measles virus-induced disruption of the glial-fibrillary-acidic

JOURNAL OF VIROLOGY,0022-538X/00/$04.0010

Apr. 2000, p. 3874–3880 Vol. 74, No. 8

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Measles Virus-Induced Disruption of the Glial-Fibrillary-AcidicProtein Cytoskeleton in an Astrocytoma Cell Line (U-251)

W. PAUL DUPREX,1* STEPHEN MCQUAID,2 AND BERT K. RIMA1

School of Biology and Biochemistry, The Queen’s University of Belfast, Belfast BT9 7BL,1 and NeuropathologyLaboratory, Royal Group of Hospitals Trust, Belfast BT12 6Bl,2 Northern Ireland, United Kingdom

Received 20 October 1999/Accepted 9 December 1999

A recombinant measles virus which expresses enhanced green fluorescent protein (MVeGFP) has been usedto infect two astrocytoma cell lines (GCCM and U-251) to study the effect of virus infection on the cytoskeleton.Indirect immunocytochemistry was used to demonstrate the cellular localization of the cytoskeletal compo-nents. Enhanced green fluorescent protein autofluorescence was used to identify measles virus-infected cells.No alteration of the actin, tubulin, or vimentin components of the cytoskeleton was observed in either cell type,whereas a disruption of the glial-fibrillary-acidic protein filament (GFAP) network was noted in MVeGFP-infected U-251 cells. The relative amounts of GFAP present in infected and uninfected U-251 cells werequantified by image analysis of data sets obtained by confocal microscopy by using vimentin, another inter-mediate filament on which MVeGFP has no effect, as a control.

The advent of reverse genetics for negative-stranded RNAviruses provides new opportunities for the examination andreassessment of various aspects of the virus infection process.Measles virus (MV) is a Morbillivirus which belongs to theParamyxoviridae. Like the other members of this family, MVhas a single-stranded negative-sense RNA genome which isencapsidated by nucleoprotein (N). Six structural genes areencoded by the genome. The polymerase (L) and phosphopro-tein (P) associate with the N protein to generate the helicalribonucleocapsid structure. Two glycoproteins, fusion (F) andhemagglutinin (H), are embedded in the pleomorphic virionenvelope, and these mediate cell entry and fusion (9, 13, 39,55). The matrix protein (M) associates with both the glycop-roteins and the ribonucleocapsid structure and plays a key rolein virion assembly (8, 41).

Many viruses have been shown to cause alterations to thecytoskeleton during in vitro infection (6, 10, 43, 47). For areview, see the work of Cudmore et al. (12). The MorbillivirusCanine distemper virus (CDV), has been reported to cause atotal reorganization of the cytoskeleton, with the most notablealterations being in the microtubule and intermediate-filamentnetworks (26). Vesicular stomatitis virus (VSV) infection, first,causes disassembly of the actin filaments and, second, altersthe distribution of the microtubules and intermediate filaments(44, 47). Respiratory syncytial virus (RSV) also causes a disrup-tion of the cytoskeleton (7, 21, 52). The effect of MV on theactin cytoskeleton is less clear. One group has reported astriking decrease in the overall number of actin bundles inhuman fibroblasts infected with MV. They also show a similardisruption upon infection with other Paramyxoviridae (16, 17).Contrary to this, a second group has not been able to demon-strate alterations to the actin cytoskeleton in MV-infectedVero cells (2).

