Mumps Virus Is Released from the Apical Surface of PolarizedEpithelial Cells, and the Release Is Facilitated by a Rab11-MediatedTransport System

Hiroshi Katoh, Yuichiro Nakatsu, Toru Kubota, Masafumi Sakata, Makoto Takeda, Minoru Kidokoro

Department of Virology III, National Institute of Infectious Diseases, Tokyo, Japan

ABSTRACT

Mumps virus (MuV) is an airborne virus that causes a systemic infection in patients. In vivo, the epithelium is a major replica-tion site of MuV, and thus, the mode of MuV infection of epithelial cells is a subject of interest. Our data in the present studyshowed that MuV entered polarized epithelial cells via both the apical and basolateral surfaces, while progeny viruses were pre-dominantly released from the apical surface. In polarized cells, intracellular transport of viral ribonucleoprotein (vRNP) com-plexes was dependent on Rab11-positive endosomes, and vRNP complexes were transported to the apical membrane. Expressionof a dominant negative form of Rab11 (Rab11S25N) reduced the progeny virus release in polarized cells but not in nonpolarizedcells. Although in this way these effects were correlated with cell polarity, Rab11S25N did not modulate the direction of virusrelease from the apical surface. Therefore, our data suggested that Rab11 is not a regulator of selective apical release of MuV,although it acts as an activator of virus release from polarized epithelial cells. In addition, our data and previous studies on Sen-dai virus, respiratory syncytial virus, and measles virus suggested that selective apical release from epithelial cells is used bymany paramyxoviruses, even though they cause either a systemic infection or a local respiratory infection.

IMPORTANCE

Mumps virus (MuV) is the etiological agent of mumps and causes a systemic infection. However, the precise mechanism bywhich MuV breaks through the epithelial barriers and achieves a systemic infection remains unclear. In the present study, weshow that the entry of MuV is bipolar, while the release is predominantly from the apical surface in polarized epithelial cells. Inaddition, the release of progeny virus was facilitated by a Rab11-positive recycling endosome and microtubule network. Ourdata provide important insights into the mechanism of transmission and pathogenesis of MuV.

Mumps is a common childhood illness characterized by pain-ful swelling of the parotid glands and is often accompanied

by severe complications such as orchitis, aseptic meningitis, pan-creatitis, and deafness (1). However, despite the prevalence andseriousness of the disease, the molecular basis of the pathogenesisof mumps is still poorly understood.

Mumps virus (MuV), which belongs to the genus Rubulaviruswithin the family Paramyxoviridae, is the causative agent ofmumps (2). The virus infection of cells is initiated by the bindingof the hemagglutinin-neuraminidase (HN) protein to sialic acidsof the cell surface (3). After receptor binding, the fusion (F) pro-tein induces pH-independent fusion of the viral envelope with thehost plasma membrane, and the viral genomic RNA is releasedinto the cytoplasm (4). The viral genomic RNA encapsidated bythe nucleocapsid (N) protein forms an active template for RNAreplication and transcription, a viral ribonucleoprotein (vRNP),with viral polymerases composed of the phosphoprotein (P pro-tein) and large (L) protein (5). Viral structural components syn-thesized in the cytoplasm are transported to the plasma mem-brane. At the plasma membrane, the matrix (M) protein organizesthe assembly of vRNP complexes (N, P, L, and genomic RNA) andenvelope proteins (F and HN), leading to efficient budding andrelease of progeny virions from the infected cells (6).

MuV is transmitted through aerosols or by direct contact withcontaminated respiratory secretions (7, 8). Following exposure,the virus initially infects the upper respiratory tract epithelia (8).After breaking through the epithelial barrier, the virus accesses theregional lymph nodes and exploits mononuclear cells for further

dissemination (9). During the viremia phase, MuV preferably in-fects epithelial cells in targeted organs, such as the parotid gland,testis, choroid plexus, pancreas, and kidney, resulting in the vari-ous symptoms described above. Although infection of the kidneyleads to viruria (10), virus shed in saliva is thought to play a moreimportant role in person-to-person transmission (7). Based onthe current knowledge of MuV pathogenesis (11), epithelial cellsare the primary targets of MuV in humans.

One of the characteristic features of epithelial cells is the pres-ence of distinct plasma membrane domains, the apical and baso-lateral membranes, which are separated by tight junctions (12).The apical membrane faces the lumen, whereas the basolateralmembrane abuts the underlying stratum of the epithelial cells.Because these membranes are exposed to different physiologicalenvironments, they have distinct profiles and distinct machineriesfor sorting proteins and lipids (13). The specialized properties of

Received 12 August 2015 Accepted 13 September 2015

Accepted manuscript posted online 16 September 2015

Citation Katoh H, Nakatsu Y, Kubota T, Sakata M, Takeda M, Kidokoro M. 2015.Mumps virus is released from the apical surface of polarized epithelial cells, andthe release is facilitated by a Rab11-mediated transport system. J Virol89:12026 –12034. doi:10.1128/JVI.02048-15.

Editor: D. S. Lyles

Address correspondence to Hiroshi Katoh, kato0704@nih.go.jp.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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epithelial cells can also influence virus infections. While the direc-tion of virus entry is strongly dependent on the distribution ofviral receptors, virus release is determined by the localization ofthe viral membrane proteins and/or the matrix protein (14–20).In the cases of paramyxoviruses, the entry of Nipah virus (NiV) isbipolar, because the entry receptor of NiV, ephrin-B2/-B3, is ex-pressed on both the apical and basolateral surfaces (21). On theother hand, the basolateral expression of nectin-4, which is anepithelial cell receptor for measles virus (MV), restricts entry ofthe virus to the basolateral surface (22). Unlike the polarity ofentry, selective apical releases have been commonly reported inparamyxovirus infections, including those with NiV (21), MV(23), Sendai virus (SeV) (24), and respiratory syncytial virus(RSV) (25). Small GTPase Rab11-mediated transport of the vRNPcomplex has been reported to facilitate the apical release ofparamyxoviruses as well as influenza A virus (IAV) (family Ortho-myxoviridae) (26–30).

