

+ + + + +

100 12.5 24.6 39.9 48.4

± ± ± ± ±

11.4 3.2 6.7 3.8 8.6

a

DBD and AD: DNA binding domain and transcription activation domain of the Gal4 protein, respectively. Percentage respect to the average of the activities of extracts of colonies transformed with pAS-CI177e + pACT-CI177e. A number ranging from 14 to 24 yeast colonies, from four independent experiments, were analyzed for each plasmid combination. b

DD3,4AA, which completely blocks PPV cell-to-cell spread, reduced the activity to 12.5% compared to that of the cells transformed with non-mutated plasmids (Table 3). The mutation KK101,102AA, which also disturbs PPV movement to some extent, caused a decrease in ␤-galactosidase activity only slightly higher than that caused by the RQ123,125AA mutation. Also the mutation KS91,92AA appeared to reduce the strength of the CI–CI interaction, but owing to the direct effect of the mutation on the NTPase activity of the protein (that does not depend on CI oligomerization, M.G.de C., L.O. and J.A.G., unpublished results) it was not possible to assess the effect of loosening of the CI–CI interaction on CI functions. 4. Discussion Most positive-stranded RNA viruses infecting plants and animals have at least a protein with a NTP binding (NTPB) domain (Gorbalenya and Koonin, 1993; Kadar´e et al., 1996). NTPase and helicase activities have been demonstrated for RNA virusencoded proteins of superfamilies (SF) 1 and 2 (Goregaoker and Culver, 2003; G´omez de Cedr´on et al., 1999; Kalinina et al., 2002; Kim et al., 1995; La´ın et al., 1990; Seybert et al., 2000; Warrener and Collet, 1995), but only NTPase activity has been demonstrated for those of SF3 (Mirzayan and Wimmer, 1994; Rodriguez and Carrasco, 1993). Most of the helicase-like proteins encoded by positivestranded RNA viruses are thought to be involved in viral RNA replication, although only in some cases it has been possible to associate this function with the ATPase or RNA helicase activities of the protein (Davenport and Baulcombe, 1997; Fern´andez et al., 1997; Grassmann et al., 1999; Matusan et al., 2001; Peters et al., 1994; Pfister and Wimmer, 1999). A widespread idea is that a helicase activity is needed to facilitate the RNA polymerase labor by disrupting putative large dsRNA stretches of replicative intermediates. However, the scarce experimental data available seem to suggest that dsRNA regions formed in the replicative intermediates might affect a limited number of nucleotides (Garnier et al., 1980). Thus, it is tempting to think that the role played by RNA helicases during viral replication could be very different to that of classical DNA helicases. Roles as chaperones or maturases that facilitate a correct RNA folding, or as RNPases that disrupt or rearrange RNA-protein interactions to form proper functional complexes have been proposed for RNA helicases (Linder, 2004; Schwer, 2001; Tanner and Linder,

2001). In the same line, it has been suggested that NTPase activity of viral SF3 helicase-like proteins, such as poliovirus 2C, could be involved in accurate organization of replication complexes (Pfister and Wimmer, 1999) or in their vesicular traffic (Rodriguez and Carrasco, 1993) rather than in unwinding RNA duplexes during RNA replication. An NTPB domain is also present in some plant virus proteins that are dispensable for RNA replication but participate in virus cell-to-cell movement (Kadar´e and Haenni, 1997; Morozov and Solovyev, 2003). RNA helicase activity has been demonstrated for several of these proteins (Kalinina et al., 2002). Viruses with a helicase-like protein involved in movement also have a replicative NTPB protein. The fact that the potyviral CI protein is required for RNA replication (Fern´andez et al., 1997; Klein et al., 1994) and for virus movement (Carrington et al., 1998) suggests that it could integrate in a single molecule the functions of the two types of viral helicase-like proteins. In agreement with previous reported data on TEV CI, the movement and replication functions of PPV CI are genetically separable. Like the equivalent TEV CI mutations (Carrington et al., 1998), the N-terminal region mutations DD3,4AA, KK101,102AA and RQ123,125AA in PPV CI (Fig. 1) appeared not to affect the function of the protein in RNA replication (Fig. 3). Also in agreement with TEV data, the DD3,4AA mutation restricted PPV to the first infected cells, completely blocking its cell-to-cell spread (Fig. 4). However, the effect in virus movement of PPV CI KK101,102AA and RQ123,125AA mutations differed from that of the comparable mutations of TEV CI. KR100,101AA and RE122,124AA mutations (Fig. 1) blocked the cell-to-cell and the systemic spread of TEV, respectively, suggesting that CI could play independent roles in shortand long-distance movement (Carrington et al., 1998). In contrast, the RQ123,125AA mutation had no noticeable effect on PPV infection (Fig. 4), and PPV KK101,102AA mutation resembled TEV RE122,124AA more than KR100,101AA mutation, since it affected mainly the systemic spread of the virus (Figs. 4 and 5A). However, a thorough analysis of PPVGFPKK101,102AA infection, showed that the systemic effect of this mutation was probably a pleiotropic result of a primary deficiency in the CI function in cell-to-cell propagation (Fig. 5B, and Tables 1 and 2). The differences in the effects caused by the TEV and PPV mutations could be due to intrinsic differences between the two CI proteins, but also to differences in virus susceptibility of the

