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Department of Plant Pathology, University of Kentucky, Lexington, KY 40546, USA
Correspondence
Michael M. Goodin
mgoodin{at}uky.edu
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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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; Reed et al., 2005; Revill et al., 2005; Tsai et al., 2005; Dietzgen et al., 2006).
Nucleorhabdoviruses share many of the structural features of animal rhabdoviruses such as vesicular stomatitis virus (VSV; Jackson et al., 2005). They are consequently composed of an infectious nucleocapsid ‘core’ surrounded by a phospholipid membrane. The core can be purified by density-gradient centrifugation of non-ionic detergent-treated virions (Wagner et al., 1996). In the case of Sonchus yellow net virus (SYNV), the core is a ribonucleoprotein (RNP) complex that consists of the negative-strand genomic RNA (Jackson & Christie, 1977) encapsidated by three associated proteins, namely the nucleocapsid (N), phospho- (P) and polymerase (L) proteins (Heaton et al., 1987; Zuidema et al., 1987; Choi et al., 1992). The membrane fraction of mature virions contains a glycoprotein (G) that protrudes from the surface of the virion (Goldberg et al., 1991). A sixth protein, sc4, which localizes to the periphery of cells, may play a role in virus cell-to-cell movement (Melcher, 2000; Goodin et al., 2002; Huang et al., 2005). The matrix (M) protein (Hillman et al., 1990) is believed to associate with G, presumably condensing the core during virion maturation (Jayakar et al., 2004; Jackson et al., 2005). Electron microscopy (EM) studies suggest that, during morphogenesis, the condensed cores acquire the G protein and a host-derived lipid envelope as they bud through the inner nuclear membrane (INM) and accumulate as mature particles in the perinuclear space (van Beek et al., 1985; Martins et al., 1998). The relationship between the sites of nucleocapsid assembly and viral morphogenesis has not been determined definitively for plant-adapted rhabdoviruses. However, biochemical characterization of purified cores and virus suggests that SYNV is structurally similar to the Indiana strain of VSV, for which the estimated numbers of molecules per infectious virus particle are: N (1000–2000); P (100–300); M (1500–4000); G (500–1500); L (20–50) (Tordo et al., 2005). For the cognate proteins of SYNV, this represents roughly a 10 : 1 molar ratio of N : P within particles. In contrast, purified complexes have been shown to be equimolar or in a 2 : 1 ratio with respect to the N and P proteins of VSV (Masters & Banerjee, 1988). Results from purification of N–P complexes of SYNV are consistent with these data (Goodin et al., 2001; R. Wang & M. M. Goodin, unpublished data). According to current models for the assembly of VSV, the excess P is removed during maturation of the nucleocapsid (Green et al., 2000, 2006).
In addition to coordination of the activities of viroplasm-associated proteins, infection of plants with members of the genus Nucleorhabdovirus results in marked alterations in nuclear membranes (Martins et al., 1998; Goodin et al., 2005). In the case of SYNV, there is an invagination of the INM into the nucleus. Thus, single membranes surround sites at which virions accumulate (Martins et al., 1998). These alterations of nuclear membranes can be observed by live-cell imaging of rhabdovirus-infected Nicotiana benthamiana ‘16c’ plants, which express endomembrane-targeted green fluorescent protein (GFP) (hereafter referred to as mGFP5-ER plants; Haseloff et al., 1997; Brigneti et al., 1998; Goodin et al., 2005). However, it was unclear from our initial study (Goodin et al., 2005), as well as previous EM studies (Martins et al., 1998), whether the membrane-bound sites of virion accumulation remain contiguous with the endomembrane system. Determination of this relationship profoundly impacts proposed models for rhabdoviral morphogenesis and systemic movement (Jackson et al., 2005). If the intranuclear membranes are not contiguous with the endomembrane system, then virion maturation may be a terminal process in plants that does not contribute to systemic movement of these viruses. Alternatively, if the intranuclear membranes are contiguous with the endomembrane system, then mature or partially budded virions may participate in cell-to-cell movement by associating with components of the endomembrane system, which are contiguous with desmotubules that pass through plasmodesmata connecting adjacent cells (Scholthof, 2005; Lucas, 2006). The hypothesis that the endomembrane system of a host cell may play a role in rhabdovirus cell-to-cell movement comes from studies that show the presence of mature particles of maize mosaic virus (MMV) in endoplasmic reticulum (ER) tubules in cells of its insect vector (Herold & Munz, 1965). Experimental support for either of the models described above requires the characterization of the virus-induced intranuclear membranes, as well as determination of their relationship to sites of SYNV protein accumulation. Therefore, we conducted experiments using fluorescence recovery after photobleaching (FRAP) and total internal reflection fluorescence microscopy (TIRFM). Given that mGFP5-ER plants accumulate soluble GFP in the lumen of the ER and nuclear membranes (Brigneti et al., 1998; Ruiz et al., 1998; Turner et al., 2004; Goodin et al., 2005), we reasoned that FRAP should be rapid if virus-induced nuclear membranes are contiguous with the ER. Alternatively, if the sites of virus accumulation are completely membrane-bound, then no FRAP should occur, as GFP is not membrane-permeable (Collings et al., 2000; Sbalzarini et al., 2005). Moreover, the biological relevance of the rhabdovirus-induced intranuclear membranes in infected mGFP5-ER plants would be enhanced if their linkage with the viroplasm could be determined. We investigated the relationship between intranuclear membranes and viroplasm by transiently expressing autofluorescent protein (AFP) fusions of the SYNV N, P, sc4, M and G proteins in virus-infected cells.
