Correspondence to: Roger N. Beachy, Donald Danforth Plant Science Center, 7425 Forsyth Blvd., Box 1098, Clayton, MO 63130. Tel:(314) 935-9852 Fax:(314) 935-8605 E-mail:rnbeachy{at}danforthcenter.org.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Little is known about the mechanisms of intracellular targeting of viral nucleic acids within infected cells. We used in situ hybridization to visualize the distribution of tobacco mosaic virus (TMV) viral RNA (vRNA) in infected tobacco protoplasts. Immunostaining of the ER lumenal binding protein (BiP) concurrent with in situ hybridization revealed that vRNA colocalized with the ER, including perinuclear ER. At midstages of infection, vRNA accumulated in large irregular bodies associated with cytoplasmic filaments while at late stages, vRNA was dispersed throughout the cytoplasm and was associated with hair-like protrusions from the plasma membrane containing ER. TMV movement protein (MP) and replicase colocalized with vRNA, suggesting that viral replication and translation occur in the same subcellular sites. Immunostaining with tubulin provided evidence of colocalization of vRNA with microtubules, while disruption of the cytoskeleton with pharmacological agents produced severe changes in vRNA localization. Mutants of TMV lacking functional MP accumulated vRNA, but the distribution of vRNA was different from that observed in wild-type infection. MP was not required for association of vRNA with perinuclear ER, but was required for the formation of the large irregular bodies and association of vRNA with the hair-like protrusions.
Key Words: in situ hybridization, tobacco mosaic virus, virus replication, endoplasmic reticulum, cytoskeleton
SPECIFIC virushost interactions determine the capability of viruses to successfully replicate in a cell and spread local and systemically in the host. Plant viruses, like those of animals, compartmentalize viral RNA synthesis by association with the cellular endomembrane system (e.g.,
Tobacco mosaic virus (TMV)1 is the type member of the tobamovirus group. The genome of TMV consists of one single-stranded 6.4-kb RNA containing four open reading frames. The genomic RNA serves as messenger RNA (mRNA) for the production of a 126-kD protein and, by readthrough of an amber termination codon, a 183-kD protein (
Cytological (
Cytological analyses of TMV-infected cells showed virus replication complexes associated with cytoplasmic inclusions, called viroplasms, which expanded throughout the infection. These inclusions contained ribosomes, tubules, and 126/183-kD replication proteins (
Since the MP binds single-stranded nucleic acids in vitro (
In many different biological systems, the coordinated activities of components of the cytoskeleton are responsible for the specific transport of RNAs, as well as the anchoring of RNAs at their final locations (e.g.,
In this work, nonradioactive methods of in situ hybridization were used to localize the intracellular distribution of TMV RNA during virus infection. The combined use of immunostaining and in situ hybridization techniques allowed examination of the relationship between the sites of vRNA accumulation and the distribution of selected host (microtubules, ER) and viral (MP, replicase) components. The pattern of vRNA accumulation after treatment with oryzalin and cytochalasin D gave new insights about the role of actin filaments and microtubules in vRNA transport and replication. Furthermore, the use of TMV derivatives lacking a functional MP allowed us to determine the role of the MP in establishing the sites of vRNA infection and association with host components.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasmids
Diagrams of the infectious clones used in this study are shown in Fig 1 A (below). The plasmids pU3/12 (M-RV (
M), respectively. The plasmids pT-MfCP (MP:GFP-CP) and pTMV-M:GfusBr (MP:GFP-
C) (
C does not contain the CP open reading frame (see Fig 1 A).
|
The plasmid pTR447 was used as template to produce the nonradioactive (digoxigenin or fluorescein) labeled RNA probes used in Northern blot and in situ hybridization experiments. To create pTR447, the XbaIHindIII fragment of the TMV replicase coding sequence (comprising nucleotides 999 to 1446) was removed from pU3/12 M-RV and subcloned into the plasmid vector Bluescript SK+ (Stratagene Inc.) previously digested with the same restriction endonucleases.