144

M. G´omez de Cedr´on et al. / Virus Research 116 (2006) 136–145

host plants used in each study. In this regard, it is important to remark that the KK101,102AA mutation affected PPV infection in N. clevelandii more severely than in N. benthamiana. It is possible that the more drastic effect of TEV CI KR100,101AA and RE122,124AA mutations might be due to the fact that N. tabacum, the plant used by Carrington et al. (1998), is usually less permissive to viral infections than N. clevelandii or N. benthamiana. As with their RNA replication function, very little is known about how NTPB proteins participate in viral movement. A model in which the protein uses the energy from ATP hydrolysis to facilitate translocation of the viral RNA from cell-to-cell can be envisaged, especially in the case of potyviruses, in which the CI protein forms specialized structures that can be localized at both ends of plasmodesmata connecting neighboring cells and that appears to be traversed by viral RNA molecules (Rodr´ıguezCerezo et al., 1997). An RNPase activity, such as that described for the NPH-II RNA helicase (Jankowsky et al., 2001), could be involved in this process. The ability of the RNA helicase TGBp1 to produce energy-dependent conformational changes in PVX virions could also be related to a similar translocation mechanism (Atabekov et al., 2000; Rodionova et al., 2003). Nevertheless, the possibility exists that these proteins could play other kind of roles, including interference with defense mechanisms blocking virus spread. In relation with this possibility, TGBp1 is a well-known suppressor of the RNA silencing mechanism, and this activity could contribute to its movement function (Voinnet et al., 2000). Also connecting defense mechanisms and virus movement, the absence of visible signals of infection in C. foetidum inoculated with PPV KK101,102AA (Table 1), could be due to a possible effect of the reduction of virus movement competence on the efficiency of the hypersensitive response defense mechanism that limits PPV infection in this host. Although it is likely that the ATPase and RNA helicase activities of PPV CI contribute to its movement function, this is difficult to demonstrate since suppression of these activities would always have a primary effect on RNA replication that would hide the possible effect on viral movement. Anyway, it is clear that the defect caused by the N-terminal region mutations of PPV CI that we have analyzed are not the result of deficiencies in the enzymatic activities of the protein since the protein with the mutation that most drastically affects PPV movement, DD3,4AA, shows levels of enzymatic activities similar to or higher than those of proteins with mutations with lower or no effect on virus movement (Fig. 2). Considering that the function of the CI protein in virus movement appears to be associated to the well-defined organization of the cylindrical inclusions (Rodr´ıguez-Cerezo et al., 1997) and that a self-interaction domain has been mapped to the first 177 aa of PPV CI, it is appealing to think that the N-terminal region mutations might affect a structural feature of the protein required to transport the virus to the neighboring cell, which could be related with its self-assembly ability. Since previous results have shown that several sequences scattered across the N-terminal 177 aa domain (CI177) contributed to the CI–CI interaction (L´opez et al., 2001), it is not unexpected that none of the point mutations that we have analyzed abolish the self-interaction of PPV CI177 in the yeast Two-

