1 Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
2 A.N. Belozersky Institute of Physico-chemical Biology, Moscow State University, Moscow 119899, Russia
3 Dept of Electronic Engineering and Physics, University of Dundee, Dundee DD1 4NH, UK
4 Horticulture Research International-East Malling, ME19 6BJ, UK
Correspondence
Peter Palukaitis
ppaluk{at}scri.sari.ac.uk
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ABSTRACT |
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INTRODUCTION |
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The capsid protein (CP), but not virus particles, was also shown to be essential for the cell-to-cell movement of CMV (Kaplan et al., 1998), and mutants in the CP also have host-specific effects on CMV movement (Kaplan et al., 1998
; Ryu et al., 1998
; Takahashi et al., 2001
; Takeshita et al., 2001
; Wong et al., 1999
). It was speculated that the CP might have a role in altering the conformation of the 3a MP, facilitating its interaction with plasmodesmata (Ryabov et al., 1999
). It is conceivable that the C-terminal region of the 3a MP is involved in altering the RNA-binding affinity, because this region was shown to be adjacent to the region involved directly in RNA binding (Vaquero et al., 1997
) and was found to be dispensable for infection (Kaplan et al., 1995
; Nagano et al., 1997
, 2001
). If this were the case, then the CP might be required to alter the stability of MPRNA complexes, while the C-terminal region of the 3a MP would be expected to have some effect on the binding of MP to RNA. No evidence could be obtained for a direct interaction between the CP and MP in vitro (Nagano et al., 2001
; D. Szilassy, T. Canto & P. Palukaitis, unpublished data). Therefore, in this study we tested the hypothesis that deletion of the C-terminal 33 aa of the MP might alter its binding to viral RNA, and hence CP would not be required to effect the stability of the mutant 3a proteinRNA complexes. Specifically, we examined the role of the C-terminal 33 aa in RNA binding and the biological properties of the proteinRNA complexes.
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METHODS |
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A plasmid containing the gene encoding the tobacco mosaic virus (TMV) 30K MP fused to sequences encoded six histidine residues at the N terminus of the 30K protein was obtained from Sean Chapman (SCRI). The TMV 30K MP was overexpressed, purified and renatured as described above for the CMV MP.
In vitro RNA binding assays.
32P-labelled RNA transcripts of CMV RNA 3 were prepared from linearized pFny309 using 5 µCi of -[32P]UTP (Amersham) and the mMESSAGE mMACHINE T7 kit (Ambion). The labelled transcripts (4 ng) were mixed with different amounts of purified protein, either wt 3a MP or the truncated 3a MP, in 15 µl of binding buffer (50 mM Tris/HCl, pH 7·0, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 1 mg BSA ml-1 and 10 % glycerol). After incubation on ice for 30 min, the mixtures were subjected to electrophoresis in non-denaturing gels of 1 % agarose. The gels were dried and subjected to autoradiography. Treatment of MPRNA complexes with RNase A (200 ng) and electrophoresis was done as described previously (Li & Palukaitis, 1996
). The formation of complexes in different concentrations of NaCl and the detection of labelled RNA in complexes by filtration of the solutions and liquid scintillation counting were done as described previously (Li & Palukaitis, 1996
).
Atomic force microscopy (AFM).
TMV RNA, total CMV RNA or purified RNA transcripts from plasmid pF:GFP/CP were used for the formation of MPRNA complexes visualized by AFM. RNA (0·1 µg) was denatured by heating at 95 °C for 30 s and quenching on ice. The denatured RNA was mixed on ice with 20 µl of MP in RNase-free water, at different weight ratios of MP : RNA, for 30 min. The complexes were diluted 10-fold in RNase-free water before image analysis. MPRNA complexes (5 µl) were placed onto freshly cleaved mica strips for 5 min. The mica strips were then rinsed with deionized water and vacuum dried at room temperature. Sometimes AP-mica strips were used instead of untreated mica (which contains a negative surface charge), prepared as described by Lyubchenko et al. (1993a, b)
. That is, the mica strips were incubated in an atmosphere of 3-aminopropyltriethoxylane (APTES) to cover the surface with amino groups. Imaging of the complexes and measuring of heights were done as described previously (Nurkiyanova et al., 2001
).
