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1 Department of Virology and A. N. Belozersky Institute of Physico-Chemical Biology, Faculty of Chemistry of Moscow State University, Moscow 119992, Russia
2 Faculty of Physics, Faculty of Chemistry of Moscow State University, Moscow 119992, Russia
Correspondence
J. G. Atabekov
atabekov{at}genebee.msu.su
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). There are 8.9 subunits per turn of primary helix. The PVX RNA contains five genes (Huisman et al., 1988; Skryabin et al., 1988). The 5'-proximal gene encodes the 165 kDa replicase; the 3'-proximal coat protein (CP) gene is preceded by three partially overlapping genes termed the ‘triple-gene block’ (TGB) encoding three movement proteins (MPs) referred to as TGBp1, TGBp2 and TGBp3, respectively. The TGB-encoded MPs, together with CP, are involved in cell-to-cell movement of PVX (Chapman et al., 1992; Verchot et al., 1998; reviewed by Batten et al., 2003; Morozov & Solovyev, 2003; Verchot-Lubicz, 2005). TGBp2 and TGBp3 are membrane-binding proteins; however, there also is evidence that, in PVX infection, they may interact with TGBp1, CP or certain cellular proteins (Yang et al., 2000; Ju et al., 2005). The 25 kDa TGBp1 is multifunctional protein that has RNA helicase activity and a moderate RNA-binding activity (Kalinina et al., 1996, 2002; Morozov et al., 1999), increases the plasmodesmal (PD) size-excluding limit (Howard et al., 2004; Yang et al., 2000) and suppresses RNA silencing (Lough et al., 2001; Voinnet et al., 2000). The RNA-binding activity of PVX TGBp1 is dramatically lower than that of the tobacco mosaic virus (TMV) MP (Karpova et al., 1997).
The 25 kDa CP of PVX exists as a 1.8S monomer in the presence of disaggregating agents (Dementjeva et al., 1970; Miki & Knight, 1968). After removal of the disaggregating agent, the 3–5S aggregate was a major component of PVX protein preparations and a minor, 10–15S component was also revealed (Dementjeva et al., 1970). Further aggregation of protein with production of a limited number of single- and double-layered discs and short stacks of discs was shown by electron microscopy (EM) and optical-diffraction patterns (Kaftanova et al., 1975). All attempts to repolymerize PVX CP into long, helical particles have heretofore failed.
Different models have been proposed for the nature of the infectious potexvirus transport form that moves from cell to cell. It has been suggested (Allison & Shalla, 1974; Oparka et al., 1996; Santa Cruz et al., 1998) that filamentous virions are involved in cell-to-cell movement of PVX. On the other hand, it has been reported that in vitro-assembled non-virion RNP (CP–RNA–TGBp1) complexes move from cell to cell in microinjection experiments in vivo (Lough et al., 1998) and that RNA encapsidation is not needed for potexvirus spread (Lough et al., 2000). It should be noted, however, that the exact structure of these RNPs has not yet been studied. Here, we examined the conditions of assembly and the structure of complexes assembled in vitro from PVX RNA, TGBp1 and CP. Single-tailed particles (STPs) with the 5'-proximal region of PVX RNA encapsidated in a helical, head-like structure and TGBp1 bound to the end of the head were revealed.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) was used. RNA was isolated with phenol and analysed by 1 % agarose-gel electrophoresis; bacterially expressed (His)6-TGBp1 MP was isolated as described previously (Karpova et al., 1997).
RNA–protein and protein–protein incubations.
The incubations were performed in 10–20 µl 10 mM Tris/HCl, pH 7.6, at 20 °C. The molar RNA : TGBp1, RNA : CP and TGBp1 : CP ratios and time used in separate experiments are indicated in the figure legends. In separate experiments, the following reagents were added to incubation mixtures singly or together: 1 mM EDTA, 5 mM MgCl2, 1 mM dithiothreitol, 150 mM NaCl. The results of experiments did not depend on addition of the reagents listed.
In vitro translation.