Treatment of MV-infected cells with the actin-depolymeriz-ing agent cytochalasin B (CB) results in the inhibition of virusmaturation. This suggests that microfilaments play a role in the

release of budding virions (2, 48, 51). Actin filaments havebeen shown to have a role in the movement of MV glycopro-teins on the surfaces of infected cells (14). The involvement ofactin filaments in the budding of MV has been examined byelectron microscopy (4, 5). Again, a close association existsbetween actin filaments from the outer part of the cytoskeletalnetwork and budding virus, with the filaments protruding intothe particles. It has been suggested that budding is possibly theresult of a vectorial growth of actin filaments (4). CB inhibitsthe production of infectious virus particles of other paramyxo-viruses (7, 11, 24). Interestingly, CB has no effect on the mat-uration of VSV (23), which has been unequivocally shown todisrupt the actin cytoskeleton (44, 47). Recently the essentialrole of cellular actin in the gene expression and morphogenesisof RSV has been described. In this instance RSV infectioncauses a gross disruption of the actin cytoskeleton (7). Thus,there appears to be confusion in the literature. Additionally, itis not clear whether these alterations are active, i.e., induced tofacilitate virus growth, or passive, i.e., simply caused as a resultof infection but playing no formal role in virus replication.

A number of virus genomes, such as Simian virus 5, Mousehepatitis virus, Human herpesvirus, and Simian varicella-zostervirus, have been engineered to express green fluorescent pro-tein (GFP) (18, 19, 25b, 32). Recently the gene encoding en-hanced GFP (EGFP) has been introduced into the MV ge-nome, and a recombinant virus (MVeGFP) has been rescued(25a). We have demonstrated that EGFP is detectable in cellsin the early stages of infection (13a). In all cases diffuse EGFPautofluorescence was detectable before viral antigen was de-tected by immunocytochemistry. Therefore, EGFP appears tobe an ideal indicator of early MV cell infection and the recom-binant virus appears to be very useful for in vitro studies andmay also be beneficial for in vivo investigations. The diffusenature of EGFP autofluorescence makes MVeGFP an idealcandidate for assessing the effects of virus replication on thecytoskeletons of MVeGFP-infected cells. As tubulin has beenshown to stimulate MV RNA synthesis in vitro (36) and thefate of actin within MV-infected cells remains unclear (2, 16,17), we decided to use MVeGFP to investigate the effects ofMV infection on the cytoskeleton. Confocal scanning lasermicroscopy (CSLM) was used to gain maximal resolution indually labeled specimens.

* Corresponding author. Mailing address: School of Biology andBiochemistry, The Queen’s University of Belfast, Medical BiologyCentre, 97 Lisburn Rd., Belfast BT9 7BL, Northern Ireland, UnitedKingdom. Phone: 01232 272060. Fax: 01232 236505. E-mail: [email protected].

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MVeGFP infects astrocytoma cells. MV infection of thecentral nervous system (CNS) is a rare event (27, 30). Oligo-dendrocytes and neurons are the predominantly infected celltypes, although infected astrocytes have also been described (1,30, 31, 34). In this study we have used two astrocytoma celllines, the first being GCCM cells, which have been used tostudy MV cell-to-cell spread (13a). This cell line was derivedfrom an anaplastic astrocytoma (grade IV). The second cellline used was U-251 MG cells, which were established from ahuman glioma (3). These cells have been used to examine theinduction of inflammatory cytokines upon MV infection (22,45). Both cell lines were maintained in RPMI 1640 mediumsupplemented with 5% fetal calf serum. MVeGFP virus wasrescued from a full-length antigenomic clone (25a) by using acell line which expresses T7 RNA polymerase and the N and Pproteins of MV (42). MVeGFP was propagated in Africangreen monkey kidney cells (Vero). The gene encoding EGFP ispresent in an additional transcription unit (ATU) which isinserted before the N gene in the MV genome in the mostpromoter-proximal position. Due to this location, largeamounts of EGFP are produced in infected cells. No majoreffects on the replication of the virus and the type of cell-pathogenic effect generated was observed (25a). Autofluores-cence was readily observed during virus rescue and propaga-tion by UV microscopy. Cells which showed none of the well-characterized signs of MV-induced cell-pathogenic effectswere frequently observed. This demonstrates the strength ofusing MVeGFP for these experiments in that observations ofthe cytoskeletons of cells in the very early stages of virus in-fection can be made.

