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A functional role for intermediate filaments in the formation of frog virus 3 assembly sites
VIROLOGY
162,264-269
(1988)
A Functional
Role for intermediate
Filaments
K. G. MURTI,’ Department
in the Formation
of Frog Virus 3 Assembly
Sites
R. GOORHA, AND M. W. KLYMKOWSKY*
of Virology and Molecular Biology, St. Jude Children’s Research Hospital, 332 N. Lauderdale, P. 0. Box 3 18, Memphis, Tennessee 38 10 1, and *Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, Colorado 80309-0347 Received
June 29,
1987; accepted
September
22,
1987
During the course of frog virus 3 (FV3) infection in baby hamster kidney 21 (BHK) cells, vimentin-type intermediate filaments reorganize to surround the virus’s cytoplasmic assembly sites. To determine whether the association between vimentin filaments and viral assembly sites has a functional role in the virus life-cycle, we treated cells with the antimicrotubule drugs taxol or colchicine, or injected them with monoclonal-antivimentin antibodies prior to FV3 infection. Each of these reagents caused the collapse of the normally extended BHK intermediate filament system. In the case of taxol-treated or antivimentin-injected cells, the collapsed vimentin filaments were unable to reorganize around the newly forming viral assembly sites. The viral assembly sites that did form were aberrant and there was a significant reduction in the number of mature virions present. Colchicine, which also caused the collapse of vimentin filament organization, did not block the reorganization of vimentin filaments in response to viral infection and viral assembly sites appeared normal. These results suggest that intermediate filaments play an important role in maintaining the structural and functional integrity of FV3 assembly sites. 0 1988Academic PWSS. IIJC.
The function of intermediate filaments, a major component of the cytoskeleton in higher eukaryotes, remains obscure (I). In a number of different types of cultured cells, the antibody-induced disruption of intermediate filament organization appears to have no apparent effect on any aspect of cellular morphology or behavior (2-5, 22). Whether this is due to the subtlety of intermediate filament function, the artificiality of the culture situation, or a failure to look in an appropriate manner for a defect remains unclear. One possible approach to the question of intermediate filament function is to study the effect of the experimental disruption of intermediate filament organization in a system in which intermediate filaments appear to be associated with a biological event. One such system is the association between intermediate filaments and the viral assembly sites formed during the course of frog virus 3 (FV3) infection. FV3 is an icosahedral DNA virus that grows in a variety of cultured cells (6). Viral genomes, synthesized in the nucleus, and viral proteins, made in the cytoplasm, are transported to morphologically distinct viral assembly sites (7) which form within 4 to 8 hr of infection. These viral assembly sites are composed of viral proteins, genomes, and assembled virions and are devoid of cellular organelles, ribosomes, and cytoskeletal elements (8, 9, 17). The formation of these viral assembly sites is preceded by a dramatic reorganization of intermediate filaments, which come to encircle ’ To whom 0042-6822188
requests
for reprints
$3.00
CopyrIght 0 1988 by Academic Press, Inc. All rights of reproducbon in any form reserved.