Infectivity assays.
RNA transcripts of CMV RNAs 1 and 2 were mixed with RNA 3 transcribed from either pFL:3aC33/GFP or pL:3a/GFP and inoculated directly onto Carborundum-dusted leaves of Nicotiana benthamiana or tobacco (N. tabacum cv. Samsun NN). Plants were maintained in a shaded greenhouse for the times indicated. Infection was detected by fluorescence due to GFP expression, using either a confocal microscope or a hand-held UV lamp, as described previously (Ryabov et al., 1999
).
Fny-CMV RNAs (50 ng) or TMV RNA (100 ng) were mixed with E. coli-expressed MP at different MP : RNA ratios from 1·25 : 1 to 100 : 1 for CMV RNA and 5 : 1 to 120 : 1 for TMV RNA, on ice for 1 h, in 20 to 40 µl of deionized water. CMV RNAs either pre-incubated with water alone or with various amounts of MP were inoculated to opposite half-leaves of Chenopodium amaranticolor plants. TMV RNA pre-incubated with water or with various amounts of MP was inoculated to opposite half-leaves of either non-transgenic tobacco (N. tabacum cv. Xanthi XHFD8) or transgenic tobacco expressing the R-CMV CP gene (line CP-R.9A). The numbers of local lesions were scored at 4 days post-inoculation (p.i) for both sets of experiments. The above inoculations (CMV and TMV) were each done in four separate experiments. The mean and standard error values for the percentage reduction in infectivity at each MP : RNA ratio were calculated.
In vitro translation and analysis of proteins.
CMV RNA 3 transcripts (0·5 µg), derived from plasmid pF:GFP/CP, were denatured at 95 °C for 30 s, quenched on ice and incubated at 0 °C for 30 min with different amounts of either the wt 3a MP or the truncated 3a MP. Free or MP-complexed RNA was translated in vitro in rabbit reticulocyte cell-free lysate, using the Transcend tRNA non-radioactive detection system (Promega), following the manufacturer's instructions. The translation products were fractionated by 10 % SDS-PAGE. The proteins were transferred onto a PVDF membrane (Bio-Rad) and the membrane was processed to detect the biotinylated GFP using alkaline-phosphatase-conjugated streptavidin followed by incubation with BCIP/NBT. Denaturing gels containing the complexes were stained with Coomassie blue to visualize the MP (Sambrook & Russell, 2001).
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RESULTS |
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To test whether the Fny-CMV wt 3a MP interfered with the movement of CMV expressing the mutant 3aC33 MP, CMV RNAs expressing both the mutant 3a
C33 MP and the GFP were inoculated to transgenic tobacco expressing high levels of the wt Fny-CMV 3a protein (Kaplan et al., 1995
). The transgenically expressed wt 3a MP did not inhibit cell-to-cell movement of the CMV mediated by the 3a
C33 MP (Fig. 2E
).
Binding kinetics, nuclease sensitivity and stability of the proteinRNA complexes
E. coli-expressed and solubilized MPs were used to determine whether the mutant 3aC33 MP showed differences in RNA binding from the wt 3a MP. The wt 3a MP bound to CMV RNA 3 in a cooperative fashion (Fig. 3
A), as described previously for wt 3a MP solubilized by a different procedure (Li & Palukaitis, 1996
). About half of the labelled RNA was bound to protein and formed complexes with 200 ng of wt 3a MP, while in the presence of 400 ng of wt 3a MP, all of the available RNA was associated with 3a MP (Fig. 3A
). The mutant 3a
C33 MP also bound RNA cooperatively, but much less protein was required to convert all of the free RNA to proteinRNA complexes than for wt 3a MP (Fig. 3B
vs Fig. 3A
). About half of the viral RNA was associated with proteinRNA complexes when between 20 and 50 ng of the 3a
C33 MP was available, and in the presence of 100 ng of 3a
C33 MP, all of the RNA was bound to protein (Fig. 3B
). This suggested that the mutant MP did not bind with the same degree of cooperativity (i.e. saturation) and/or it had a greater affinity for RNA. In the former situation, more protein would be available to bind to other RNA molecules. In the latter case, the MPRNA complexes formed between 3a
C33 MP and RNA might be expected to be more stable than those formed between wt 3a MP and RNA. The same results were obtained using different viral RNAs (data not shown).