MVeGFP has no effect on the actin-, tubulin-, or vimentin-based cytoskeletons of astrocytoma cells. GCCM and U-251cells were grown to a confluence of 80% on glass coverslips.Cells were infected with MVeGFP at multiplicity of infectionof 0.01 for 1 h at 37°C, after which time the inoculum wasremoved and maintenance medium, RPMI 1640 containing2% fetal calf serum, was added. Infections were carried out for50 h at 37°C. During this time the cells attained 95 to 100%confluence. Cells were permeabilized and fixed by using freshlyprepared 4% paraformaldehyde. The cytoskeletons of the cellswere visualized with a monoclonal antibody specific for eithertubulin (Sigma) or vimentin (Dako). Antivimentin and antitu-bulin antibodies were diluted in phosphate-buffered saline(PBS) containing 0.5% Triton X-100 (1:100 and 1/1,500, re-spectively). The detergent was included to increase the perme-ability of the paraformaldehyde-fixed cells. Antibodies wereincubated on the coverslips for 20 h at 4°C. Unbound antibod-ies were removed by three washes in PBS, each lasting 5 min.CY3-conjugated sheep anti-mouse immunoglobulin G (Sigma)was used as a secondary antibody. Dilutions (1:40) were madein PBS containing 0.5% Triton X-100, and the antibody wasincubated on the coverslips for 3 h at 37°C. Unbound antibodywas removed as described above. Tetramethyl rhodamine iso-thiocyanate (TRITC)-conjugated phalloidin (Sigma), a fluo-rescently conjugated phallotoxin from Amanita phalloideswhich specifically binds to F-actin, was used to directly stainthe microfilaments. TRITC-conjugated phalloidin (200 ng/ml)in PBS was incubated on the coverslips for 2 h at 37°C. Excessphalloidin was removed by a single PBS wash. Coverslips weremounted with Citifluor (Amersham). A Leica TCS/NT confo-cal microscope equipped with a krypton-argon laser as thesource for the ion beam was used to examine the samples forfluorescence. CY3-stained samples were imaged by excitationat 568 nm with a 564- to 596-band-pass emission filter. EGFPwas visualized by virtue of its autofluorescence by excitation at488 nm with a 506- to 538-band-pass emission filter. Data sets

were collected by dual excitation, and image stacks were accu-mulated every 0.5 mm through an optical plane of 5 mm. Com-posite images were generated for the separate EGFP (green)and TRITC (red) channels in single-excitation mode to pre-vent spillover artifacts. Images were accumulated from regionsof the monolayer which contained uninfected and infectedcells and thereby permitted direct comparison of their cy-toskeletal networks.

MVeGFP infection of GCCM and U-251 cells led to exten-sive fusion. Syncytia which are typical of MV-infected cellswere observed. Nuclei clustered in the centers of the syncytia,and possibly due to a nonspecific accumulation of EGFP, thesewere brightly autofluorescent, as is shown for both cell types inFig. 1. EGFP was present diffusely throughout the cytoplasm,and no overlap was observed between the green and red chan-nels. This is particularly important as the most readily detect-able MV antigens, N and P, produce a pronounced punctatestaining pattern in extensively fused syncytia. Under these con-ditions, any overlap between channels by standard, dual-label-ing indirect immunofluorescence may give the impression thatalterations have occurred in the cytoskeleton. This effect isabsent when MVeGFP infection is used in conjunction withCSLM, and thus this combined technology provides an excel-lent approach for examination of the effects of MV on thecytoskeleton. Using this approach, we investigated the effectsof infection on microtubule, intermediate-filament, and micro-filament components of the cytoskeletons of U-251 andGCCM cells.