Protoplast Infection, RNA Extraction, and Northern Hybridization Analysis
Infectious RNAs were obtained by in vitro transcription of TMV clones using the MEGAscript T7 kit (Ambion Inc.). Protoplasts were transfected by electroporation as described by
To monitor plus- and minus-strand accumulation, the RNAs were denatured with glyoxal following the procedure described by
Prehybridization, hybridization, and colorimetric detection were performed as previously described by
Immunofluorescent Labeling
Protoplasts were fixed in paraformaldehyde and spun onto polylysine-coated slides (Sigma Chemical Co.) as described by -tubulin and antiactin antibodies (Amersham Corp.) were diluted 1:100 in PBS-TE. The antibody against the rabbit antiER lumenal binding protein (BiP), kindly provided by R.S. Boston (North Caroline State University, Raleigh, NC), was used at 1:100 dilution in PBS-TE. To immunolocalize TMV replicase, a polyclonal antibody against the 126/183-kD replicase protein (
In Situ Hybridization
Before hybridization, the samples were treated with proteinase K (1 µg/ml) for 5 min, washed, and refixed with paraformaldehyde at room temperature for 30 min. After fixation, the samples were washed again and immersed in 0.25% acetic anhydride, 100 mM triethanolamine-HCl, pH 8.0, for 10 min to prevent nonspecific binding of the probe to positively charged amino groups. After acetylation, the samples were dehydrated by 10 min washes in 70, 80, 90, 95, and 100% ethanol solutions. The samples were then hybridized overnight at 55°C in a moist chamber with the hybridization solution containing 50% (vol/vol) of deionized formamide, 4x SSC, 0.1% (wt/vol) SDS, 8% (wt/vol) dextran sulphate, and 10 ng/µl of fluorescein-RNA (fluor-RNA) probe. The fluor-RNA probes that recognized plus or minus strand of the genomic TMV RNA were obtained by in vitro transcription of pTR447 as described above. Probes were labeled with fluorescein-12-UTP (Boehringer Mannheim Biochemicals) following the recommendations of the manufacturer. After hybridization, the samples were washed two times in 2x SSC for 10 min at room temperature and two times in 0.1x SSC for 10 min at 50°C, air-dried, and mounted as described above.
Treatment with Cytoskeleton Depolymeration Agents
To disrupt microtubules, infected protoplasts were treated with 10 µM oryzalin (ChemService) for 2 h. To disrupt microfilaments, infected protoplasts were treated with 100 µM cytochalasin D (Calbiochem Corp.) for 2 h. Stock solutions of oryzalin and cytochalasin D were prepared in DMSO so that the final concentration of DMSO added to the protoplasts did not exceed 0.1% (vol/vol). After these treatments, the protoplasts were fixed and processed for in situ hybridization following the procedure described above.
Confocal Microscopy
Confocal imaging was performed as previously described (
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Northern Hybridization of Protoplasts Infected with TMV
Before in situ hybridization analysis, the specificity of the RNA probe and the accumulation of viral RNA of the different TMV constructs used in this study (Fig 1 A) were analyzed by Northern blot hybridization (Fig 1 B). Total RNAs from equal numbers of protoplasts were extracted at different times after infection and subjected to Northern hybridization with a probe labeled with digoxigenin (dig-RNA). The RNA probe contains a fragment of the replicase sequence (see details in Materials and Methods). The probe hybridized with an RNA of 6.4 kb, representing the full-length, plus-strand genome of TMV (Fig 1 B, w.t.). As anticipated, vRNA accumulated to approximately the same level in cells infected with wt TMV RNA and in cells infected with TMV RNA that lacked the MP (
M), confirming previous studies showing that the MP is not required for replication (
Time-course analysis showed that vRNA accumulated to detectable levels at 4 hpi, increased until 14 hpi, and then gradually decreased to a lower level through 30 hpi (Fig 1 B). The levels of accumulation of vRNA in mutants that lack the CP sequence and express the MP:GFP fusion protein (MP:GFP-C in Fig 1) were somewhat lower than in wt infection, especially at 30 hpi (Fig 1 B). Since MP:GFP-
C produced no CP, the lower levels of vRNA in these samples are presumed to reflect the lack of encapsidated vRNA. Protoplasts infected with RNAs derived from constructs containing the CP open reading frame and MP:GFP fusion protein (MP:GFP-CP) showed lower levels of vRNA than those observed in wt infection (compare wt and MP:GFP-CP in Fig 1 B).