should
the nascent assembly sites. This circumferential shell of intermediate filaments remains associated with the viral assembly sites throughout infection (9). In order to study the functional significance of the association between-intermediate filaments and viral assembly sites, we disrupted the organization of intermediate filaments in BHK cells either by drugs or by antivimentin antibody injection and studied the effect of such disruption on FV3 assembly. Of all the drugs that have been reported to affect intermediate filament organization, those that act through effects on microtubule organization appear to be the most specific. The primary effect of the drug colchicine is to depolymerize microtubules (10). In response to microtubule depolymerization, the intermediate filament system collapses (I 1, 12). The collapse of intermediate filament organization is presumably due to its dependence upon an intact microtubule system (13). The primary effect of the drug taxol appears to be to promote microtubule assembly (14, 15), and while doing so the drug induces a dramatic reorganization of the microtubule and the intermediate filament systems (16). We treated BHK cells with colchicine or taxol under a variety of conditions and examined them by immunofluorescence using antitubulin or antivimentin antibodies. We found that colchicine at 40 pg/ml completely depolymerized the microtubules and collapsed the intermediate filament organization within 2 hr. Cells treated with 50 &” taxol for 2 hr revealed many disorganized bundles of microtubules and the intermediate
be addressed. 264
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filaments either were collapsed near the nucleus or formed networks of thick bundles. These drug-induced cytoskeletal changes persisted as long as the drugs were present in the cuture media. We next examined the effect of the above drug-induced alterations of intermediate filament organization on FV3 replication. BHK cells were treated for 2 hr with 40 pg/ml colchicine or 50 @‘IA taxol and then infected with FV3 in the presence of the drug for 8 hr. The cells
265
were processed for immunofluorescence with antivimentin and anti-FV3 antibodies to determine whether or not (1) assembly sites formed and (2) intermediate filaments reorganized as in normal infection. Typical results from these experiments are shown in Fig. 1. BHK cells, not drug treated and infected with FV3 for 8 hr (controls), showed a reorganization of intermediate filaments around virus assembly sites as previously described (9). The cells treated with colchicine and
FIG. 1. lmmunofluorescence analysis of FV3 assembly and intermediate filament reorganization in cells treated with colchicine and taxol. BHK cells were grown, infected with FV3, and processed for indirect immunofluorescence as described (9). Intermediate filaments were labeled with a monoclonal antibody (No. 814318, Boehringer-Mannheim, Indianapolis, IN) and FV3 antigens were identified with an anti-FV3 polyclonal (mouse) antibody made by us (9). Fluorescein-conjugated goat anti-mouse antibodies were purchased from Miles Laboratories (Elkhart, IN). Colchicine was bought from Sigma Chemical Co. (St. Louis, MO) and taxol was a kind gift of Dr. Matthew Suffness (NIH, Bethesda, MD). In cells treated for 2 hr with 40 pg/ml of colchicine and infected for 8 hr with FV3 in the presence of the drug, the intermediate filaments surrounded the assembly sites (a) and FV3 assembly sites were normal (b). In cells treated for 2 hr with taxol and infected for 8 hr with FV3 in the presence of the drug, the intermediate filaments did not encircle the assembly sites (c) and the assembly sites were atypical (d). AS, virus assembly sites; IF, intermediate filaments; N, nucleus. Xl 500.
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then infected with FV3 also revealed a normal reorganization of intermediate filaments around assembly sites (Fig. la) and a distribution of FV3 antigens (Fig. 1 b) comparable to that of the control (9). By contrast, the cells treated with taxol and then infected with FV3 exhibited an abnormal distribution of intermediate filaments and atypical FV3 assembly sites. In these cells, the intermediate filaments were found as aggregates in the cytoplasm or around the nucleus (Fig. lc) but not encircling the assembly sites. In taxol-treated cells labeled with anti-FV3 antibodies, there was substantial fluorescence due to FV3 antigens (Fig. Id). However, viral proteins were not as concentrated as in normal assembly sites and the fluorescence was distributed in many clumps throughout the cytoplasm (compare Figs. 1 b and Id). The fluorescent clumps may represent abnormal assembly sites. Thus, the drug studies suggest that colchicine at concentrations as high as 40 pg/ml had no effect on the reorganization of intermediate filaments around the FV3 assembly site nor on FV3 assembly, whereas taxol at 50 pLIL1affected both events. To determine if the fluorescent clumps seen in taxol-treated and anti-FV3-labeled cells (Fig. 1 d) represent atypical viral assembly sites, we processed some of these samples for electron microscopy. The assembly sites in these cells when seen in the electron microscope were not as clearly demarcated as control
cells and were recognizable only by their lower electron density than the surrounding cytopsm (Fig. 2a). The interior of these assembly sites contained cell components such as microtubules, polysomes, and ribosomes (data not shown). In the normal assembly sites, the above cell components were never seen in the interior of the assembly sites by either immunofluorescent or electron microscopy (8, 9, 17). Many of these assembly sites also contained fewer assembling virions than those in controls (Fig. 2b). The results above suggested that virus assembly sites and virus assembly were normal in colchicinetreated cells but not in taxol-treated cells. To obtain a more accurate comparison of virus growth in untreated and drug-treated cells, we quantitated virus production in these cells by plaque assay. The results are shown in Table 1. The virus production was unaffected by 10 pg/ml or 40 pg/ml of colchicine. The virus production was unaffected by 10 PALMtaxol, but 50 PAA taxol reduced the virus yield by about 80%. The plaque assay results confirmed the immunofluorescence and electron microscopic studies which showed normal virus assembly in the presence of colchicine and atypical virus assembly in 50 &I taxol. The reduction of virus growth observed in cells treated with 50 PAA taxol may be due to some secondary effect of the drug either on the synthesis of viral genomes or on viral gene expression. To examine this,
FIG. 2. Electron micrographs of assembly sites in taxol-treated (a) and normal (b) cells. described in the legend of Fig. 1 and processed for electron microscopy as described (9).