In addition to integrating localization data for SYNV-encoded proteins into models for rhabdovirus assembly and morphogenesis, our data underscore the importance of conducting protein-localization studies in the context of viral infection.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) were conducted as described previously (Goodin et al., 2005; Senthil et al., 2005). Note that mGFP5-ER is targeted to the ER via an amino-terminal signal sequence derived from the Arabidopsis thaliana basic chitinase and a carboxy-terminal HDEL ER-retention signal. Because of the contiguity of the outer nuclear membrane with the ER, GFP–HDEL also accumulates in the lumen of the nuclear envelope (Haseloff et al., 1997; Collings et al., 2000).
Construction of binary vectors for expression of fluorescent protein fusions in plant cells.
Binary vectors employed in this study for transient expression of AFP fusions in plant cells were derivatives of the pSAT series described by Tzfira et al. (2005) and Chung et al. (2005). Following confirmation by PCR screening, recombinant binary vectors were transformed into Agrobacterium tumefaciens as described previously (Goodin et al., 2002), except that strain LBA4404 was used instead of C58C1. Primers for PCR were designed according to the SYNV sequences deposited into GenBank. The GenBank accession numbers for each SYNV gene were L32603
[GenBank]
(G, N), AY971951
[GenBank]
(P), M35689
[GenBank]
(M), L32604
[GenBank]
(sc4) and M87829
[GenBank]
(L). The construction of correct in-frame fusions between AFP and SYNV genes was validated by DNA sequencing and immunodetection (data not shown).
Transient expression of proteins in plant cells by using agroinfiltration.
Suspensions of A. tumefaciens were infiltrated into leaves of N. benthamiana as described previously (Goodin et al., 2002; Tsai et al., 2005). In order to express proteins in SYNV-infected cells, symptomatic leaves of plants were infiltrated at the peak of symptom expression, typically 14 days post-inoculation (p.i.). Leaves were examined by confocal microscopy after incubation for 48 h under constant illumination.
In order to mark the nuclear envelope, we expressed GFP fused to the first 238 aa of the human lamin B receptor (LBR), as described by Irons et al. (2003).
Preparation of protoplasts.
Protoplasts were prepared from mock-inoculated or virus-infected leaves of mGFP5-ER plants essentially as described by Panaviene et al. (2003). Prior to preparation of protoplasts, leaf samples were examined by epifluorescence microscopy to confirm the presence of virus-induced intranuclear GFP (Goodin et al., 2005). Protoplasts were used immediately for microscopy or kept in 10x35 mm Petri plates in protoplast culture medium (Panaviene et al., 2003) at room temperature in the dark for up to 24 h.
TIRFM.
TIRFM was conducted by using a Nikon TE2000E inverted microscope equipped with CFI Plan ApoTIRF 60x-NA1.45 and CFI Plan ApoTIRF 100x-NA1.45 oil-immersion objectives. Excitation of GFP was accomplished by using the 488 nm line of a multi-line argon laser. Controlling software for image acquisition was METAMORPH version 6.2 (Molecular Devices Corporation).