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Architecture of the proteinRNA complexes
The architecture of MPRNA complexes has been examined before by AFM, both for the wt CMV 3a MP and the 30K MP encoded by TMV (Kiselyova et al., 2001; Nurkiyanova et al., 2001
). In the case of the wt 3a MP, large aggregates of proteinRNA complexes were formed. Using a slightly different sample treatment for the AFM (see Methods) allowed visualization of non-aggregated proteinRNA complexes (Fig. 5
A). Most of the RNA within these complexes appeared coated (Fig. 5A
, panels 1 and 2), but at higher resolution the complexes formed between wt 3a MP and RNA showed alternating thicker and thinner regions (Fig. 5A
, panel 3), with an apparent periodicity of approximately 15 nm (Fig. 5A
, panel 4). These complexes had some similarities to the beads-on-a-string complexes described for the TMV MP (Kiselyova et al., 2001
), although the string was not naked RNA but, based on its thickness, was also coated with MP. All of the various complexes formed between the wt 3a MP and RNA showed the same pattern and periodicity. By contrast, the complexes observed between the mutant 3a
C33 MP and RNA were not uniform (Fig. 5B
, panels 14) and showed three structural elements, which may be present on the same RNA molecule (Fig. 5B
, panel 1): (a) protein forming thick beads on a coated string (Fig. 5B
, panel 5), similar to what was observed for the wt 3a MP (Fig. 5A
, panel 3), with an apparent periodicity of 20 nm (Fig. 5B
, panel 7); (b) a more densely coated string (Fig. 5B
, panel 6), with an apparent periodicity of 20 or 30 nm, but with a low amplitude between the bead units (Fig. 5B
, panel 8); and (c) structures that may be described as white nodules (asterisks in Figs 5B
, panels 1, 2 and 4). The same results were obtained using various viral RNAs to form the proteinRNA complexes.
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Infectivity of proteinRNA complexes
If the mutant 3aC33 MP binds more efficiently to viral RNA than the wt 3a MP, then complexes formed in vitro between CMV RNA and the two MPs would be expected to differ in their infectivities. Therefore, CMV RNAs were incubated with different amounts of the two MPs and were inoculated to C. amaranticolor, to assess the effect of the MPs on the infectivity of the viral RNAs. The infectivity of CMV RNAs pre-incubated without protein was assessed on opposite half leaves in four separate experiments. There was considerable variation in the number of local lesions obtained for the same concentration of inocula in different experiments and on different half leaves (Fig. 6
A), but this is typical of CMV (Habili & Francki, 1974
; Lakshman & Gonsalves, 1985
; Nagano et al., 2001
; Rao & Francki, 1981
). Nevertheless, the data show that at high ratios of MP : RNA, the infectivity of both types of MPRNA complex was inhibited. However, the ratio of MP : RNA required to inhibit the infectivity of the viral RNA was less for the mutant 3a
C33 MP than for the wt 3a MP (Fig. 6A
). That is, the wt 3a MP showed a drop in relative infectivity to 50 % at a MP : RNA ratio of about 25 : 1. By contrast, the relative infectivity of complexes formed between the mutant 3a
C33 MP and CMV RNA dropped to 50 % at a MP : RNA ratio of about 15 : 1 (Fig. 6A
).