The cells were
treated
with
taxol
and infected
as
SHORT TABLE
1
GROWTH OF FV3 IN UNTREATED AND DRUG-TREATED Treatment
BHK CELLS
Plaque-forming
Background No drug 10 pg/ml colchicine 40 pg/ml colchicine 10 &I tax01 50 pM taxol Note. The virus cells as described
was titrated (23).
COMMUNICATIONS
5.3 1.7 1.3 1.6 1.6 2.9 by plaque
assay
units/ml x x x X X x
lo4 10’ 10’ 10’ 10’ lo6
on fathead
minnow
we compared viral DNA and protein synthesis in untreated and taxol-treated cells and found no significant differences (data not shown). At this point, two possibilities could explain taxol’s effect on FV3 assembly site formation. Either taxol’s effect could be due to the inability of intermediate filaments in taxol-treated cells to reorganize around FV3 assembly sites or the effect could be the result of the taxol-induced stabilization of microtubules. As an independent test of the role of intermediate filament reorganization in the formation and/or maintenance of FV3 assembly sites, we examined the effect of antibodyinduced disruption of intermediate filaments on FV3 infection. We, and others, have previously shown that
267
the intracellular injection of antivimentin antibodies causes vimentin-type intermediate filaments to collapse in a variety of cell types (2-5). These studies also showed that the antibody-induced collapse of intermediate filament organization has no apparent effect on the extent or organization of the microtubule system. To test the effect of antivimentin-induced collapse of intermediate filaments on FV3 assembly, cells were infected with FV3 2 hr after antibody injection. Some of the antibody-injected cells were processed for immunofluorescence while others were used for electron microscopy. When examined by immunofluorescence, the cells injected with antivimentin antibodies showed collapsed bundles of intermediate filaments near the nucleus (Fig. 3a). In cells labeled with anti-FV3 antibodies, there were several loose aggregates of FV3 antigens (Fig. 3b) and these did not look like typical assembly sites (compare Figs. 3b and 1 b). The aggregates resembled those seen in taxol-treated cells (see Fig. Id). The cells injected with mouse IgGs showed normal reorganization of intermediate filaments around assembly sites and normal FV3 assembly (data not shown). When the antivimentin-injected and FV3-infected cells were examined by electron microscopy, the assembly sites were seen as irregular fibrous structures attached to the periphery of the larger masses of col-
FIG. 3. lmmunofluorescence analysis of cells that were microinjected with antivimentin antibodies and then infected clonal antibodies, antivimentin34g5 and antivimentin BM (No. 814318, Beohringer-Mannheim, Indianapolis, IN), were tion studies and both gave similar results. Microinjection of antivimentin antibodies into BHK cells was done as described were infected with FV3 for 8 hr and then examined by immunofluorescence. When labeled for intermediate filaments, juxtanuclear caps or collapsed masses of intermediate filaments (a). When labeled with anti-FV3 antibodies, these cells assembly sites (b). Xl 500.
with FV3. Two monoused in the microinjec(22). The injected cells these cells showed revealed many atypical
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lapsed intermediate filaments (Fig. 4a). The sites were not clearly demarcated from the surrounding cytoplasm and were identified as assembly sites because of the aggregation at these sites of virions at various stages of assembly (Fig. 4b). As in taxol-treated cells,
these assembly sites also contained microtubules (Fig. 4c) and polysomes (Fig. 4d). These assembly sites also showed fewer virions than the serum-injetted controls. To quantitate virus production in antivimentin-injected cells and normal serum-injected
FIG. 4. Electron micrographs of FV3 assembly sites of cells microinjected with antivimentin antibodies and infected with FV3. The cells, injected and infected as in the legend of Fig. 3. were processed for electron microscopy as described (9). The assembly sites (arrowheads) were seen attached to collapsed masses of intermediate filaments (a) and were not clearly demarcated from the surrounding cytoplasm (b). The sites also contained aggregates of microtubules (c) and polysomes (d). IF, intermediate filaments; v, virions; m, mitochondrion, mt, microtubules; p, polysomes.