The levels of ribosomal RNAs in each sample were similar, as visualized by staining with methylene blue before hybridization (not shown).
When samples were hybridized with a probe to detect minus-strand RNA, the signal was observed only at early stages of infection (48 hpi) and in much lower amounts than plus strands (not shown). Similar results were described by
These studies show that the nonradioactive RNA probes were sufficiently sensitive and specific for use in situ hybridization procedures. They also indicate that vRNA produced by wt, M, and MP:GFP virus infections accumulate to approximately the same degree throughout infection.
Subcellular Distribution of TMV Genomic RNA in Infected Protoplasts
The intracellular accumulation of TMV RNA during infection was visualized by in situ hybridization. Protoplasts isolated from BY-2 tobacco cells were transfected with wt RNA, collected at different times after infection, and hybridized with fluor-RNA probes. The fluorescent signals were visualized by confocal microscopy after collecting optical sections with a focal depth of 0.8 µm. Since infection of BY-2 protoplasts is apparently not synchronous (
|
At midstages of infection (Fig 2, DI), vRNA was associated with large irregularly shaped bodies (Fig 2 D, arrowheads). High magnification of the image revealed that these bodies were, in many cases, associated with a weakly fluorescent reticulated network (Fig 2 E) that resembled the cortical ER. Short fluorescent strands of tubules, small fluorescent circles, and vesicles of variable size and shape were also observed (Fig 2 F, arrows). During the middle stage of infection, most of the large bodies were localized around the nucleus, although some were dispersed throughout the cytoplasm (Fig 2 D) and in some cells occupied much of the cytoplasm (Fig 2 G). Clear localization of vRNA in filamentous structures was visible during this period (Fig 2H and Fig I); most of the filaments appeared to be associated with the fluorescent bodies (Fig 2H and Fig I). At late stages of infection (Fig 2J and Fig K), vRNA was localized to small, intensely labeled spots around the nucleus, throughout the cytoplasm, and at the periphery of the cell (Fig 2 J). The number and intensity of these spots decreased over time. By 40 hpi, 15% of infected protoplasts showed vRNA localized to elongated structures that appeared to protrude through the plasma membrane (Fig 2 K, arrowheads). The protrusions resembled those previously described as binding the MP:GFP fusion protein (
We conclude from these studies that vRNA accumulates in different subcellular compartments throughout infection. This compartmentalization most likely reflects the roles of vRNA in replication, translation, and assembly in viral particles.
Mock-inoculated protoplasts identically processed and imaged did not show fluorescent signals (Fig 2 L). In addition, no signal was observed when either the fluor-RNA probe was omitted from the hybridization reaction or when the samples were treated with RNAse A before the hybridization reaction (not shown). Furthermore, 9095% of the inoculated protoplasts showed hybridization signal, coinciding with the percentage of protoplasts that are infected after inoculation with wt vRNA.
As anticipated from the Northern hybridization studies described above and those described by
|
Taken together, these results indicate that the fluorescent signal obtained after in situ hybridization of U1-infected protoplasts reflects the intracellular distribution of TMV RNA.
Spatial Relationships between vRNA and Viral Proteins
Viral RNA Colocalizes with the Replicase.
To examine the relationship between the sites of vRNA accumulation and the location of the viral replicase, protoplasts infected with wt TMV RNA were collected at early stages of infection, first immunostained with antibody raised against the TMV replicase protein (
|
In double labeling experiments, the fluorophores were scanned independently to minimize crossover between the two channels. Similar patterns of distribution of replicase were observed when the samples were processed only by immunostaining with the replicase antibody, indicating that the results were not due to artifacts produced as a result of the in situ hybridization procedure.