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It is conceivable that the CP is able to interact with the wt 3a MP associated with the viral RNA, disrupting the MPRNA complex. However, CMV CP could not be added to such complexes in vitro, since CP capsomers isolated from virions are insoluble. Therefore, transgenic tobacco plants expressing high levels of CP, but not showing resistance to infection by CMV (Jacquemond et al., 2001), were used as a source of CP to determine whether the presence of CP had any effects on the inhibition of viral infectivity by MP. Unfortunately, as CMV RNA coated with wt CMV MP retained some basal level of infection even at MP : RNA ratios of 50 to 100 : 1 (Fig. 6A
), yielding three to ten local lesions on C. amaranticolor (data not presented), tobacco, a systemic host for CMV, could not be used to assess quantitative effects on infection. However, these transgenic tobacco plants also contained the N gene, which gives a hypersensitive response after infection by TMV. Thus, to quantify the effects of CP, as well as different levels of MP on the infection of the viral RNAs, TMV RNA was combined with different amounts of wt 3a MP or mutant 3a
C33 MP and was inoculated to transgenic tobacco plants expressing the CMV CP, as well as to non-transgenic plants of the same tobacco cultivar. Inoculation of non-transgenic tobacco plants with TMV complexed with either the wt 3a MP or the mutant 3a
C33 MP resulted in the formation of local lesions and a reduction in the specific infectivity of the TMV RNA with increasing amounts of MP (Fig. 6B
). Moreover, the mutant 3a
C33 MP again showed greater inhibition of infection at lower protein : RNA ratios than did the wt 3a MP, with infectivity reduced to about 50 % by a protein : RNA ratio of 15 : 1 for the mutant 3a
C33 MP vs 50 : 1 for the wt 3a MP (Fig. 6B
). When tobacco plants transgenic for the CMV CP were inoculated with the same proteinRNA complexes, TMV RNA complexed with either the mutant 3a
C33 MP or the wt 3a MP showed the same effects on inhibition of infection as shown in the non-transgenic tobacco plants (Fig. 6B
). These data indicate that the transgenically expressed CMV CP was not able to destabilize the proteinRNA complexes formed in vitro using either the wt 3a MP or the mutant 3a
C33 MPs.
Inhibition of translation of CMV RNA 3 in MPRNA complexes
To determine whether complexes formed between CMV RNAs and MPs were able to interfere with translation of the RNA, CMV RNA 3 was mixed with various amounts of either wt 3a MP or mutant 3aC33 MP and assayed for translatability. The CMV RNA used for these assays contained the gene encoding the GFP in place of the native 3a gene. Analysis of the translation products showed that at low ratios of MP : RNA there was no inhibition of translation (Fig. 7A and B
), while as the amount of MP increased (shown in Fig 7C and D
), translation of the GFP gene was inhibited (Fig. 7A and B
). Inhibition of translation by the wt 3a MP occurred over a broad range of MP : RNA ratios, starting between 20 : 1 and 30 : 1, with complete inhibition occurring at 60 : 1 (Fig. 7A
). By contrast, inhibition of translation by the mutant 3a
C33 MP occurred sharply between MP : RNA ratios of 10 : 1 and 20 : 1 (Fig. 7B
). Therefore, MPRNA complexes formed in vitro inhibited gene expression of the coated viral RNAs, and somewhat less MP was required for the mutant 3a
C33 MP than for the wt 3a MP to confer this inhibition.
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DISCUSSION |
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The wt 3a MP showed a distinctive, but repetitive pattern in the coating of the viral RNA, in which complexes appeared as beads on a thick string (Fig. 5A). There were also gaps present on the string, which allowed access to RNase giving only smaller protected fragments (Fig. 4A
). By contrast, the mutant 3a
C33 MP was able to interact with the viral RNA to produce several types of complexes, at both high and low ratios of protein : RNA. These included complexes that were thicker than those produced by the wt 3a MP, in which the MP appeared to have more layers, as well as complexes in which more of the RNA was exposed (Fig. 5B
). The former may also have led to more stable complexes (Fig. 4B
), as well as complexes that offered better protection against RNase (Fig. 4A
) and did not require complete coating of the viral RNA to affect inhibition of several processes. This is consistent with the observations that lower concentrations of the mutant MP were required to inhibit infection than for the wt MP (Fig. 6
).