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(control) cells, five assembly sites from each group of cells were serially sectioned and the total number of virus particles in the assembly sites was counted in the electron microscope. The assembly sites of antivimentin-injected cells contained 281 mature virions while those of serum-injected cells had 953 mature virions. Thus, there appears to be a 70% inhibition of virus production in antivimentin-injected cells. In general, the effect of antivimentin injection on viral assembly site morphology and virus growth was very much like that observed in taxol-treated cells. The taxol and microinjection studies described above have shown that the intermediate filaments play a crucial role in FV3 assembly. Both taxol and antivimentin microinjection prevented the reorganization of intermediate filaments around FV3 assembly sites. The failure of intermediate filaments to surround the assembly site led to (1) intrusion of cell components into the assembly site, (2) reduced accumulation of viral proteins, and (3) 70 to 80% inhibition of virus growth. Thus, intermediate filaments appear to be necessary to produce and/or maintain the structural integrity of the viral assembly sites, but they are not strictly required for the assembly of mature virions. The disorganized assembly sites that are formed in the absence of the circumferential intermediate filament system do produce mature virions, but they do so with dramatically lower efficiency than normal, intermediate filament-associated assembly sites. This implies that in FV3-infected BHK cells, intermediate filaments appear to help organize a region of the cytoplasm, the viral assembly site, into a more efficient three-dimensional ensemble of interacting viral components. A recent study has suggested that in murine 3T3 cells the intermediate filaments compartmentalize lipid globules for adipogenesis (19). A variety of animal viruses are shown to interact with the cytoskeleton for their assembly and transport (for a review, see (18)). Two viruses of the family Reoviridae, which assemble in cytoplasmic factories, have also been shown to interact specifically with the intermediate filaments (20, 21). The functional interaction between FV3 and intermediate filaments demonstrated here, however, constitutes the first direct experimental evidence that the intermediate filaments play an active role in virus assembly.
269
ACKNOWLEDGMENTS We express our appreciation to Dick McIntosh for his helpful comments on the manuscript; we thank Mrs. Kathy Troughton and Mrs. Ramona Tirey for their expert technical assistance, and Ms. Glenith D. White for typing the manuscript. This work was supported by grants from the American Cancer Society to K.G.M., the NIH to R.G. and M.W.K., and the American Lebanese Syrian Associated Charities. M.W.K. is a Pew Biomedical Scholar. A portion of this work was presented at the 26th Annual Meeting of the American Society for Cell Biology, 7-l 1 December 1986, Washington, D.C.
REFERENCES 1. TRAIJB, P., In “Intermediate Filaments.” Springer-Verlag, Berlin, 1985. 2. GAWLITTA, W., OSBORN, M., and WEBER, K., Eur. /. Cell Biol. 26, 83-90 (1981). 3. LIN, 1. J.-C., and FERAMISCO, J. R., Cell24, 185-193 (1981). 4. LANE, E. B., and KLYMKOWSKY, M. W., ColdSpring Harbor Symp. &ant. Biol. 46, 387-402 (1982). 5. KLYMKOWSKY, M. W., Nature (London) 291, 249-251 (1981). 6. GOORHA, R., and GRANOFF, A., Comp. Viral. 14,347-399 (1979). 7. GOORHA. R., MURTI, G., GRANOFF. A., and TIREY, R., virology 64, 31-50 (1978). 8. MURTI, K. G., PORTER, K. R., GOORHA, R., ULRICH, M., and WRAY, G., Exp. Cell Res. 154, 270-282 (1984). 9 MURTI, K. G., and GOORHA, R., /. Cell Biol. 92, 1248-1257 (1983). 10. OLMSTED, J. B., and BORISY, G. G.. Annu. Rev. Biochem. 43, 507-539 (1973). 11. GOLDMAN, R. D., and KNIPE, D. M., Cold Spring Harbor Symp. Quanr. B/o/. 37, 523-533 (1972). 12. BLOSE, S. H., and CHACKO, S., /. Cell Biol. 70, 459-466 (1976). 13. GOLDMAN, R. D.. MILSTED, A., SCHLOSS. J. A.. STARGER, J.. and YERNA. M. J., Annu. Rev. Physiol. 41, 703-722 (1979). 14. SCHIFF, P. B., and HORWITZ, S. B., Proc. Nat/. Acad. SC;. USA 77, 1561-1565 (1980). 