Viral RNA Colocalizes with the MP.
The patterns of vRNA accumulation over time resembled the pattern of synthesis, accumulation, and degradation of the MP during TMV infection (C, a mutant virus that expresses the MP:GFP fusion and lacks the CP sequence (not shown).
The hybridization procedure was incompatible with simultaneous detection of MP:GFP due to bleaching of GFP fluorescence during the treatment of the samples. Therefore, to compare the accumulation of both the MP and vRNA in the same cell, protoplasts infected with vRNA-MP:GFP-C were harvested at midstages of infection, fixed, and spun onto poly-lysinecoated slides (see Materials and Methods). Cells showing the characteristic fluorescence pattern of MP:GFP accumulation as previously described (
After hybridization with the fluor-RNA probe, the samples were examined to detect the products of hybridization (vRNA). As shown in Fig 5, there was a striking coincidence between the distribution of MP and the sites of vRNA accumulation. vRNA was also found in small cytoplasmic patches that lacked detectable MP (arrows). Similar patterns of distribution of replicase, MP, and vRNA distribution confirmed the hypothesis that the replication of vRNA and the accumulation of the MP occur at the same subcellular sites.
|
Role of the MP in Establishing the Sites of RNA Accumulation in Infected Cells.
As shown above, MP colocalizes with the main sites of vRNA accumulation during TMV infection. To clarify the role of the MP in establishing the sites of virus replication, protoplasts infected with vRNA-M were processed for in situ hybridization. At early stages of infection, a similar pattern of fluorescence was observed in protoplasts infected with wt vRNA or vRNA-
M. The fluorescent signal was localized in vesicle-like structures surrounding the nucleus and in small cytoplasmic patches (Fig 6 A). In contrast to infection by wt TMV, vRNA-
M also accumulated in stacks of tubular structures near the nucleus (Fig 6 B, arrowheads). Higher magnification revealed vesicles of different sizes (25 µm diameter) around the nucleus as well as throughout the cytoplasm (Fig 6 C, arrowheads). At midstages of infection, the most striking feature that distinguished this pattern of vRNA-
M distribution from that of wt vRNA was the absence of large fluorescent bodies. Instead, smaller fluorescent spots were distributed throughout the cell, interconnected by fewer fluorescent reticulated strands than in wt infection (Fig 6 D, arrowheads). We often observed stacks of large lamellar cisternae that were up to 10 µm in length and 45 µm in depth (Fig 6 E, arrows). At late stages of infection, the fluorescent signal was observed only as small, intensely labeled spots around the nucleus and scattered throughout the cytoplasm (Fig 6 F). Cells infected with vRNA-
M did not exhibit fluorescent protrusions from the surface of the cells as in cells infected with wt vRNA (e.g., Fig 2 K).
|
Similar distribution of vRNA was observed when protoplasts were infected with vRNA-M-GFP-
C, a virus construct that lacks CP and MP and produces free GFP (not shown).
Spatial Relationship between Viral RNA and Host Components
Viral RNA Colocalizes with the Endoplasmic Reticulum.
As noted above, the reticulated pattern of fluorescence that represents the location of wt vRNA resembled the distribution of the ER. Furthermore, previous studies demonstrated colocalization of TMV MP and the replicase with ER on the fluorescent irregularly shaped bodies (
|
In protoplasts treated at early stages of infection with 50 µg/ml of Brefeldin A (BFA), a fungal metabolite that disrupts the endomembrane system in plant cells (
|
Viral RNA Colocalizes with Microtubules.