Cell-free translation in wheatgerm extract (WGE) was carried out as described previously (Rodionova et al., 2003) in the presence of [35S]methionine for 60 min at 25 °C. The concentration of RNA was 40 µg ml–1.
EM.
Speciments were prepared and examined as reported previously (Kaftanova et al., 1975). The assembled material was subjected to differential centrifugation [2 h at 100 000 g; the pellets were resuspended in 0.01 M Tris/HCl (pH 7.5) and clarified at 1300 g for 10 min] before staining with uranyl acetate or shadowing with platinum.
Immunoelectron microscopy (IEM).
The single-tailed complexes were adsorbed onto Formvar film attached to 200-mesh nickel EM grids. Speciments were blocked in 1 % BSA in PBS for 20 min and floated on 0.1 µg TGBp1 µl–1 for 20 min. Grids were washed with PBS, incubated with polyclonal antibodies to TGBp1 (AbTGBp1) and then with secondary gold-conjugated antibodies (AbSEC). The size of gold particles was 10 nm. After immunolabelling, the grids were washed with distilled water and air-dried. Finally, the specimens were rotary-shadowed with platinum. In control (lacking TGBp1) mixtures, the labelling of complexes with AbSEC was entirely absent.
Atomic-force microscopy (AFM).
AFM measurements were carried out by using a NanoScope IIIa multimode-scanning probe microscope (Digital Instruments) in tapping mode in air, as described previously (Kiselyova et al., 2001, 2003). AFM imaging in air (rather than in liquid) was used, as our previous data showed that, with the sample-preparation technique used (Kiselyova et al., 2003), no difference in AFM images of PVX particles could be observed, irrespective of whether they were imaged in air or in liquid. Tapping-mode imaging was performed with tapping mode-etched silicon cantilevers (Nanosensors), with a resonant frequency of 300–350 kHz and a length of 125 µm. Image processing was performed with the help of FemtoScan SPM image-processing software, Advanced Technologies Center, Moscow, Russia (http://www.nanoscopy.net). The substrates for imaging (APS–mica) were freshly prepared immediately before the experiments by treating freshly cleaved mica with aminopropylsilatran solution as described by Shlyakhtenko et al. (1998). Sample solution (a 5 µl droplet) was applied onto the substrate, left for 5 min for adsorption, rinsed with tridistilled water and dried in airflow at room temperature.
RT-PCR.
RT-PCR was done according to the manufacturer's protocol (Promega) with some modifications (Rodionova et al., 2003).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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(lanes 5–8) shows that the complexes contained both CP and TGBp1, revealed with antibodies to CP (Fig. 1a) and TGBp1 (Fig. 1b), respectively. These data indicated that, in the absence of RNA, interaction of the low-molecular-mass form of PVX CP (presumably, the 3–5S aggregates or/and monomers) with TGBp1 led to insoluble CP–TGBp1 complex formation.
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, lane 3) or, otherwise, PVX RNA was preincubated with TGBp1 and then CP was added (Fig. 1, lane 4). These data provided initial evidence that the production of soluble TGBp1–RNA–CP complexes is mediated by viral RNA involved in the assembly. Furthermore, the assembly of soluble triple complexes was examined more closely in two types of experiments.
Firstly, the particles presented in Figs 2(a) and 3(a–c) were assembled when PVX RNA was preincubated with TGBp1 and CP was then added in excess over TGBp1. The STPs revealed contained head-like structures of varying length (Fig. 2b) located at only one end of the RNA. Apparently, the RNA tails of STPs presented in Fig. 2(a) resemble the thick, highly condensed TMV RNA fibres described by Kuznetsov et al. (2005). In many cases, the RNA tail did not exist as an extended thread, but appeared as a random, disorganized coil (Fig. 3a, b). Occasionally, in electron micrographs of specimens shadowed with platinum, the RNA strand appeared as a long, extended chain (Fig. 3c). In control experiments, EM examination showed that RNase treatment removed the tails from STPs. The height of heads measured by AFM corresponded to that of native PVX virions (13.5 nm), independently of the head length. The helical structure of the rod-like heads could not be resolved by means of AFM; however, it was visualized on electron micrographs with negative staining (Fig. 3e).