15. DEBRABANDER, M., GUENS, G., NUYDENS, R., WILLEBRORDS, R., and DEMEY, J., Proc. Nat/. Acad. Sci. USA 78, 5608-5612 (1981). 16. GEUENS, G., DEBRABANDER, M., NUYDENS, R., and DEMEY, J., Cell B/o/. lnt. Rep. 7, 35-47 (1983). 17. DARLINGTON. R. W., GRANOFF, A., and BREEZE, D. C., Virology29, 149-156 (1966). 18. LUFTIG, R. B., /. Theor. Biol. 99, 173-l 91 (1982). 19. FRANKE, W. W., HERGT, M., and GRUND, C., Cell49, 131-141 (1987). 20. SHARPE, A. H., CHEN, L. B., and FIELDS, B. N., Virology 120, 399-411 (1982). 21. EATON, B. T.. HYATT, A. D., and WHITE, 1. R., Virology 157, 107-116 (1987). 22. KLYMKOWSKY. M. W., MILLER, R. H., and LANE, B., /. Cell Biol. 96, 494-509 (1983). 23. NAEGELE, R. F., and GRANOFF, A., Virology44, 286-295 (1971).
162,264-269
(1988)
A Functional
Role for intermediate
Filaments
K. G. MURTI,’ Department
in the Formation
of Frog Virus 3 Assembly
Sites
R. GOORHA, AND M. W. KLYMKOWSKY*
of Virology and Molecular Biology, St. Jude Children’s Research Hospital, 332 N. Lauderdale, P. 0. Box 3 18, Memphis, Tennessee 38 10 1, and *Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, Colorado 80309-0347 Received
June 29,
1987; accepted
September
22,
1987
During the course of frog virus 3 (FV3) infection in baby hamster kidney 21 (BHK) cells, vimentin-type intermediate filaments reorganize to surround the virus’s cytoplasmic assembly sites. To determine whether the association between vimentin filaments and viral assembly sites has a functional role in the virus life-cycle, we treated cells with the antimicrotubule drugs taxol or colchicine, or injected them with monoclonal-antivimentin antibodies prior to FV3 infection. Each of these reagents caused the collapse of the normally extended BHK intermediate filament system. In the case of taxol-treated or antivimentin-injected cells, the collapsed vimentin filaments were unable to reorganize around the newly forming viral assembly sites. The viral assembly sites that did form were aberrant and there was a significant reduction in the number of mature virions present. Colchicine, which also caused the collapse of vimentin filament organization, did not block the reorganization of vimentin filaments in response to viral infection and viral assembly sites appeared normal. These results suggest that intermediate filaments play an important role in maintaining the structural and functional integrity of FV3 assembly sites. 0 1988Academic PWSS. IIJC.
The function of intermediate filaments, a major component of the cytoskeleton in higher eukaryotes, remains obscure (I). In a number of different types of cultured cells, the antibody-induced disruption of intermediate filament organization appears to have no apparent effect on any aspect of cellular morphology or behavior (2-5, 22). Whether this is due to the subtlety of intermediate filament function, the artificiality of the culture situation, or a failure to look in an appropriate manner for a defect remains unclear. One possible approach to the question of intermediate filament function is to study the effect of the experimental disruption of intermediate filament organization in a system in which intermediate filaments appear to be associated with a biological event. One such system is the association between intermediate filaments and the viral assembly sites formed during the course of frog virus 3 (FV3) infection. FV3 is an icosahedral DNA virus that grows in a variety of cultured cells (6). Viral genomes, synthesized in the nucleus, and viral proteins, made in the cytoplasm, are transported to morphologically distinct viral assembly sites (7) which form within 4 to 8 hr of infection. These viral assembly sites are composed of viral proteins, genomes, and assembled virions and are devoid of cellular organelles, ribosomes, and cytoskeletal elements (8, 9, 17). The formation of these viral assembly sites is preceded by a dramatic reorganization of intermediate filaments, which come to encircle ’ To whom 0042-6822188
requests
for reprints
$3.00
CopyrIght 0 1988 by Academic Press, Inc. All rights of reproducbon in any form reserved.