TMV RNA complexed with TMV MP was found to be infectious on plants, but not translatable (Karpova et al., 1997). By contrast, the CMV MP was able to inhibit infection of either CMV RNAs or TMV RNA in plants (Fig. 6
), as well as translation (Fig. 7
), whereas the TMV MP also did not inhibit the infection of CMV RNAs in plants (Fig. 6A
). For TMV, this has been explained by the observation that at high TMV MP : RNA ratios, translation of TMV RNA was inhibited, but transfer of the associated RNA from one cell to another was not (Karpova et al., 1997
). At high TMV MP : RNA ratios, the complexes were found to adopt a thickened string architecture (Kiselyova et al., 2001
). In the case of the CMV MP, the thickened string structure only was observed for complexes involving the truncated 3a MP, and not the wt 3a MP (Figs 5B
vs 5A).
In contrast to the formation of the TMV beads-on-a-string complexes, in which binding of the TMV MP to RNA was done under non-cooperative-binding conditions (Kiselyova et al., 2001), CMV 3a MPRNA complexes were formed under cooperative interaction conditions (Fig. 3A
). Moreover, in contrast to the TMV complexes, in which the RNA was fully accessible to RNase (Kiselyova et al., 2001
), the CMV 3a MPRNA complexes were partially resistant to RNase, producing linear fragments 30 to 50 nm in length. The 3a
C33 MPRNA complexes produced similar RNase resistant fragments, as well as fragments with a length of approximately 120 nm. These two different sized fragments could have originated from structural elements described as resembling beads on a string and more densely packed beads, respectively, and may correspond to the two types of RNase-protected complexes observed in Fig. 4(A)
(lanes 3 and 4). Interestingly, the length of the former (30 to 50 nm) corresponds to two or three units of the uniform structure of the wt 3a MPRNA complexes of 15 nm periodicity (Fig. 5A
, panel 3). Such segments are separated presumably by short gaps of free RNA, accessible to RNase, but not resolvable by AFM.
Contrary to our initial expectations, the 3aC33 MP bound RNA more strongly than the wt 3a MP. During infection, the MP has to compete with the virus replicase, the CP and ribosomes for binding to the viral RNA. Thus, to promote virus movement, the CMV MP may need to bind RNA either very strongly, or with a higher affinity early during infection. It is conceivable that the CMV CP could alter the MP conformation to increase its binding efficiency. Thus, deleting the C terminus of the MP might have the same effect on the overall conformation as adding CP. This effect of the CP could not be mimicked in vivo after inoculation of pre-formed MPRNA complexes to tobacco plants expressing CP (Fig. 6B
). This might be because MP had already formed a complex, and the CP did not affect the stability of such complexes. Thus, we propose that the role of the CMV CP would not be to destabilize the MPRNA complexes already formed, as originally envisaged, but rather would be one of modifying the type of complexes initially formed, by altering the conformation of the MP available for complex formation. CP may also not interact directly with the MP, but via some host protein, which may explain the failure to detect direct interactions between MP and CP.
The studies of Nagano et al. (2001) showed that CMV containing the 3a
C33 MP was able to infect N. benthamiana systemically, in the presence but not in the absence of CP. Therefore, if the C terminus of the MP is not required for cell-to-cell or long-distance movement, why is it maintained? These sequences may have other host-dependent functions that are not obvious from the limited plant species so far inoculated. In addition, differences in the binding affinities between the wt 3a MP and 3a
C33 MP to RNA may have different consequences for infection in different plant backgrounds. Future studies should allow us to distinguish between such possibilities.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Canto, T. & Palukaitis, P. (1999a). The hypersensitive response to cucumber mosaic virus in Chenopodium amaranticolor requires virus movement outside the initially infected cell. Virology 265, 7482.[CrossRef][Medline]
Canto, T. & Palukaitis, P. (1999b). Are tubules generated by the 3a protein necessary for cucumber mosaic virus movement? Mol Plant Microbe Interact 12, 985993.