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Secondly, likewise, STPs were produced when RNA and CP, taken at various molar ratios, were preincubated and TGBp1 was then added. Furthermore, the possibility of STP assembly was examined in a series of experiments where viral RNA was mixed with CP or TGBp1 and the second protein was then added without preincubation: (i) PVX CP was added to RNA and TGBp1 was then added immediately or, otherwise, (ii) PVX TGBp1 was first added to RNA and, immediately afterwards, the CP was added. It is particularly noteworthy that no structural differences were revealed between the STPs produced under the conditions of any type of experiment described above. Consequently, production of the STP heads occurs rapidly and is not inhibited by the presence of TGBp1.
It was also important that no RNA–TGBp1 complexes could be revealed by AFM when viral RNA and TGBp1 were incubated at the molar RNA : TGBp1 ratios of 1 : 100 and even as high as 1 : 2000. Presumably, stability of the PVX RNA–TGBp1 complexes was rather low and the TGBp1 molecules were bound loosely to PVX RNA (see Discussion).
IEM location of TGBp1 in STPs
IEM was used to visualize the location of TGBp1 in STPs. Fig. 4 shows that TGBp1 molecules were located at the extremity of the helical heads of single-tailed TGBp1–RNA–CP complexes. In a control mixture lacking TGBp1, the labelling of complexes with secondary gold-conjugated antibodies was entirely absent (data not shown). The same was the case in a control mixture lacking antibodies to TGBp1. Not unexpectedly, the RNA tails were not resolved by IEM in Fig. 4.
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). Assuming that 45 nt are bound to each turn of primary PVX CP helix with a pitch of 3.6 nm (8.9 protein subunits per turn of helix, and 5 nt associated with each subunit), PVX RNA fragments about 300, 600 and 2000 nt in length should be incapsidated in STPs. To obtain the PVX RNA fragments coated with CP within TGBp1-free STPs, the preparations were treated with micrococcus RNase (MR) under conditions when the encapsidated part of the RNA was protected from MR attack. After MR treatment, the RNase-inaccessible RNA was isolated and characterized. It was found that RNAs isolated from the helical heads of STPs assembled at the RNA : CP ratio of 1 : 700 produced three bands on agarose-gel electrophoresis, corresponding to 400–500, 700–800 and about 2000 nt (Fig. 5a). Translation in WGE of RNAs protected from MR resulted in the production of a heterogeneous set of polypeptides (Fig. 5b, lane 4). The absence of the full-size 165 kDa replicase encoded by the 5'-proximal gene of PVX RNA indicated that long, 5'-terminal fragments were absent from these preparations. It was reasonable to suggest that polypeptides encoded by RNA fragments protected in STPs from MR attack represented the N-coterminal, C-truncated portions of the 165 kDa replicase.
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, lanes 2 and 5). It was also shown (negative control) that translation of heterologous (TMV) RNA was not arrested by this oligodeoxyribonucleotide (Fig. 5b, lanes 6–7). These results suggested that MR-inaccessible RNA from STPs represented the 5'-terminal fragments of the PVX genome.
Furthermore, RT-PCR was used to demonstrate that the 5'-terminal fragments of PVX RNA were encapsidated in STPs. Fig. 5(c, lanes 1 and 2) shows that RT-PCR products corresponding only to 5'-terminal fragments of PVX RNA (lane 2) could be revealed in particles reassembled at the ratio of 1 : 350. No amplification products representing the 3'-terminal domain of PVX RNA were observed (lane 1). Likewise, the overwhelming majority of RT-PCR amplification products formed with RNA encapsidated in STPs assembled at the ratios of 1 : 700 and 1 : 2100 corresponded to the 5'-terminal domain of PVX RNA (Fig. 5c, lanes 4 and 6, respectively). Only negligible amounts of the amplification products corresponding to the 3'-terminal domain of PVX RNA could be detected in the particles assembled at RNA : CP ratios of 1 : 700 (lane 3) and 1 : 2100 (lane 5). Taken together, these data indicate that the 5'-terminal regions of viral RNA were encapsidated selectively in the heads of STPs.