Hybrid system. This is also in agreement with the observation that oligomerization of PPV CI appears to be required for an efficient RNA helicase activity (M.G.de C. and J.A.G., unpublished results). The fact that CI–CI interactions appear to be affected in the Two-Hybrid assays by the four mutations analyzed, but the strongest effect is found for the mutation which completely abolishes viral spread, could suggest that all of the mutated residues could contribute to the oligomerical structure involved in movement, but some loosening of the interactions would not have a critical effect on the movement function, the tolerance depending on the viral protein and the host. This would explain the distinct effects of the KK101,102AA/KR100,101AA and RQ123,125AA/RE122,124AA mutations in the different plant/virus systems. On the other hand, the fact that loosening of the CI–CI linkage did not affect the helicase activity of the proteins with mutations at their N-terminal region suggests that interactions required for RNA replication and virus movement could be somewhat different. It is clear, however, that a detailed structural analysis of the cylindrical inclusions formed in the cells infected with the different mutants is required to draw definitive conclusions. Acknowledgements We wish to thank Elvira Dom´ınguez for technical assistance, Carmen Sim´on-Mateo and Josefa M. Alamillo for critical reading of the manuscript and Caroline Coope for correcting its English. This work was supported by Grants BIO2004-02687 from the Spanish Ministry of Education and Science, and QLG22002-01673 and QLK2-2002-01050 from the European Union. References Atabekov, J.G., Rodionova, N.P., Karpova, O.V., Kozlovsky, S.V., Poljakov, V.Y., 2000. The movement protein-triggered in situ conversion of potato virus X virion RNA from a nontranslatable into a translatable form. Virology 271, 259–263. Beachy, R.N., Heinlein, M., 2000. Role of P30 in replication and spread of TMV. Traffic 1, 540–544. Carrington, J.C., Jensen, P.E., Schaad, M.C., 1998. Genetic evidence for an essential role for potyvirus CI protein in cell-to-cell movement. Plant J. 14, 393–400. Carrington, J.C., Kasschau, K.D., Mahajan, S.K., Schaad, M.C., 1996. Cellto-cell and long distance transport of viruses in plants. Plant Cell 8, 1669–1681. Caruthers, J.M., McKay, D.B., 2002. Helicase structure and mechanism. Curr. Opin. Struc. Biol. 12, 123–133. Davenport, G.F., Baulcombe, D.C., 1997. Mutation of the GKS motif of the RNA-dependent RNA polymerase from potato virus X disables or eliminates virus replication. J. Gen. Virol. 78, 1247–1251. Dolja, V.V., 2003. Beet yellows virus: the importance of being different. Mol. Plant Pathol. 4, 91–98. Eagles, R.M., Balmori-Meli´an, E., Beck, D.L., Gardner, R.C., Forster, R.L.S., 1994. Characterization of NTPase, RNA-binding and RNA-helicase activities of the cytoplasmic inclusion protein of tamarillo mosaic potyvirus. Eur. J. Biochem. 224, 677–684. Edwardson, J.R., Christie, R.G., 1996. Cylindrical inclusions. Florida Agric. Exp. Station Bull., 894. Fern´andez, A., Guo, H.S., S´aenz, P., Sim´on-Buela, L., G´omez de Cedr´on, M., Garc´ıa, J.A., 1997. The motif V of plum pox potyvirus CI RNA helicase is involved in NTP hydrolysis and is essential for virus RNA replication. Nucleic Acids Res. 25, 4474–4480.