Laser-scanning confocal microscopy.
All microscopy was performed on an Olympus FV1000 laser-scanning confocal microscope. Cyan fluorescent protein (CFP), GFP and red fluorescent protein (RFP) were excited by using 440, 488 and 543 nm laser lines, respectively. When using multiple fluors simultaneously, images were acquired sequentially, line by line, in order to reduce excitation and emission crosstalk. The primary objective used was an Olympus water immersion PLAPO60xWLSM-NA1.0 (hereafter referred to as the x60 objective). Image acquisition was conducted at a resolution of 512x512 pixels and a scan rate of 10 µs pixel–1, except where noted. Control of the microscope and image acquisition and export as TIFF files were conducted by using Olympus Fluoview software version 1.5. Exposure settings that minimized oversaturated pixels in the final images were used. Figures of micrographs were assembled by using Adobe Photoshop 7.0 and Deneba Canvas 8.0 software.
FRAP.
We conducted FRAP experiments by using leaf tissue harvested 10–14 days p.i. from SYNV-infected mGFP5-ER plants. Mock-inoculated plants of similar ages were used as controls. FRAP experiments were performed by using the Olympus FV1000 microscope described above. Briefly, 5x5 mm square sections of leaf tissue were mounted on glass slides in water and covered with a glass coverslip. Imaging for FRAP experiments was conducted by using a x60 objective and 488 nm laser line from a multi-line argon laser set at 0.3 % power. Regions of interest (ROIs) were photobleached for 50 ms using a 405 nm diode laser set at full power, which was delivered via the FV1000 Simultaneous (SIM) scanner. Images for FRAP analyses were acquired at a resolution of 512x512 pixels and a scan rate of 2 µs pixel–1, which was necessary to monitor fast protein dynamics. Two images were acquired prior to photobleaching, followed by an additional seven images to monitor fluorescence recovery. Quantitative fluorescence data in Microsoft Excel format and confocal images in TIFF format were exported by using Olympus Fluoview software. FRAP experiments were repeated three times for each ROI, with 2 min between bleaching events in order to allow full recovery of fluorescence. For proteins that did not show FRAP, such as SYNV M, independent ROIs were used for each experiment. Replicated fluorescence intensity data were averaged and these data were normalized across experiments. Means and SD for fluorescence intensity at each time point were calculated and plotted by using Microsoft Excel software.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In order to rule out the possibility that mGFP5-ER localization was not affected per se in virus-infected cells, we confirmed membrane-associated fluorescence in the mGFP5-ER line by examination of protoplasts, derived from mock-inoculated or SYNV-infected leaves, using both TIRFM and confocal microscopy. In both cases, GFP fluorescence was restricted to ER tubules, with no detectable differences of fluorescence in the cytoplasm (Fig. 1a, b). Given that the outer nuclear membrane is contiguous with the ER, the lumen of the nuclear envelope fills with mGFP5-ER (Fig. 1c) (Collings et al., 2000). As we have reported previously, mGFP5-ER accumulates in nuclei of SYNV-infected cells (Fig. 1d) (Goodin et al., 2005).
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). Condensed nucleocapsids of SYNV bud through the INM and accumulate as mature virions in the perinuclear space (van Beek et al., 1985; Martins et al., 1998; Goodin et al., 2005). We determined the relationship between intranuclear membranes by FRAP analysis. No significant difference in the diffusion rates of GFP was observed when either the nuclear envelope or any ROI of intranuclear membranes was photobleached (Fig. 2a, b). Fluorescence intensity for all ROIs returned to 94–100 % of pre-bleach values within the course of these experiments. The mean values for 50 % recovery of fluorescence (t1/2) were approximately 2.3 s in all cases.
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; Irons et al., 2003; Makatsori et al., 2004). It has been determined that the amino-terminal 238 aa of LBR are sufficient to target GFP to the nuclear envelope in plant cells (Irons et al., 2003). We therefore used this fusion to determine whether SYNV induced the intranuclear accumulation of single membranes, as suggested by EM (Martins et al., 1998). Expression of LBR–GFP in mock-inoculated leaves resulted in accumulation of this marker primarily on the nuclear envelope (Fig. 3a–c). In contrast, expression of LBR–GFP in SYNV-infected leaves resulted in accumulation of this marker on both the nuclear envelope and intranuclear membranes (Fig. 3d–f). The relative fluorescence intensity of GFP on the nuclear envelope, measured in a 0.196 µm2 ROI, which spans the mean width of the nuclear envelope, was 1416±222 units. In equivalent areas of the intranuclear membranes, the fluorescence intensity was found to be 757±110 units, a 47 % reduction compared with that of the nuclear envelope (Fig. 3g).