TGBp1-triggered conversion of STPs from a non-translatable into a translatable form
It could be presumed that even partial encapsidation of PVX RNA within STPs prevents it from translation and replication in the case that the 5'-terminal region of RNA is coated with CP. In a series of experiments, the translatability of STPs in WGE was studied (Fig. 6). Three control samples were used to check that (i) encapsidated RNA of native PVX was non-translatable in vitro (lane 1), but (ii) could be translationally activated by TGBp1 (lane 2), and that (iii) no inhibition of protein-free PVX RNA translation was caused by TGBp1 (lane 4). Fig. 6 shows that translatability of PVX RNA was reduced after incubation with the CP at the molar RNA : CP ratio of 1 : 700 (lane 5). The translation level observed in this case (lane 5) can be attributed to the fact that a minor proportion of PVX RNA molecules remained free of CP and, therefore, were translatable. On the other hand, translation was abolished completely after RNA : CP incubation at the molar ratio of 1 : 1500 (Fig. 6, lane 7), but was restored fully by TGBp1 addition (cf. lanes 5–6 and 7–8). Translatability of RNA was retained in experiments where viral RNA was preincubated with TGBp1 and then the CP was added to the incubation mixture (lanes 9–10). This was in line with observations that TGBp1 did not prevent viral RNA from translation (Fig. 6, lane 4) and that assembly of STPs occurred at TGBp1 excess.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Oparka et al., 1996; Santa Cruz et al., 1998) or a particular type of RNP complex different from native virions (Lough et al., 1998, 2000). Microinjection studies provided evidence that in vitro-assembled non-virion RNPs consisting of potexvirus RNA, CP and TGBp1 moved from cell to cell (Lough et al., 1998, 2000). Thus, the preparations assembled in vitro from potexvirus RNA, CP and TGBp1 contained infectious material that translocated from cell to cell. The authors concluded that RNA encapsidation was not needed for potexvirus spread and proposed that non-virion (CP–RNA–TGBp1) RNP complexes assembled in vitro moved from cell to cell in vivo. Unfortunately, the exact structure of PVX RNA–CP–TGBp1 complexes is not yet known. Here, we examined the conditions of assembly and the structure of complexes assembled in vitro from PVX RNA, TGBp1 and CP. Remarkably, no RNP complexes with non-virion structure were found; the STPs that were readily revealed contained head-like structures of varying length, located at only one end of the RNA.
TGBp1 forms soluble complexes with helically assembled CP
In most PVX hosts, TGBp1 is the primary viral protein to increase PD permeability (reviewed by Howard et al., 2004; Verchot-Lubicz, 2005). TGBp1 can move from cell to cell in the absence of other PVX-encoded proteins (Howard et al., 2004; Yang et al., 2000). In contrast, the CP accumulates in PD of infected cells, but does not gate PD (Oparka et al., 1996). Our data showed that incubation of TGBp1 with disaggregated PVX CP invariably resulted in insoluble TGBp1–CP agglomerate production. Therefore, it was hardly possible that TGBp1 could interact directly with unassembled PVX CP upon ternary (TGBp1–RNA–CP) complex formation. In contrast, TGBp1 readily formed soluble complexes with helically assembled CP of native PVX virions and of artificial, polar, helical PVX-like particles (PVXRNA-DEG) lacking intact RNA (Rodionova et al., 2003). In addition, the IEM data presented here show that TGBp1 binds to one end of the STP heads. It is not unlikely that direct interaction of unassembled CP and TGBp1 does not occur in vivo. However, biolistic bombardment experiments showed that PVX TGBp1 was restricted to single cells in CP-transgenic tobacco lines (Yang et al., 2000), which could indicate that, under these conditions, inhibition of TGBp1 trafficking was due to intracellular formation of non-functional (perhaps insoluble) CP–TGBp1 complexes similar to those formed in vitro. Conceivably, the CP moves from cell to cell only when it is part of a helical RNP complex (e.g. STP or PVX virion) associated with TGBp1 and, presumably, some cellular proteins.