At early and midstages of infection, vRNA was associated with fluorescent cytoplasmic filaments (Fig 2H and Fig I). To determine if vRNA is associated with elements of the cytoskeleton, infected protoplasts that were immunostained with a monoclonal antibody to -tubulin were processed for in situ hybridization. A variety of different experimental conditions was evaluated before selecting conditions that permitted visualization of both signals. The procedure that was used involved treating fixed protoplasts with 1 µg/ml of proteinase K for 5 min to partially digest cross-linked proteins and subsequent refixation to avoid disintegration of the cells. These procedures improved the accessibility of the probe while preserving the integrity of the microtubules. Fig 9 shows confocal micrographs of tubulin (in red) and vRNA (in green) in an infected protoplast processed at midstage of infection. Merging the images clearly showed the coalignment of both signals (merged image). Most of the green fluorescent filaments corresponding to sites of vRNA accumulation coaligned with the cytoplasmic strands of microtubules (Fig 9 A, yellow in merged image). Furthermore, some of the bright fluorescent spots that contained vRNA accumulated in tracks that were aligned with microtubule filaments (Fig 9 B).
|
Protoplasts infected with vRNA-M that were identically processed and imaged did not show fluorescent signals associated with cytoskeleton (except in cytochalasin Dtreated protoplasts, see below). The results indicate that colocalization of vRNA with microtubules was not due to artifactual aggregation that occurred during fixation procedures. These conclusions are based on replicated data, either in the number of experiments or in the number of protoplasts analyzed per experiment.
In companion studies, we attempted to stain with antiactin antibodies and with the probe for vRNA. However, we did not resolve a typical pattern of filamentous actin distribution (not shown). Most of the cells showed very short fluorescent strands of actin dispersed throughout the cytoplasm. In some cells, we observed a coincidence between the short strands of TRITC-fluorescent signal and vRNA (not shown). It is possible that under the conditions of these experiments, the integrity of the microfilaments was not maintained. A relationship between actin filaments and vRNA can not be ruled out since disruption of microfilaments by treating infected protoplasts with cytochalasin D altered the pattern of vRNA distribution (see below).
Effects of Cytoskeletal Inhibitors on Localization of vRNA.
The results provide strong evidence for colocalization of vRNA with microtubules and suggest that cytoskeletal elements may be involved in distribution of vRNA in protoplasts. To clarify the role of the cytoskeleton in vRNA distribution, protoplasts infected with wt vRNA were treated with specific cytoskeletal inhibitors and vRNA accumulation was examined by in situ hybridization. Representative examples are shown in Fig 10. Treatment of protoplasts at early stages of infection with oryzalin, a plant microtubule depolymerizing agent (
|
In protoplasts treated during early stages of infection with cytochalasin D, there was a clear delay in the formation of the cytoplasmic bodies compared with nontreated protoplasts. Interestingly, the pattern of vRNA accumulation in cytochalasin Dtreated cells was similar to that observed in cells infected with vRNA-M (compare Fig 10, vRNA/Cytoch D, with Fig 6). At midstages of infection, at a time when the large cytoplasmic bodies were formed, the vRNA in cytochalasin Dtreated protoplasts (not shown) was detected in enlarged bodies that appeared as more elongated structures than those observed in nontreated cells.
In protoplasts infected with vRNA-M and treated with cytochalasin D at early stages of infection, vRNA-
M was associated with filaments resembling microtubules (Fig 10,
M/Cytoch D). In these protoplasts, vRNA-
M also accumulated at the periphery of the cell, but no fluorescence was observed around the nucleus or in small bodies throughout the cytoplasm, as in the case of nontreated, vRNA-
Minfected protoplasts (see Fig 6 for comparisons). The effects of cytochalasin D were more dramatic in infections with vRNA-
M than with wt vRNA, suggesting an influence of the MP in the localization of vRNA in cytochalasin Dtreated protoplasts.