should
the nascent assembly sites. This circumferential shell of intermediate filaments remains associated with the viral assembly sites throughout infection (9). In order to study the functional significance of the association between-intermediate filaments and viral assembly sites, we disrupted the organization of intermediate filaments in BHK cells either by drugs or by antivimentin antibody injection and studied the effect of such disruption on FV3 assembly. Of all the drugs that have been reported to affect intermediate filament organization, those that act through effects on microtubule organization appear to be the most specific. The primary effect of the drug colchicine is to depolymerize microtubules (10). In response to microtubule depolymerization, the intermediate filament system collapses (I 1, 12). The collapse of intermediate filament organization is presumably due to its dependence upon an intact microtubule system (13). The primary effect of the drug taxol appears to be to promote microtubule assembly (14, 15), and while doing so the drug induces a dramatic reorganization of the microtubule and the intermediate filament systems (16). We treated BHK cells with colchicine or taxol under a variety of conditions and examined them by immunofluorescence using antitubulin or antivimentin antibodies. We found that colchicine at 40 pg/ml completely depolymerized the microtubules and collapsed the intermediate filament organization within 2 hr. Cells treated with 50 &” taxol for 2 hr revealed many disorganized bundles of microtubules and the intermediate
be addressed. 264
SHORT
COMMUNICATIONS
filaments either were collapsed near the nucleus or formed networks of thick bundles. These drug-induced cytoskeletal changes persisted as long as the drugs were present in the cuture media. We next examined the effect of the above drug-induced alterations of intermediate filament organization on FV3 replication. BHK cells were treated for 2 hr with 40 pg/ml colchicine or 50 @‘IA taxol and then infected with FV3 in the presence of the drug for 8 hr. The cells
265
were processed for immunofluorescence with antivimentin and anti-FV3 antibodies to determine whether or not (1) assembly sites formed and (2) intermediate filaments reorganized as in normal infection. Typical results from these experiments are shown in Fig. 1. BHK cells, not drug treated and infected with FV3 for 8 hr (controls), showed a reorganization of intermediate filaments around virus assembly sites as previously described (9). The cells treated with colchicine and
FIG. 1. lmmunofluorescence analysis of FV3 assembly and intermediate filament reorganization in cells treated with colchicine and taxol. BHK cells were grown, infected with FV3, and processed for indirect immunofluorescence as described (9). Intermediate filaments were labeled with a monoclonal antibody (No. 814318, Boehringer-Mannheim, Indianapolis, IN) and FV3 antigens were identified with an anti-FV3 polyclonal (mouse) antibody made by us (9). Fluorescein-conjugated goat anti-mouse antibodies were purchased from Miles Laboratories (Elkhart, IN). Colchicine was bought from Sigma Chemical Co. (St. Louis, MO) and taxol was a kind gift of Dr. Matthew Suffness (NIH, Bethesda, MD). In cells treated for 2 hr with 40 pg/ml of colchicine and infected for 8 hr with FV3 in the presence of the drug, the intermediate filaments surrounded the assembly sites (a) and FV3 assembly sites were normal (b). In cells treated for 2 hr with taxol and infected for 8 hr with FV3 in the presence of the drug, the intermediate filaments did not encircle the assembly sites (c) and the assembly sites were atypical (d). AS, virus assembly sites; IF, intermediate filaments; N, nucleus. Xl 500.