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, 2002, 2005; Tsai et al., 2005) or by immunological methods using light microscopy or EM (van Beek et al., 1985; Martins et al., 1998). However, these studies, which focused primarily on the N and P proteins, did not determine the localization of SYNV proteins in the context of infected cells. Therefore, we expressed the SYNV N, P, sc4, M and G proteins as fusions to a monomeric RFP in virus-infected and mock-inoculated cells of mGFP5-ER leaves (Fig. 4). The majority of single optical sections showed that the RFP–P fusion in virus-infected nuclei accumulated in discrete, ring-shaped structures that did not colocalize with intranuclear membranes (Fig. 4a–c). In contrast, expression of this fusion in mock-inoculated cells resulted in diffuse accumulation throughout the entire nucleoplasm (Fig. 4d). RFP–N accumulated in nuclei in contiguous areas that partially overlapped the intranuclear membranes (Fig. 4e–g), whereas this fusion expressed in mock-inoculated cells was entirely localized in the nucleoplasm without any association with the nuclear envelope (Fig. 4h). RFP–M was found to be primarily colocalized with membranes in virus-infected nuclei, whereas expression of this fusion in mock-inoculated cells resulted in accumulation throughout the entire nucleoplasm (Fig. 4i–l). RFP–G expressed in virus-infected nuclei was found to be associated with intranuclear membranes (Fig. 4m–o). Expression of RFP–G outside the context of virus infection had a profound effect on nuclear membranes (Fig. 4p). In contrast to virus-infected cells, RFP–G neither induced nor accumulated on intranuclear membranes. However, large aggregates of this protein formed in membrane-associated bodies on the nuclear envelope (Fig. 4p). Expression of RFP alone showed that this protein was excluded from intranuclear membranes in virus-infected nuclei and accumulated diffusely in nuclei of mock-inoculated cells (Fig. 4q–t). Expression of RFP fused to the 240 kDa SYNV L protein did not result in detectable fluorescence or protein (data not shown).
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). However, the spatial organization of viral proteins within the viroplasm has not been addressed in detail. Given the reported association of rhabdoviral N and P proteins as complexes and in association with viroplasm (Martins et al., 1998; Goodin et al., 2001; Majumdar et al., 2004), we considered it curious that the localization of the P protein did not overlap that of the intranuclear membranes, whereas that of the N protein did (Fig. 4b, f
). Therefore, in order to define further the spatial relationship between viral proteins in SYNV-infected nuclei, we coexpressed CFP and RFP fusions of the N, P, sc4, M and G proteins in pairwise combinations in virus-infected leaves of non-transgenic N. benthamiana plants. A subset of these micrographs is shown in Fig. 5. Coexpression of CFP–P and RFP–N revealed that these proteins colocalize in the viroplasm, but that the accumulation of P protein is reduced relative to N in regions immediately adjacent to the intranuclear membranes (Fig. 5a–c). Consistent with this finding are the results obtained when CFP–P and RFP–M were coexpressed. In this case, CFP–P localized adjacent to RFP–M, which was shown to localize exclusively on intranuclear membranes in virus-infected nuclei (Fig. 5d–f). Likewise, coexpression of CFP–M with RFP–N demonstrated that M colocalizes with N only on intranuclear membranes (Fig. 5g–i). Finally, RFP–N colocalized partially with CFP–G, which was found to localize exclusively on intranuclear membranes (Fig. 5j–l).