Canto, T., Prior, D. A. M., Hellwald, K.-H., Oparka, K. J. & Palukaitis, P. (1997). Characterization of cucumber mosaic virus. IV. Movement protein and coat protein are both essential for cell-to-cell movement of cucumber mosaic virus. Virology 237, 237248.[CrossRef][Medline]
Ding, S.-W., Li, W.-X. & Symons, R. H. (1995a). A novel naturally occurring hybrid gene encoded by a plant RNA virus facilitates long distance virus movement. EMBO J 14, 57625772.[Abstract]
Ding, B., Li, Q., Nguyen, L., Palukaitis, P. & Lucas, W. J. (1995b). Cucumber mosaic virus 3a protein potentiates cell-to-cell trafficking of CMV RNA in tobacco plants. Virology 207, 345353.[CrossRef][Medline]
Gal-On, A., Kaplan, I., Roossinck, M. J. & Palukaitis, P. (1994). The kinetics of infection of zucchini squash by cucumber mosaic virus indicate a function for RNA 1 in virus movement. Virology 205, 280289.[CrossRef][Medline]
Gal-On, A., Kaplan, I. & Palukaitis, P. (1995). Differential effects of satellite RNA on the accumulation of cucumber mosaic virus RNAs and their encoded proteins in tobacco vs zucchini squash with two strains of CMV helper virus. Virology 208, 5866.[CrossRef][Medline]
Habili, N. & Francki, R. I. B. (1974). Comparative studies of tomato aspermy and cucumber mosaic viruses. III. Further studies on relationship and construction of a virus from parts of the two viral genomes. Virology 61, 443449.[Medline]
Hellwald, K.-H. & Palukaitis, P. (1995). Viral RNA as a potential target for two independent mechanisms of replicase-mediated resistance against cucumber mosaic virus. Cell 83, 937946.[Medline]
Jacquemond, M., Teycheney, P.-Y., Carrère, I., Navas-Castillo, J. & Tepfer, M. (2001). Resistance phenotypes of transgenic tobacco plants expressing different cucumber mosaic virus (CMV) coat protein genes. Mol Breed 8, 8594.[CrossRef]
Kaplan, I. B., Shintaku, M. H., Li, Q., Zhang, L., Marsh, L. E. & Palukaitis, P. (1995). Complementation of virus movement in transgenic tobacco expressing the cucumber mosaic virus 3a gene. Virology 209, 188199.[CrossRef][Medline]
Kaplan, I. B., Gal-On, A. & Palukaitis, P. (1997). Characterization of cucumber mosaic virus. III. Localization of sequences in the movement protein controlling systemic infection in cucurbits. Virology 230, 343349.[CrossRef][Medline]
Kaplan, I. B., Zhang, L. & Palukaitis, P. (1998). Characterization of cucumber mosaic virus. V. Cell-to-cell movement requires capsid protein but not virions. Virology 246, 221231.[CrossRef][Medline]
Karpova, O. V., Ivanov, K. I., Rodionova, N. P., Dorokhov, Y. L. & Atabekov, J. G. (1997). Nontranslatability and dissimilar behavior in plants and protoplasts of viral RNA and movement protein complexes formed in vitro. Virology 230, 1121.[CrossRef][Medline]
Kiselyova, O. I., Yaminsky, I. V., Karger, E. M., Frolova, O. Y., Dorokhov, Y. L. & Atabekov, J. G. (2001). Visualization by atomic force microscopy of tobacco mosaic virus movement proteinRNA complexes formed in vitro. J Gen Virol 82, 15031508.
Lakshman, D. K. & Gonsalves, D. (1985). Genetic analysis of two large-lesion isolates of cucumber mosaic virus. Phytopathology 75, 758762.