The results of these studies demonstrate that disruption of the cytoskeleton produces changes in the pattern of vRNA localization and suggest an important role of microtubules and microfilaments in the distribution of vRNA during virus infection.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Early Stages of TMV Infection
The replication of many positive-strand RNA viruses occurs in close association with membranes (e.g.,
Minus-strand RNA was also localized to structures (presumed to be ER) that surround the nucleus, with the exception of a discrete region. The absence of minus-strand RNA from this region could reflect compartmentalization of the perinuclear ER, or it may indicate that the ER is divided into subdomains with specific morphological or functional properties (
vRNA and viral replicase were colocalized in small patches that are distributed throughout the cytoplasm. As previously indicated in poliovirus infection (
At early stages of infection, vRNA was associated with fluorescent filaments that resembled elements of the cytoskeleton (not shown). Based on the effects of oryzalin and cytochalasin D on the distribution of vRNA in the cytoplasm, we suggest that there is association of vRNA with the cytoskeleton at a very early stage of infection. In this scenario, vRNA exploits the cytoskeleton for transport from the cytoplasm to perinuclear positions. This hypothesis is based in part on the observation that treating protoplasts with oryzalin at the time of inoculation prevented the localization of vRNA to the perinuclear region. A recent report described a mechanism in newt lung epithelial cells by which the ER membranes attached to microtubules are transported toward the cell center through actomyosin-based retrograde flow of microtubules with ER attached as cargo (
Throughout the infection, vRNA was localized in different subcellular compartments. We suggest that this reflects movement of vRNA to different compartments, but cannot eliminate the possibility that compartmentalization represents the synthesis and subsequent degradation of vRNA rather than movement of vRNA from one compartment to another.
When protoplasts were infected with vRNA-M, a mutant of TMV that does not produce MP, vRNA-
M was localized to vesicle-like structures around the nucleus and in small patches in the cytoplasm. These results indicate that at an early stage of infection, association with the ER is an intrinsic property of vRNA and/or the replicase and does not require MP. It is possible that vRNA contains sequences that target to the ER. Some cellular mRNAs are known to contain specific signals that direct them to the rough ER for translation (
Midstages of Infection: Virus Accumulation and Intracellular Transport
At midstages of infection, vRNA was localized in fluorescent, irregularly shaped bodies, some of which were vesicle-like in appearance. Furthermore, the replicase (Fig 4) and MP (Fig 5) colocalized with vRNA on these bodies. Since such structures were not observed in protoplasts infected with vRNA-M, we conclude that MP is required for the formation and/or stabilization of the bodies. These structures may correspond to the previously described "viroplasms or amorphous inclusions" induced by TMV infection (
Over time, the cytoplasmic bodies become elongated structures, often associated with fluorescent filaments, directed toward the periphery of the cell. Immunostaining with antitubulin antibody and in situ hybridization reactions provide clear evidence of colocalization of vRNA with microtubules. Treatment of protoplasts at midstage of infection with oryzalin prevented the dispersion of the bodies to the periphery of the cell, suggesting that microtubules play a role in intracellular distribution of vRNA. Two different microtubule-based mechanisms, membrane sliding and tip attachment complexes, participate in the movement of ER from the cell center to the periphery of newt lung epithelial cells (
Treatment of infected protoplasts with cytochalasin D clearly altered the pattern of vRNA distribution and caused a delay in the appearance of the large bodies, suggesting a role of microfilaments in their formation and/or stabilization. However, depolymerization of microtubules can also lead to disruption of microfilaments (
vRNA-M was not associated with elements of the cytoskeleton unless the cells were treated at early stages of infection with cytochalasin D. Such treatment resulted in accumulation of vRNA-
M in fluorescent spots at the periphery of the cell, as well as on filaments that were similar in appearance to microtubules. These results suggest that vRNA was associated with microtubules when both MP and microfilaments were absent. The transport of mRNAs along cytoskeletal components has been described in a variety of biological systems, especially in relation to cell differentiation and development (
M vs. wt vRNA. These data suggest some influence of the MP in localization of vRNA in cytochalasin Dtreated protoplasts. The implications of MP and microfilaments in the formation and anchoring of the cytoplasmic bodies are discussed below.
Late Stages of Infection: Virus Spread and/or Degradation?