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; Albertini et al., 2006; Green et al., 2006; Mavrakis et al., 2006). It has been shown previously that coexpression of SYNV N and P results in colocalization of these proteins and that a soluble N–P complex can be isolated from cells coexpressing these proteins (Goodin et al., 2001). Therefore, in order to obtain insight into the nature of the N protein in virus-infected cells, we compared the FRAP kinetics of RFP–N expressed in virus-infected nuclei and mock-inoculated cells in which CFP–P was also expressed. We were able to clearly distinguish membrane- and viroplasm-associated forms of N in virus-infected nuclei (Fig. 6a, b). Consistent with the prediction that the majority of N in the viroplasm should exist as an N–P complex is the finding that RFP–N FRAP in viroplasm of virus-infected nuclei was not significantly different from FRAP of RFP–N coexpressed with CFP–P in mock-inoculated cells (Fig. 6a, b). FRAP for N expressed in mock-inoculated cells or associated with the viroplasm ranged from 70 to 80 %, respectively, with t1/2 values of approximately 2.3 s. FRAP kinetics for RFP–N expressed in the absence of P were faster than those for this fusion in the presence of P (data not shown).
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). However, the fate of virions that accumulate in the perinuclear space, as well as the mechanism by which plant-adapted rhabdoviruses move systemically in plants, are poorly understood. It has been proposed that sc4 is the putative movement protein for SYNV (Goldberg et al., 1991; Melcher, 2000). Indeed, P3, the rice yellow stunt virus (RYSV) homologue of sc4, has been shown to complement a movement-deficient mutant of potato virus X (Huang et al., 2005). It was also shown that P3 interacts with the RYSV nucleocapsid protein and thus proposed that this virus moves as a protein–nucleocapsid complex. Similar data for sc4 are lacking; however, this protein localizes to punctate loci on cell walls, consistent with plasmodesmal targeting expected for virus movement proteins (Fig. 8a–d). Intriguingly, we found that expression of RFP–M, which is entirely nuclear-localized in mock-inoculated cells (Fig. 4l) (Goodin et al., 2002) or associated with intranuclear membranes in virus-infected cells (Fig. 4i–k), could also be found in complexes that appeared to bud from nuclei (Fig. 8e–i). Examination of the M-containing complexes showed that they were surrounded by, and moved on, ER membranes (Fig. 8j–s).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 2002; Tsai et al., 2005) by using fluorescence microscopy, or in virus-infected cells for a limited number of proteins (Martins et al., 1998). Furthermore, the relationship between the localization of viral proteins and relocalized host-cell and nuclear membranes has not been determined. By expressing AFP fusions in SYNV-infected cells, we have revealed a novel insight into protein and membrane dynamics in the SYNV–N. benthamiana pathosystem.
By using TIRFM and confocal microscopy, we did not observe GFP in any cellular loci other than those contiguous with the endomembrane system. Therefore, the source of GFP in virus-infected nuclei should be the ER and lumen of the nuclear envelope. This contention is supported by FRAP analyses of GFP in the nuclear envelope and intranuclear membranes. As there was no statistically significant difference in the FRAP kinetics of GFP in any of these loci, we conclude that the intranuclear membranes remain contiguous with the ER and are not confined to covalently closed intranuclear membranes. This finding is significant because it allows for the possibility that the intranuclear membranes are bona fide sites of virion assembly, and not simply alterations in host membranes that do not participate in viral biology per se. Contiguity of the intranuclear membranes with the ER is essential for delivery of the glycosylated SYNV G protein to the INM from the ER and Golgi. It is noteworthy that the G protein did not, on its own, induce formation of intranuclear membranes; neither did coexpression of M and G, which, for some negative-strand RNA viruses, results in the budding of empty particles from transfected cells (Swenson et al., 2004). Therefore, we suspect that formation of the intranuclear membranes may require additional viral proteins and, perhaps, RNA. Furthermore, whilst overexpression of viral glycoproteins commonly results in adverse cytopathic effects, such effects seen in mock-inoculated cells were absent or markedly reduced in virus-infected cells. Following budding into the perinuclear space, contiguity with the ER might provide rhabdoviruses with a continuous conduit by which to travel from cell to cell. Although the current models for cell-to-cell movement, developed primarily from studies with positive-strand RNA viruses, do not favour virus movement through ER tubules, such a mechanism cannot be ruled out for plant-adapted rhabdoviruses. Indeed, MMV has been observed in ER tubules in cells of its insect vector (Herold & Munz, 1965). An alternative means for cell-to-cell movement that also requires contiguity of the ER with virus-induced intranuclear membranes is budding of mature virions from the perinuclear space to release the core particle, which could function as a movement complex. This mechanism would be akin to the ‘bud-in, bud-out’ envelopment and de-envelopment of herpesvirus particles, which allow this virus to move from sites of replication and assembly in the nucleus to the cytoplasm and, subsequently, by additional budding events, to exit the infected cell (Mettenleiter, 2004, 2006).