M. G´omez de Cedr´on et al. / Virus Research 116 (2006) 136–145 Fern´andez, A., La´ın, S., Garc´ıa, J.A., 1995. RNA helicase activity of the plum pox potyvirus CI protein expressed in Escherichia coli. Mapping of an RNA binding domain. Nucleic Acids Res. 23, 1327–1332. Fern´andez-Fern´andez, M.R., Mouri˜no, M., Rivera, J., Rodr´ıguez, F., PlanaDur´an, J., Garc´ıa, J.A., 2001. Protection of rabbits against rabbit hemorrhagic disease virus by immunization with the VP60 protein expressed in plants with a potyvirus-based vector. Virology 280, 283–291. Garnier, M., Mamoun, R., Bov´e, J.M., 1980. TYMV RNA replication in vivo: replicative intermediate is mainly single stranded. Virology 104, 357–374. Gorbalenya, A.E., Koonin, E.V., 1993. Helicases: amino acid sequence comparisons and structure-function relationships. Curr. Opin. Struc. Biol. 3, 419–429. Goregaoker, S.P., Culver, J.N., 2003. Oligomerization and activity of the helicase domain of the tobacco mosaic virus 126- and 183-kilodalton replicase proteins. J. Virol. 77, 3456–3549. Grassmann, C.W., Isken, O., Behrens, S.E., 1999. Assignment of the multifunctional NS3 protein of bovine viral diarrhea virus during RNA replication: an in vivo and in vitro study. J. Virol. 73, 9196–9205. Guo, H.S., Cervera, M.T., Garc´ıa, J.A., 1998. Plum pox potyvirus resistance associated to transgene silencing that can be stabilized after different number of plant generations. Gene 206, 263–272. G´omez de Cedr´on, M., Ehsani, N., Mikkola, M.L., Garc´ıa, J.A., Kaariainen, L., 1999. RNA helicase activity of Semliki Forest virus replicase protein NSP2. FEBS Lett. 448, 19–22. Hacker, D.L., Petty, I.T., Wei, N., Morris, T.J., 1992. Turnip crinkle virus genes required for RNA replication and virus movement. Virology 186, 1–8. Herlitze, S., Koenen, M., 1990. A general and rapid mutagenesis method using polymerase chain reaction. Gene 91, 143–147. James, P., Halladay, J., Craig, E.A., 1996. Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144, 1425–1436. Jankowsky, E., Gross, C.H., Shuman, S., Pyle, A.M., 2001. Active disruption of an RNA-protein interaction by a DExH/D RNA helicase. Science 291, 121–125. Kadar´e, G., David, C., Haenni, A.-L., 1996. ATPase, GTPase, and RNA binding activities associated with the 206-kilodalton protein of turnip yellow mosaic virus. J. Virol. 70, 8169–8174. Kadar´e, G., Haenni, A.L., 1997. Virus-encoded RNA helicases. J. Virol. 71, 2583–2590. Kalinina, N.O., Rakitina, D.V., Solovyev, A.G., Schiemann, J., Morozov, S.Y., 2002. RNA helicase activity of the plant virus movement proteins encoded by the first gene of the triple gene block. Virology 296, 321– 329. Kim, D.W., Gwack, Y., Han, J.H., Choe, J., 1995. C-terminal domain of the hepatitis C virus NS3 protein contains an RNA helicase activity. Biochem. Biophys. Res. Commun. 215, 160–166. Klein, P.G., Klein, R.R., Rodr´ıguez-Cerezo, E., Hunt, A., Shaw, J.G., 1994. Mutational analysis of the tobacco vein mottling virus genome. Virology 204, 759–769. Langenberg, W.G., 1986. Virus protein associated with cylindrical inclusions of two viruses that infect wheat. J. Gen. Virol. 67, 1161–1168. Lawson, R.H., Hearon, S.S., 1971. The association of pinwheel inclusions with plasmodesmata. Virology 44, 454–456. La´ın, S., Riechmann, J.L., Garc´ıa, J.A., 1989. The complete nucleotide sequence of plum pox potyvirus RNA. Virus Res. 13, 157–172. La´ın, S., Riechmann, J.L., Garc´ıa, J.A., 1990. RNA helicase: a novel activity associated with a protein encoded by a positive strand RNA virus. Nucleic Acids Res. 18, 7003–7006. Lesemann, D.E., 1988. Cytopathology. In: Milne, R.G. (Ed.), The Plant Viruses, 4. Plenum Press, New York & London, pp. 179–235. Linder, P., 2004. The life of RNA with proteins. Science 304, 694–695.