Li, Q. & Palukaitis, P. (1996). Comparison of the nucleic acid- and NTP-binding properties of the movement protein of cucumber mosaic cucumovirus and tobacco mosaic tobamovirus. Virology 216, 7179.[CrossRef][Medline]
Li, Q., Ryu, K. H. & Palukaitis, P. (2001). Cucumber mosaic virusplant interactions: identification of 3a protein sequences affecting infectivity, cell-to-cell movement, and long-distance movement. Mol Plant Microbe Interact 14, 378385.[Medline]
Lyubchenko, Y. L., Oden, P. I., Lampner, D., Lindsay, S. M. & Dunker, K. A. (1993a). Atomic force microscopy of DNA and bacteriophage in air, water and propanol: the role of adhesion forces. Nucleic Acids Res 21, 11171123.[Abstract]
Lyubchenko, Y. L., Shiyakhtenko, L., Harrington, R., Oden, P. & Lindsay, S. (1993b). Atomic force microscopy of long DNA: imaging in air and under water. Proc Natl Acad Sci U S A 90, 21372140.[Abstract]
Nagano, H., Okuno, T., Mise, K. & Furusawa, I. (1997). Deletion of the C-terminal 33 amino acids of cucumber mosaic virus movement protein enables a chimeric brome mosaic virus to move from cell to cell. J Virol 71, 22702276.[Abstract]
Nagano, H., Mise, K., Furusawa, I. & Okuno, T. (2001). Conversion in the requirement of coat protein in cell-to-cell movement mediated by the cucumber mosaic virus movement protein. J Virol 75, 80458053.
Nurkiyanova, K. M., Ryabov, E. V., Kalinina, N. O., Fan, Y., Andreev, I., Fitzgerald, A. G., Palukaitis, P. & Taliansky, M. (2001). Umbravirus-encoded movement protein induces tubule formation on the surface of protoplasts and binds RNA incompletely and non-cooperatively. J Gen Virol 82, 25792588.
Rao, A. L. N. & Francki, R. I. B. (1981). Comparative studies on tomato aspermy and cucumber mosaic viruses. VI. Partial compatibility of genome segments for the two viruses. Virology 114, 573575.
Rizzo, T. M. & Palukaitis, P. (1990). Construction of full-length cDNA clones of cucumber mosaic virus RNAs 1, 2 and 3: generation of infectious RNA transcripts. Mol Gen Genet 222, 249256.[Medline]
Ryabov, E. V., Roberts, I. M., Palukaitis, P. & Taliansky, M. (1999). Host-specific cell-to-cell and long-distance movements of cucumber mosaic virus are facilitated by the movement protein of groundnut rosette virus. Virology 260, 98108.[CrossRef][Medline]
Ryu, K. H., Kim, C.-H. & Palukaitis, P. (1998). The coat protein of cucumber mosaic virus is a host range determinant for infection of maize. Mol Plant Microbe Interact 11, 351357.
Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Suzuki, M., Kuwata, S., Kataoka, J., Masuta, C., Nitta, N. & Takanami, Y. (1991). Functional analysis of deletion mutants of cucumber mosaic virus RNA3 using an in vitro transcription system. Virology 183, 106113.[Medline]
Takahashi, H., Suzuki, M., Natsuaki, K., Shigyo, T., Hino, K., Teraoka, T., Hosokawa, D. & Ehara, Y. (2001). Mapping the virus and host genes involved in the resistance response in cucumber mosaic virus-infected Arabidopsis thaliana. Plant Cell Physiol 42, 340347.
Takeshita, M., Suzuki, M. & Takanami, Y. (2001). Combination of amino acids in the 3a protein and the coat protein of cucumber mosaic virus determines symptom expression and viral spread in bottle gourd. Arch Virol 146, 697711.[CrossRef][Medline]
Vaquero, C., Liao, Y.-C., Nähring, J. & Fischer, R. (1997). Mapping of the RNA-binding domain of the cucumber mosaic virus movement protein. J Gen Virol 78, 20952099.[Abstract]
Wong, S., Thio, S. S., Shintaku, M. H. & Palukaitis, P. (1999). The rate of cell-to-cell movement in squash of cucumber mosaic virus is affected by sequences of the capsid protein. Mol Plant Microbe Interact 12, 628632.
Zhang, L., Hanada, K. & Palukaitis, P. (1994). Mapping local and systemic symptom determinants of cucumber mosaic cucumovirus in tobacco. J Gen Virol 75, 31853191.[Abstract]
Received 19 August 2003;
accepted 10 October 2003.