Actin. No disruption of the actin-based cytoskeleton wasobserved upon MVeGFP infection of either cell type (Fig. 1A).Actin is present in two forms within cells, a globular, mono-meric form (G-actin) and a polymerized, filamentous form(F-actin). It is the latter form which contributes to the cytoskel-eton and is detected by phalloidin (29). The distributions ofF-actin in GCCM and U-251 cells were similar to that observedin a previous study (20). In uninfected cells F-actin was presentin long parallel stress fibers. These ran along the long axes ofthe cells. Cortical filaments outlined the peripheries of bothcell types. In MVeGFP-infected cells which formed syncytia,the microfilaments were integrated into a larger, but organi-zationally similar, network. Extended fibers, which weregreater in length than those of the single cells, spanned thesyncytia (Fig. 1A, GCCM), indicating that actin polymerizationdoes not seem to be inhibited by virus infection. Clumping ofactin was noted in the U-251 cells (Fig. 1A). Equivalentamounts of F-actin appear to be present in both uninfectedand infected cells. Actin bundles were more prevalent inMVeGFP-infected GCCM cells (Fig. 1A) than in U-251 cells.These observations confirm what was previously shown fornonrecombinant MV-infected Vero cells (2) and contrast withthe results of two studies (16, 17) which observed severe actindisruption during MV infection of a human lung cell line. Wehave confirmed that MVeGFP infection of Vero cells causesno disruption of the actin cytoskeleton (data not shown). In arecent report, (24) colocalization of human parainfluenza virustype 3 (HPIV3) ribonucleoprotein (RNP) and actin microfila-ments was observed in infected CV-1 cells by confocal micros-copy with an HPIV3 polyclonal antiserum. The extent of co-localization was striking and demonstrates the usefulness ofCSLM in this type of investigation. In MV-infected cells wehave never observed a close relationship between MV RNPand the actin cytoskeleton using either polyclonal or monoclo-nal antibodies (anti-P or anti-N antibodies) for staining. Thisresult is in spite of the fact that actin is known to associate withMV nucleocapsid (36). Rather, a punctate perinuclear stainingpattern in which antigen is detected in MV-induced cytoplas-

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FIG. 1. Effect of MVeGFP infection on the cytoskeletons of GCCM and U-251 astrocytoma cells. Astrocytoma cells were infected with MVeGFP at a multiplicityof infection of 0.01 for 50 h. Cells were fixed and examined by CSLM for autofluorescence and immunoreactivity. Micrographs represent an 8- to 10-mm-deep compositeoptical section, and all images were obtained in double-excitation mode. EGFP was detected by virtue of its autofluorescence (green). (A) Actin microfilaments inGCCM and U-251 cells were visualized using TRITC-labeled phalloidin (red). The arrow indicates a single actin stress fiber in a GCCM cell. The arrowhead indicatesa nonfibrillary aggregation of actin in a U-251 cell. (B) Tubulin was visualized using a monoclonal antibody and a CY3-conjugated secondary antibody (red). The arrowindicates a tubulin-rich astrocytic process from a GCCM cell. The arrowhead in the U-251 panel indicates tubulin accumulation around the nucleus of an unaffectedcell. (C) Vimentin was visualized using a monoclonal antibody and a CY3-conjugated secondary antibody (red). The arrowhead indicates an astrocytic process froma U-251 cell. Magnification, 3400.

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mic inclusion bodies is observed. The HPIV3 system appearsto be unique in that the actin microfilaments seem to be di-rectly involved in viral-RNA synthesis in vivo (24). Interest-ingly, no disruption of the actin cytoskeleton was observedupon HPIV3 infection.