Throughout infection, vRNA was localized around the nucleus, although at mid and late stages, vRNA was also dispersed throughout the cytoplasm and at the periphery of the cell. Accumulation of vRNA around the nucleus may facilitate association with host components that are required for virus infection. However, the accumulation of the much larger bodies that surround the nucleus may imply a different biological role in replication. Recently, a model was proposed by which misfolded proteins that escape the proteosome-mediated pathway of degradation can aggregate to form large structures, referred to as "aggresomes." Aggresomes are transported on microtubules from peripheral sites to a ubiquitin-rich nuclear location at the microtubule-organizing center, where they are entangled with collapsed intermediate filaments (
At late stages of infection, vRNA was localized in structures that protrude from the surface of the cell. These protrusions were not disrupted by oryzalin or cytochalasin D. Furthermore, in other studies, we observed that the protrusions were stained with DiOC6(3) (3, 3'-dihexyloxacarbocyanine iodide), a vital fluorescent stain of ER (not shown). Other studies indicate that these ER-containing structures are not induced by virus infection per se, but may be stimulated by infection (P. Más and R.N. Beachy, manuscript in preparation). We propose that the protruding structures are related to desmotubules, the appressed ER that comprises the central component of plasmodesmata (M was not associated with the protrusions, indicating a role of the MP in localization of vRNA with these structures. Together, these results may imply that at least two different types of ER are involved in TMV infection. One type of ER is involved in vRNA replication and does not require the presence of the MP. A second type corresponds to the filamentous protrusions that may be involved in the intercellular spread of the virus and requires a functional MP.
Model of TMV Infection
The data presented here and in previous publications are consistent with a model of TMV infection (Fig 11) in which the replication of vRNA takes place in close association with membranes of the ER (Fig 11, Fig 1). In this model, cytoskeletal elements are involved in targeting vRNA/replicase complexes to the perinuclear ER, perhaps via a retrograde flow of microtubules with ER attached as cargo. The ER-associated nascent vRNAs in replication complexes function as mRNAs for the synthesis of MP (Fig 11, Fig 2). The MP remains associated with vRNA in the complex, resulting in the formation of large ER-derived structures (Fig 11, Fig 3). At this point, the distribution of vRNA would be determined by a balance between the formation and anchoring of the large structures and their spread towards the periphery of the cell. Our results are consistent with a model in which MP and microfilaments participate in the formation and anchoring of the ER-derived structures (Fig 11, Fig 3), while microtubules are involved in the transport to their final destinations; i.e., to the periphery for intercellular spread, or toward the nucleus for degradation (Fig 11, Fig 4).
|
Several types of experimental evidence support this model. First, there is a dramatic effect on distribution of vRNA-M in cytochalasin Dtreated protoplasts. Not only were bodies not found in these protoplasts, in contrast to wt vRNA, but vRNA-
M was located on or near the cell periphery but not in the protrusions from the plasma membrane. Since in nontreated protoplasts vRNA-
M was not localized at the periphery in early stages of infection, the intracellular spread that normally occurs later in infection was apparently accelerated in the absence of microfilaments. Second, the clear association of vRNA-
M with microtubules would explain the role of microtubules in the intracellular spread of the virus towards the periphery of the cell. Third, at late stages of infection, the close relationship between the ER and microtubules explains the association of vRNA in the presence of the MP to a precursor of the plasmodesmata (Fig 11, Fig 5) that would result in the cell-to-cell spread of the virus in leaf tissues.
![]() |
Footnotes |
---|
Roger N. Beachy's current address is Donald Danforth Plant Science Center, Clayton, MO 63130.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We thank Drs. S. Halpain, C. Reichel, and B. Cooper for critical comments of this manuscript. We are also grateful to the Olympus Co. and Drs. S. Kay and S. Halpain (Department of Cell Biology, The Scripps Research Institute, La Jolla, CA) for use of the confocal laser scanning microscope.
This research was supported by National Science Foundation grant MCB 9631124 and by The Scripps Family Chair. P. Más was supported by fellowship from Ministerio de Educación y Cultura, Spain.
Submitted: 7 July 1999
Revised: 23 September 1999
Accepted: 5 October 1999
BFA, Brefeldin A; BiP, lumenal binding protein; CP, coat protein; fluor-RNA, fluorescein-RNA; GFP, green fluorescent protein; hpi, hours post-infection; MP, movement protein; TMV, tobacco mosaic virus; wt, wild-type
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|