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then infected with FV3 also revealed a normal reorganization of intermediate filaments around assembly sites (Fig. la) and a distribution of FV3 antigens (Fig. 1 b) comparable to that of the control (9). By contrast, the cells treated with taxol and then infected with FV3 exhibited an abnormal distribution of intermediate filaments and atypical FV3 assembly sites. In these cells, the intermediate filaments were found as aggregates in the cytoplasm or around the nucleus (Fig. lc) but not encircling the assembly sites. In taxol-treated cells labeled with anti-FV3 antibodies, there was substantial fluorescence due to FV3 antigens (Fig. Id). However, viral proteins were not as concentrated as in normal assembly sites and the fluorescence was distributed in many clumps throughout the cytoplasm (compare Figs. 1 b and Id). The fluorescent clumps may represent abnormal assembly sites. Thus, the drug studies suggest that colchicine at concentrations as high as 40 pg/ml had no effect on the reorganization of intermediate filaments around the FV3 assembly site nor on FV3 assembly, whereas taxol at 50 pLIL1affected both events. To determine if the fluorescent clumps seen in taxol-treated and anti-FV3-labeled cells (Fig. 1 d) represent atypical viral assembly sites, we processed some of these samples for electron microscopy. The assembly sites in these cells when seen in the electron microscope were not as clearly demarcated as control
cells and were recognizable only by their lower electron density than the surrounding cytopsm (Fig. 2a). The interior of these assembly sites contained cell components such as microtubules, polysomes, and ribosomes (data not shown). In the normal assembly sites, the above cell components were never seen in the interior of the assembly sites by either immunofluorescent or electron microscopy (8, 9, 17). Many of these assembly sites also contained fewer assembling virions than those in controls (Fig. 2b). The results above suggested that virus assembly sites and virus assembly were normal in colchicinetreated cells but not in taxol-treated cells. To obtain a more accurate comparison of virus growth in untreated and drug-treated cells, we quantitated virus production in these cells by plaque assay. The results are shown in Table 1. The virus production was unaffected by 10 pg/ml or 40 pg/ml of colchicine. The virus production was unaffected by 10 PALMtaxol, but 50 PAA taxol reduced the virus yield by about 80%. The plaque assay results confirmed the immunofluorescence and electron microscopic studies which showed normal virus assembly in the presence of colchicine and atypical virus assembly in 50 &I taxol. The reduction of virus growth observed in cells treated with 50 PAA taxol may be due to some secondary effect of the drug either on the synthesis of viral genomes or on viral gene expression. To examine this,
FIG. 2. Electron micrographs of assembly sites in taxol-treated (a) and normal (b) cells. described in the legend of Fig. 1 and processed for electron microscopy as described (9).
The cells were
treated
with
taxol
and infected
as
SHORT TABLE
1
GROWTH OF FV3 IN UNTREATED AND DRUG-TREATED Treatment
BHK CELLS
Plaque-forming
Background No drug 10 pg/ml colchicine 40 pg/ml colchicine 10 &I tax01 50 pM taxol Note. The virus cells as described
was titrated (23).
COMMUNICATIONS
5.3 1.7 1.3 1.6 1.6 2.9 by plaque
assay
units/ml x x x X X x
lo4 10’ 10’ 10’ 10’ lo6
on fathead
minnow
we compared viral DNA and protein synthesis in untreated and taxol-treated cells and found no significant differences (data not shown). At this point, two possibilities could explain taxol’s effect on FV3 assembly site formation. Either taxol’s effect could be due to the inability of intermediate filaments in taxol-treated cells to reorganize around FV3 assembly sites or the effect could be the result of the taxol-induced stabilization of microtubules. As an independent test of the role of intermediate filament reorganization in the formation and/or maintenance of FV3 assembly sites, we examined the effect of antibodyinduced disruption of intermediate filaments on FV3 infection. We, and others, have previously shown that
267
the intracellular injection of antivimentin antibodies causes vimentin-type intermediate filaments to collapse in a variety of cell types (2-5). These studies also showed that the antibody-induced collapse of intermediate filament organization has no apparent effect on the extent or organization of the microtubule system. To test the effect of antivimentin-induced collapse of intermediate filaments on FV3 assembly, cells were infected with FV3 2 hr after antibody injection. Some of the antibody-injected cells were processed for immunofluorescence while others were used for electron microscopy. When examined by immunofluorescence, the cells injected with antivimentin antibodies showed collapsed bundles of intermediate filaments near the nucleus (Fig. 3a). In cells labeled with anti-FV3 antibodies, there were several loose aggregates of FV3 antigens (Fig. 3b) and these did not look like typical assembly sites (compare Figs. 3b and 1 b). The aggregates resembled those seen in taxol-treated cells (see Fig. Id). The cells injected with mouse IgGs showed normal reorganization of intermediate filaments around assembly sites and normal FV3 assembly (data not shown). When the antivimentin-injected and FV3-infected cells were examined by electron microscopy, the assembly sites were seen as irregular fibrous structures attached to the periphery of the larger masses of col-
FIG. 3. lmmunofluorescence analysis of cells that were microinjected with antivimentin antibodies and then infected clonal antibodies, antivimentin34g5 and antivimentin BM (No. 814318, Beohringer-Mannheim, Indianapolis, IN), were tion studies and both gave similar results. Microinjection of antivimentin antibodies into BHK cells was done as described were infected with FV3 for 8 hr and then examined by immunofluorescence. When labeled for intermediate filaments, juxtanuclear caps or collapsed masses of intermediate filaments (a). When labeled with anti-FV3 antibodies, these cells assembly sites (b). Xl 500.