Tubulin. This component of the cytoskeleton has been im-plicated as having a role in the MV life cycle, possibly as asubunit of the viral RNA polymerase (35). The distribution ofthe microtubules in MV-infected cells has not been examinedpreviously by immunocytochemistry. The tubulin-based cy-toskeletons are quite similar in organization in both theGCCM and U-251 cells. The microtubule bundles are thinnerand more filamentous than F-actin stress fibers which crosssyncytia. Generally, tubulin was present throughout the cell atsimilar levels, although the processes seemed to be particularlyrich in microtubules (Fig. 1B, GCCM). There may be a slightaccumulation of tubulin around the nuclei of the U-251 cells(Fig. 1B). Tubulin distribution was examined in MVeGFP-infected GCCM cells. No disruption of the cytoskeleton wasobserved in infected cells. Filaments were longer in the syncy-tia, indicating that the dynamic process of microtubule assem-bly is not perturbed in infected cells engulfed in syncytia. Oneinvestigation has reported a thickening of the microtubules inHep-2 cells infected with the closely related Morbillivirus CDV(26). Bright foci and thick, long bundles crossing near oramong the multiple nuclei were also observed in these syncytia.We have not been able to detect any such accumulation inMV-infected astrocytoma cells. Disruption of the microtubuleshas been suggested to have a role in the bipolar budding ofSendai virus (50). A mutant virus which buds in a bipolarizedmanner has alterations in the M protein, and it has beensuggested that this protein may cause the alteration of micro-tubules. Involvement of the VSV M protein in microtubule

disruption has also been suggested (47). In that study majorchanges in tubulin distribution were detected soon after VSVinfection. As is the case for MV, a role has also been suggestedfor tubulin in VSV viral transcription (35).

Vimentin. The intermediate filament, vimentin, is a majorcomponent of the cytoskeleton. The fate of the vimentin net-work in MV-infected cells has not been examined previously.In uninfected GCCM and U-251 cells the overall structure ofthe vimentin component of the cytoskeleton was similar inorganization to the fine structure of filamentous tubulin. Itappears, however, that the filaments are less well organized inparallel arrays than either the microtubules or the microfila-ments. Astrocytic processes were particularly detectable in theU-251 cells by vimentin staining (Fig. 1C). We have previouslyshown that these processes mediate cell-to-cell spread of MVin vitro (13a). Once again no disassembly of this intermediatefilament was observed in MVeGFP-infected cells (Fig. 1C).Extended filaments which were longer than those present insingle cells were visible in syncytia, again indicating that as-sembly is not noticeably impaired within infected cells, as wasalso the case for actin and tubulin. A number of viruses havebeen shown to cause alterations in the intermediate-filament-based cytoskeletons (37, 46). RSV infection of Hep-2 cellsleads to morphological changes of vimentin and an overallreduction in abundance, possibly due to proteolytic degrada-tion (21). CDV infection of epithelial cells has been shown tolead to a disruption of the intermediate-filament network (26).

Disruption of the cytoskeleton therefore seems to dependon the virus studied. Here we clearly demonstrate that MVinfection does not perturb the microfilament-, intermediate-filament-, or microtubule-based cytoskeletons of the two astro-cytoma cell lines even when cell-pathogenic effect has pro-gressed to form large, but intact, syncytia.

FIG. 1—Continued.

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MVeGFP disrupts the GFAP-based cytoskeleton of astrocy-toma cells. Glial-fibrillary-acidic protein (GFAP) is an inter-mediate filament of the astrocytic cytoskeleton. This protein isfound almost exclusively in astrocytes and is therefore com-monly used as a marker for these cells (33). Its initial expres-sion marks the differentiation of precursor cells into astrocytes,and its up-regulation accompanies the reactive response to

CNS injury (15). Due to the contribution of GFAP to theastrocyte cytoskeleton, we examined the effects of MVeGFPinfection on the organization of this protein. Immunocyto-chemistry was carried out as described above. GFAP was de-tected using a rabbit polyclonal antiserum (Dako) at a dilutionof 1:100 in PBS containing 0.5% Triton X-100. CY3-conju-gated sheep anti-rabbit immunoglobulin G (Sigma) was used