Structural similarity between native virions and STPs
It is obvious that STPs represent the products of incomplete PVX reconstitution. PVX was the first flexuous plant virus reassembled in vitro from RNA and CP (Goodman et al., 1975; Novikov et al., 1972; Kaftanova et al., 1975). Remarkably, a great majority of the reassembled particles were shorter than native PVX and, presumably, represented STPs described here. In accordance with this, the results of AFM and EM, as well as those presented in Figs 2(b), 5(a, lanes 4–6) and 5(c, lanes 3 and 5) indicate that no or only a negligible amount of full-length virions was produced. Analysis of optical-diffraction patterns of electron micrographs showed that the helical structure of reconstituted PVX preparations (that obviously contained STPs in large excess) was identical to that of native PVX (Kaftanova et al., 1975).
Under examination by EM and AFM, the structures of STP heads containing and lacking TGBp1 were indistinguishable from each other and from that of native PVX. The diameter of particles determined by EM and AFM was in good agreement (about 13.5 nm). It is well known that, in AFM images, horizontal dimensions of objects are usually overestimated due to the effect of the geometry of the AFM tip, which has finite dimensions. As a consequence, the objects appear broader than their real dimensions (Kuznetsov et al., 2005). Therefore, the fine structure of the PVX CP helix composed of individual 25 kDa subunits was not resolved by AFM. Likewise, the presence of the 25 kDa TGBp1 molecules bound to one end of STPs could not be revealed on AFM images. Therefore, IEM was used in this work to resolve the TGBp1 in STPs.
Our previous data indicated that binding of TGBp1 to a native PVX virion resulted in a linear destabilization of the whole helical structure, suggesting that intersubunit conformational changes could be transferred from one end of a particle along the CP helix (Kiselyova et al., 2003; Rodionova et al., 2003). No TGBp1-induced changes in the geometry were detected in EM or AFM images of PVX particles containing or lacking TGBp1. Several lines of evidence suggest that the STP heads are structurally similar or identical to the CP helix of PVX. (i) Contrary to some plant viruses, encapsidated PVX RNA is completely non-translatable in vitro; however, a selective binding of PVX TGBp1 to one end of a polar CP helix converted viral RNA into a translatable form (Atabekov et al., 2000). Similarly, translation of PVX RNA was repressed within RNA–CP STPs, and STPs could be rendered translatable by a selective binding of TGBp1 to the end of the helical head. (ii) Moreover, TGBp1 bound specifically to the end of artificial, polar, helical PVX-like particles (PVXRNA-DEG) lacking intact RNA (Rodionova et al., 2003), i.e. TGBp1 molecules were capable of recognizing the ends of the polar CP helix of native PVX, PVXRNA-DEG and STPs. Apparently, the ability of TGBp1 to selectively bind the terminal protein subunits at one end in each of the three type of particles requires a structural similarity in their CP helices. (iii) Morphological similarity could be revealed by EM of negatively stained native PVX (Fig. 3d) and in vitro-assembled head (Fig. 3e). (iv) The so-called trypsin degradation test (TDT) can be applied to PVX to demonstrate the native state of the virion helix. Helically assembled PVX CP is relatively resistant to trypsin (Koenig et al., 1970). Detection after trypsin treatment of the full-size CP form (CP–Ps, about 29 kDa) and/or of the N-terminally truncated CP–Ptd form (25 kDa) in SDS-PAGE indicated that PVX CP was in a trypsin-resistant, i.e. helically assembled, state. In contrast, disappearance of the CP band from SDS-PAGE after trypsin treatment indicated that PVX CP was in a disassembled (trypsin-sensitive) form. When PVX and STPs were compared by TDT in our experiments, it appeared that the STP heads existed in an assembled form (data not shown). Collectively, these results provided strong evidence on similarity between the structure of native virions and STP heads.