Both of the movement models above require that viral proteins be delivered to the intranuclear membranes in a manner consistent with the known function of rhabdoviral proteins in assembly (Jayakar et al., 2004). Therefore, we expressed all of the SYNV proteins as RFP fusions in plant cells, except for the 240 kDa L protein. In contrast to expression in mock-inoculated cells, the localization patterns in virus-infected cells were consistent with models for rhabdovirus assembly and morphogenesis proposed by Green et al. (2000, 2006) and Jayakar et al. (2004). The first step in the budding process is formation of nucleocapsids by delivery of the N protein to nascent genomic-length RNAs via an N–P complex. The majority of P should be excluded from the RNA–N–P complex (Green et al., 2006) to form the mature nucleocapsid, which is in turn delivered to an M–G complex that has formed on membranes (Jayakar et al., 2004). The membrane-anchored nucleocapsid, as suggested by our ‘slow FRAP’ data, is condensed by M to form a core particle that buds through the INM to form a mature virion. Consistent with this model (Fig. 7), we found that the majority of the P protein did not colocalize with the intranuclear membranes. In fact, this protein appears to be excluded immediately adjacent to these membranes at loci occupied by the N and M proteins. In addition to colocalizing in part with membranes, the N protein was found in a highly mobile (‘fast FRAP’) region in the nucleoplasm, consistent in location with the viroplasm, which is the proposed site of rhabdovirus replication (Martins et al., 1998).
The fact that the intranuclear membranes upon which the N, M and G proteins associate are derived from the nuclear envelope has been established by EM (Martins et al., 1998). Consistent with these results is the demonstration that the relative fluorescence intensity of the LBR–GFP marker on SYNV-induced intranuclear membranes is almost exactly half (53 %) that on the nuclear envelope. Because the LBR–GFP marker does not contain the lamin-binding domains, the distribution of this fusion on the outer and inner nuclear membranes is expected to be the same under steady-state observations. Therefore, as predicted, the fluorescence per unit area of a single membrane was half that of a double membrane. We do not suspect that the reduction in fluorescence is due to occlusion of LBR–GFP by SYNV-encoded proteins because SYNV G does not hyperaccumulate on intranuclear membranes relative to the nuclear envelope.
During the course of our localization studies, we discovered heretofore-unreported complexes in the cytoplasm of virus-infected cells that incorporated M protein fusions to CFP or RFP. Further analyses showed that these complexes were liberated from nuclei of virus-infected cells, which then proceeded to track on ER membranes. We have not yet been able to label these complexes with fluorescent fusions of other SYNV proteins, most notably N or P, which might indicate that nucleocapsid cores were also associated with these complexes. However, given the small amounts of P protein in virus particles relative to M and N (data not shown), it may not be possible to detect these complexes in the same manner in which VSV nucleocapsids have been labelled by using GFP–P fusions (Das et al., 2006). Extensive analyses failed to reveal such complexes in mock-inoculated leaves in which RFP–M was coexpressed. Further, the finding that these complexes are ER-associated is intriguing, as it suggests that G could also be a part of the complex. However, the hypothesis that the observed M protein complex is the bona fide SYNV movement complex will require extensive characterization by EM in planned future studies. Intriguingly, one way that such complexes could arise, if they are derived from mature virions in the perinuclear space, is via budding through the outer nuclear membrane, which would release cores into the cytoplasm. Therefore, when considered with our FRAP data, which show that virus-induced nuclear membranes are contiguous with the ER, it is conceivable that SYNV moves from cell to cell via M protein condensed cores that track on ER membranes. Further investigation into the characterization of these complexes is thus warranted in future studies.
Taken together, our live-cell imaging conducted in the context of virus-infected cells revealed the spatial relationship between viral proteins, which suggests a contiguous pathway from the putative sites of virus replication to those of morphogenesis. The protein and localization data presented here could not be gleaned from studies conducted in the traditional manner of expression in virus-free cells. Therefore, the ability to express AFP fusions in the context of virus infection represents a significant advance in our ability to study plant–rhabdovirus interactions in live cells.
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ACKNOWLEDGEMENTS |
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Received 9 November 2006;
accepted 9 February 2007.
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