with FV3. Two monoused in the microinjec(22). The injected cells these cells showed revealed many atypical
268
SHORT
COMMUNICATIONS
lapsed intermediate filaments (Fig. 4a). The sites were not clearly demarcated from the surrounding cytoplasm and were identified as assembly sites because of the aggregation at these sites of virions at various stages of assembly (Fig. 4b). As in taxol-treated cells,
these assembly sites also contained microtubules (Fig. 4c) and polysomes (Fig. 4d). These assembly sites also showed fewer virions than the serum-injetted controls. To quantitate virus production in antivimentin-injected cells and normal serum-injected
FIG. 4. Electron micrographs of FV3 assembly sites of cells microinjected with antivimentin antibodies and infected with FV3. The cells, injected and infected as in the legend of Fig. 3. were processed for electron microscopy as described (9). The assembly sites (arrowheads) were seen attached to collapsed masses of intermediate filaments (a) and were not clearly demarcated from the surrounding cytoplasm (b). The sites also contained aggregates of microtubules (c) and polysomes (d). IF, intermediate filaments; v, virions; m, mitochondrion, mt, microtubules; p, polysomes.
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COMMUNICATIONS
(control) cells, five assembly sites from each group of cells were serially sectioned and the total number of virus particles in the assembly sites was counted in the electron microscope. The assembly sites of antivimentin-injected cells contained 281 mature virions while those of serum-injected cells had 953 mature virions. Thus, there appears to be a 70% inhibition of virus production in antivimentin-injected cells. In general, the effect of antivimentin injection on viral assembly site morphology and virus growth was very much like that observed in taxol-treated cells. The taxol and microinjection studies described above have shown that the intermediate filaments play a crucial role in FV3 assembly. Both taxol and antivimentin microinjection prevented the reorganization of intermediate filaments around FV3 assembly sites. The failure of intermediate filaments to surround the assembly site led to (1) intrusion of cell components into the assembly site, (2) reduced accumulation of viral proteins, and (3) 70 to 80% inhibition of virus growth. Thus, intermediate filaments appear to be necessary to produce and/or maintain the structural integrity of the viral assembly sites, but they are not strictly required for the assembly of mature virions. The disorganized assembly sites that are formed in the absence of the circumferential intermediate filament system do produce mature virions, but they do so with dramatically lower efficiency than normal, intermediate filament-associated assembly sites. This implies that in FV3-infected BHK cells, intermediate filaments appear to help organize a region of the cytoplasm, the viral assembly site, into a more efficient three-dimensional ensemble of interacting viral components. A recent study has suggested that in murine 3T3 cells the intermediate filaments compartmentalize lipid globules for adipogenesis (19). A variety of animal viruses are shown to interact with the cytoskeleton for their assembly and transport (for a review, see (18)). Two viruses of the family Reoviridae, which assemble in cytoplasmic factories, have also been shown to interact specifically with the intermediate filaments (20, 21). The functional interaction between FV3 and intermediate filaments demonstrated here, however, constitutes the first direct experimental evidence that the intermediate filaments play an active role in virus assembly.
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ACKNOWLEDGMENTS We express our appreciation to Dick McIntosh for his helpful comments on the manuscript; we thank Mrs. Kathy Troughton and Mrs. Ramona Tirey for their expert technical assistance, and Ms. Glenith D. White for typing the manuscript. This work was supported by grants from the American Cancer Society to K.G.M., the NIH to R.G. and M.W.K., and the American Lebanese Syrian Associated Charities. M.W.K. is a Pew Biomedical Scholar. A portion of this work was presented at the 26th Annual Meeting of the American Society for Cell Biology, 7-l 1 December 1986, Washington, D.C.
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