RNA-mediated STP assembly as a two-step process
Production of insoluble TGBp1–CP complexes was abolished or inhibited severely in the presence of RNA. Fig. 1 (lanes 3–4) shows that, in the presence of RNA, no insoluble CP–TGBp1 aggregates were formed. Apparently this was due to highly efficient utilization of the CP for STP assembly, i.e. the PVX CP interacted efficiently with RNA, producing soluble STPs. Thereafter, no insoluble CP–TGBp1 complexes were produced, even in an excess of TGBp1. It should be mentioned that assembly of STP heads occurred quickly when no preincubation of RNA–CP preceded TGBp1 addition and was not inhibited in the opposite situation when TGBp1 was added to RNA before the CP and immediately after the CP was added to the mixture. In other words, assembly of heads was not inhibited by the presence of TGBp1. Thus, the PVX CP competes very efficiently for RNA in the mixture with TGBp1. Remarkably, the complexes formed under either set of conditions of assembly were indistinguishable and represented STPs with rod-like heads of varying length (Fig. 2b), located at only one end of the RNA molecule (Figs 2a, 3a–c). The TGBp1 molecules bound to the terminal subunits of the head were revealed by IEM (Fig. 4).
It has been shown (AbouHaidar & Erickson, 1985; Lok & AbouHaidar, 1986; Sit et al., 1994) that reassembly of Papaya mosaic virus starts at or near the 5' end of the RNA. Recently, it was reported (Kwon et al., 2005) that the CP-binding element is located within the stem–loop region of the 5' non-translated sequence of PVX RNA. Here, we demonstrated that the 5'-terminal region of PVX RNA was encapsidated selectively in single-tailed RNA–CP particles lacking TGBp1 (Fig. 5b, c). Fast assembly of STPs was apparently due to high affinity of PVX CP for the 5'-proximal RNA region. In contrast, TGBp1–RNA binding was not efficient. The latter suggestion is supported by several lines of evidence: (i) no TGBp1–RNA binding could be revealed by using double-filter nitrocellulose-membrane filter-binding assays (Karpova et al., 1997); (ii) an indirect test for MP binding was based on the ability of the MP to block viral RNA translation in vitro. Several plant virus MPs were shown to act as efficient translational suppressors, including TMV 30 kDa, barley stripe mosaic virus TGBp1 MPs (Karpova et al., 1997) and cucumber mosaic virus 3a (Kim et al., 2004). In contrast, PVX TGBp1 did not inhibit in vitro translation of viral RNA (Karpova et al., 1997) (see also Fig. 6, lane 4). (iii) PVX TGBp1–RNA binding could be detected by North-Western binding assays (Kalinina et al., 1996; Morozov et al., 1999); however, the salt stability of the PVX TGBp1–RNA complexes was significantly lower than those formed by TGBp1 MPs of other TGB-containing viruses; (iv) finally, we failed to reveal complexes of the PVX RNA with TGBp1, whereas the structure of complexes formed under the same AFM conditions by the 30 kDa TMV MP and RNA were readily detected and examined (Kiselyova et al., 2001). Likewise, the complexes of RNA with the cucumber mosaic virus 3a MP were detected by AFM (Kim et al., 2004; Nurkiyanova et al., 2001).
Taken together, these observations lead one to conceive the production of ‘CP–RNA–TGBp1’ STPs as a two-step process. At the first step, a fast assembly of the TGBp1-lacking single-tailed RNA–CP particles with the helical heads located at the 5' end of PVX RNA occurs. This is due to the great affinity of the CP for the 5' region of PVX RNA. At the second step, the TGBp1 molecules bind to the end of the helical head of STPs.
The data presented propose a compromise between the two models for potexvirus movement discussed above. We hypothesize that: (i) PVX CP moves from cell to cell only when it is part of a complex with RNA and TGBp1; (ii) the transport form used in PVX infection should be translatable to induce infection in healthy cells; and (iii) translatable complexes of TGBp1 with the extremity of the STP CP helix or/and of PVX virions may represent the transport form of viral infection.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 1 March 2006;
accepted 20 April 2006.
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