FIG. 2. Effect of MVeGFP infection on the GFAP and vimentin cytoskeleton. U-251 cells were infected, fixed, and examined by CSLM for autofluorescence andimmunoreactivity, as described for Fig. 1. GFAP was visualized using a polyclonal antiserum and CY3-conjugated secondary antibody (red). Vimentin was visualizedusing a monoclonal antibody and a CY3-conjugated secondary antibody (red). EGFP autofluorescence (green) indicates infected cells. (A) Disruption of GFAP withinMVeGFP-infected syncytia. Magnification, 3160. The red line crossing the image indicates the region selected for quantification of green and red fluorescence usingthe TCS/NT software. (B) Lack of disruption of the vimentin cytoskeleton. The line used for subsequent quantification is shown in red. Magnification, 3160. (C)Intensity profile obtained from panel A showing a correlation between the decrease in GFAP (red) and the increase in EGFP (green) autofluorescence. (D) Intensityprofile obtained from panel B showing no alteration in vimentin (red) staining within areas of infection (green). (E to G) Severe disruption of GFAP cytoskeleton inU-251 cells. (E) Infection of cells shown by EGFP autofluorescence. (F) Vimentin staining. (G) Overlaid image. Magnification, 3400. Strongly positive GFAP cells werereadily observed (arrow c); these were not present within the syncytia. In more recently infected cells at the peripheries of the syncytia (arrow b), the intermediate-filament network was partially disrupted and the overall amount of GFAP staining was diminished. A residual amount of GFAP occasionally remained associated withthe nuclei of cells in the central areas of the syncytia (arrow a).



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as the secondary antibody. Immunocytochemical detection ofGFAP in paraformaldehyde-fixed GCCM cells proved prob-lematic (data not shown). This was disappointing, as we wishedto be consistent and continue to examine the cytoskeleton inMVeGFP-infected cells using EGFP autofluorescence as anindicator of infection. For this, paraformaldehyde fixation wasa prerequisite, as EGFP autofluorescence is rapidly lost whenall organic fixatives are used. Detection of GFAP expression inthe U-251 cells was more satisfactory. Nevertheless, it wasimportant to use the cells at a low passage number due to theoverall decrease in the levels of expression of GFAP as cellswere cultured. Not all U-251 cells stained equally for GFAP,and it proved essential to carry out incubations in the presenceof detergent to improve the detection of the fibrillary network.However, greater than 99% of cells were GFAP positive, per-mitting a satisfactory examination of the effects of MVeGFPinfection on this intermediate filament in these cells. Unin-fected U-251 cells stain brightly for GFAP, and MVeGFP wasobserved to efficiently infect GFAP-positive cells and formsyncytia (Fig. 2A). At this low magnification large numbers ofinfected and uninfected cells are shown. In the uninfected cellsGFAP was present as a fibrillary network which was similar inorganization to that of vimentin. Astrocytic processes, whichconnect the cells, also stained positive for GFAP. A severedisruption of the GFAP cytoskeletal network was seen inMVeGFP-infected cells, and the overall amount of GFAPstaining was reduced compared to that of the uninfected cells.Intensity profiles were plotted, using quantification softwareinstalled on the Leica TCS/NT confocal microscope, to assessthe overall levels of fluorescence derived from the presence ofGFAP and EGFP. A line was drawn across the composite datasets through a region of uninfected and infected cells. Theanalysis software was used to determine the total intensity ofred and green in each pixel present along the length of thisline, and the results were obtained as graphs (Fig. 2C and D).A direct correlation between infection (EGFP positive) anddecrease in GFAP levels was observed (Fig. 2C). The line fromwhich this profile was obtained is shown in red in Fig. 2A. Thistype of analysis was repeated for complete data sets collectedfrom 10 distinct syncytia present in different areas of the in-fected monolayer. Similar profiles were obtained in all cases.Duplicate coverslips, infected at the same time with the samevirus pool, were stained, as described above, for the interme-diate filament vimentin and are shown at the same magnifica-tion for comparison (Fig. 2B). No disruption of the vimentincytoskeleton had been observed previously (Fig. 1C). There-fore, this intermediate-filament protein served as the best con-trol for this type of analysis. In this case no decrease in theintensity of the vimentin staining was observed in the infectedarea (Fig. 2D). Again, the line chosen from which this profilewas produced is shown in red in Fig. 2B. Ten control data setsshowed no diminution of the amount of vimentin in infectedcells. An extreme example of GFAP disruption is shown at ahigher magnification (Fig. 2E to G). In this case GFAP wasbarely detectable within the main body of the syncytium. Pos-itive staining was observed within the syncytium, albeit at avery low level. This residual GFAP seemed to aggregatearound the nucleus. Cells on the periphery of a syncytium canbe assumed to be more recently infected than those in thecenter. GFAP staining in these infected cells was diminishedcompared to that in the uninfected cells, and the cytoskeletalnetwork showed a certain degree of reorganization. Thisseems, therefore, to represent an intermediate stage in thedisintegration of the GFAP cytoskeletal network. Associationof residual GFAP with the nucleus was also observed. Exam-ination of the GFAP fibrils demonstrated that they were much