145

L´opez, L., Urzainqui, A., Dom´ınguez, E., Garc´ıa, J.A., 2001. Identification of an N-terminal domain of the plum pox potyvirus CI RNA helicase involved in self-interaction in a yeast Two-Hybrid system. J. Gen. Virol. 82, 677–686. L´opez-Moya, J.J., Garc´ıa, J.A., 2000. Construction of a stable and highly infectious intron-containing cDNA clone of plum pox potyvirus and its use to infect plants by particle bombardment. Virus Res. 68, 99–107. Marcos, J.F., Vilar, M., P´erez-Pay´a, E., Pall´as, V., 1999. In vivo detection, RNA-binding properties and characterization of the RNA-binding domain of the p7 putative movement protein from carnation mottle carmovirus (CarMV). Virology 255, 354–365. Matusan, A.E., Pryor, M.J., Davidson, A.D., Wright, P.J., 2001. Mutagenesis of the Dengue virus type 2 NS3 protein within and outside helicase motifs: effects on enzyme activity and virus replication. J. Virol. 75, 9633–9643. Mirzayan, C., Wimmer, E., 1994. Biochemical studies on poliovirus polypeptide 2C: evidence for ATPase activity. Virology 199, 176–187. Morozov, S.Y., Solovyev, A.G., 2003. Triple gene block: modular design of a multifunctional machine for plant virus movement. J. Gen. Virol. 84, 1351–1366. Peters, S.A., Verver, J., Nollen, E.A.A., van Lent, J.W.M., Wellink, J., van Kammen, A., 1994. The NTP-binding motif in cowpea mosaic virus B polyprotein is essential for viral replication. J. Gen. Virol. 75, 3167–3176. Pfister, T., Wimmer, E., 1999. Characterization of the nucleoside triphosphatase activity of poliovirus protein 2C reveals a mechanism by which guanidine inhibits poliovirus replication. J. Biol. Chem. 274, 6992–7001. Pouwels, J., Carette, J.E., van Lent, J.W.M., Wellink, J., 2002. Cowpea mosaic virus: effects on host cell processes. Mol. Plant Pathol. 3, 411–418. Reichel, C., Mas, P., Beachy, R.N., 1999. The role of the ER and cytoskeleton in plant viral trafficking. Trends Plant Sci. 4, 458–462. Riechmann, J.L., La´ın, S., Garc´ıa, J.A., 1990. Infectious in vitro transcripts from a plum pox potyvirus cDNA clone. Virology 177, 710–716. Riechmann, J.L., La´ın, S., Garc´ıa, J.A., 1992. Highlights and prospects of potyvirus molecular biology. J. Gen. Virol. 73, 1–16. Roberts, I.M., Wang, D., Findlay, K., 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. Rodionova, N.P., Karpova, O.V., Kozlovsky, S.V., Zayakina, O.V., Arkhipenko, M.V., Atabekov, J.G., 2003. Linear remodeling of helical virus by movement protein binding. J. Mol. Biol. 333, 565–572. Rodriguez, P.L., Carrasco, L., 1993. Poliovirus protein-2C has ATPase and GTPase activities. J. Biol. Chem. 268, 8105–8110. Rodr´ıguez-Cerezo, E., Findlay, K., Shaw, J.G., Lomonossoff, G.P., Qiu, S.G., Linstead, P., Shanks, M., Risco, C., 1997. The coat and cylindrical inclusion proteins of a potyvirus are associated with connections between plant cells. Virology 236, 296–306. Schwer, B., 2001. A new twist on RNA helicases: DExH/D box proteins as RNPases. Nat. Struct. Biol. 8, 113–116. Seybert, A., van Dinten, L.C., Snijder, E.J., Ziebuhr, J., 2000. Biochemical characterization of the equine arteritis virus helicase suggests a close functional relationship between arterivirus and coronavirus helicases. J. Virol. 74, 9586–9593. Tanner, N.K., Linder, P., 2001. DExD/H box RNA helicases: from generic motors to specific dissociation functions. Mol. Cell 8, 251–262. Voinnet, O., Lederer, C., Baulcombe, D.C., 2000. A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana. Cell 103, 157–167. Warrener, P., Collet, M.S., 1995. Pestivirus NS3 (p80) protein possesses RNA helicase activity. J. Virol. 69, 1720–1726. Wetzel, T., Candresse, T., Macquaire, G., Ravelonandro, M., Dunez, J., 1992. A highly sensitive immunocapture polymerase chain reaction method for plum pox potyvirus detection. J. Virol. Methods 39, 27–37.