shorter than those in uninfected cells. Therefore, a reorgani-zation of the GFAP-based cytoskeleton occurs in many of theinfected astrocytoma cells upon MVeGFP infection. This hasnot been previously reported for MV.

The closely related Morbillivirus CDV causes a demyelinat-ing disease (reviewed by Summers and Appel [49]). CDV pri-marily infects astrocytes producing intracytoplasmic and in-tranuclear inclusion bodies (40). A small percentage (5%) ofmacrophages are also thought to be infected in the acute de-myelinating lesions (38). Double labeling-immunohistochem-istry has been used to detect viral antigen and GFAP in astro-cytes present in, or derived from, brain tissues of CDV-infected animals. In vitro-infected primary cultures have alsobeen examined (25, 54, 56). An overall decrease has beenobserved, both in vivo and in vitro, in the numbers of GFAP-positive cells, the prevalence of astrocytic processes, andGFAP staining in some cells (53, 57). Neither of these studies,however, links this diminution in GFAP staining with a reor-ganization of the cytoskeleton. Infection of primary fetal as-trocytes with a lytic varicella-zoster virus causes a down-regu-lation or modification of GFAP expression (28). Morphologicalchanges in the GFAP cytoskeleton which were very similar tothose observed for MV infection were observed (Fig. 2), dem-onstrating that an alteration of the GFAP organization is notwithout precedent, albeit, in the case of varicella-zoster virus,in a lytic virus.

In this study we set out to examine the effect of MV infectionon the cytoskeleton. MVeGFP was used to facilitate the ex-amination of the cytoskeleton by CSLM because of its ability toexpress EGFP, which produces a diffuse cytoplasmic autofluo-rescence. We observed no disruption of the actin-, vimentin-,or tubulin-based cytoskeletal networks. A disruption of theGFAP cytoskeleton was observed. A direct correlation be-tween MVeGFP infection and a decrease in GFAP amountwas confirmed using quantitative confocal fluorescence micros-copy. This is the first time that this effect has been noted for afusogenic virus. Whether this disruption is a passive or activephenomenon remains unclear. It also remains to be seen if thisdecrease in GFAP mirrors the in vivo situation. This is an invitro study which has examined effects in transformed cell linesbecause they expressed appreciable levels of GFAP. Neverthe-less, this finding is important as the effect of MV on GFAP maygive rise to an underestimation of the numbers of infectedastrocytes in MV infection of the CNS.

We are very grateful to Martin Billeter for advice and constructivecriticism throughout the course of this study. We acknowledge the helpof Uta Gassen in the critical reading of the manuscript. We thank RoyCreighton for photographic work, Paula Haddock for excellent tech-nical assistance, and Aaron Maule for advice on phalloidin staining.

This work was supported by the Wellcome Trust (grant 047245).

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