In order to address the question of how MuV infects polarizedepithelial cells, we considered that it would be of interest to ana-lyze the polarity of MuV infection. Here, we present data ondirectional MuV entry and exit pathways in polarized cells. Inaddition, we demonstrate that MuV utilizes a Rab11-mediatedrecycling endosome system for efficient virus release from po-larized cells.

MATERIALS AND METHODSCells and virus. Madin-Darby canine kidney (MDCK) II, Calu-3 (humanlung epithelial), Vero (African green monkey kidney), and 293T (humankidney) cells were maintained in Dulbecco’s modified Eagle’s minimalessential medium (DMEM) (Nacalai Tesque, Kyoto, Japan) supple-mented with 100 U/ml penicillin, 100 mg/ml streptomycin (P/S), and10% fetal bovine serum (FBS). Rabbit kidney 13 (RK-13) cells were main-tained in minimal essential medium (MEM) (Life Technologies Inc.,Rockville, MD) supplemented with P/S and 5% FBS. MDCK II and Vero/hSLAM cells constitutively expressing EGFP-Rab11WT or -Rab11S25Nwere established in a previous study (29) and cultured in DMEM with10% FBS and 5 �g/ml puromycin. Calu-3 cells were transduced with aretroviral vector expressing EGFP-Rab11WT or -Rab11S25N as describedpreviously (29). For studies of polarized cells, MDCK and Calu-3 cellswere seeded and cultured on permeable Transwell filter membranes witha 0.4-�m or 3.0-�m pore size (Corning Inc., Corning, NY). The forma-tion of an electrically tight monolayer was checked by measuring thetransepithelial resistance (TER) with an ERS-2 apparatus (Millipore, Bed-ford, MA).

The MuV strain Odate, which was isolated from a patient who devel-oped aseptic meningitis (31), and the vaccinia virus strain LC-16 (32)were used in this study.

Plasmids and transfection. Plasmids encoding enhanced green fluo-rescent protein (EGFP)-tagged wild-type Rab11 and dominant negativeRab11 (with the mutation S25N) were provided by Tadaki Suzuki. 293Tcells were transfected with the plasmids by using TransIT LT1 (Mirus,Madison, WI) and subsequently superinfected with MuV. To assess thetransfection efficiency, the number of EGFP-positive cells was countedand expressed as a percentage of the total number of cells that were stainedwith 4=,6-diamidino-2-phenylindole (DAPI). The percentages of 293Tcells expressing EGFP-Rab11WT and -Rab11S25N were �95% for both.

Reagent and antibodies. Nocodazole was purchased from Sigma (St.Louis, MO). Anti-N (clone 23D), -M (clone 79D), and -HN (clone 78)mouse monoclonal antibodies (MAbs) and anti-MuV N rabbit polyclonalantibody (PAb) were prepared as described previously (33–35). Anti-�-actin (clone AC-15) and anti-�-tubulin (clone DM1A) mouse MAbs werepurchased from Sigma. Anti-EGFP mouse MAb and anti-ZO-1 rabbit

PAb (ab59720) were purchased from Clontech (Mountain View, CA) andAbcam (Cambridge, United Kingdom), respectively.

Virus titration. Infectious titers of MuV were determined in triplicateby plaque assay using Vero cells in 12-well plates. After 1 to 2 h of virusadsorption, the cells were cultured in DMEM with 5% FBS and 1% aga-rose. At 6 days postinoculation, the cells were stained with neutral redsolution (Sigma), and the plaque counts were determined. Infectious ti-ters of vaccinia virus were determined in triplicate by plaque assay usingRK13 cells in 12-well plates. After 1 h of virus adsorption, the cells werecultured in MEM with 5% FBS and 1.5% carboxymethyl cellulose. At 2days postinoculation, the cells were stained with crystal violet, and theplaque counts were determined.

Cell extracts and immunoblotting. For the preparation of cell ex-tracts, cells were washed twice with cold phosphate-buffered saline (PBS)and then lysed in cell lysis buffer (20 mM Tris-HCl [pH 7.5], 135 mMNaCl, 1% Triton X-100, and protease inhibitor cocktail [Complete Mini;Roche, Mannheim, Germany]). For immunoblotting, the cell lysate wasboiled in sodium dodecyl sulfate (SDS) sample buffer and subjected toSDS-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins weretransferred to polyvinylidene difluoride membranes (Millipore) and in-cubated with the appropriate antibodies. Each protein was visualized withSuperSignal West Femto maximum-sensitivity substrate (Life Technolo-gies Inc.) and detected by use of an LAS-3000 image analyzer system (FujiFilm, Tokyo, Japan).

Immunofluorescence microscopy. Cells were fixed in 4% parafor-maldehyde in PBS for 15 min at room temperature. Then, the cells werepermeabilized with 0.2% Triton X-100 in PBS for 10 min, blocked withPBS containing 2% bovine serum albumin (BSA) for 30 min at roomtemperature, and incubated with the appropriate antibodies. F-actin andnuclei were stained with Alexa 594-conjugated phalloidin (Life Technol-ogies Inc.) and 4=,6-diamidino-2-phenylindole (DAPI), respectively. Thesamples were examined under an FV1000D confocal laser-scanning mi-croscope (Olympus, Tokyo, Japan).

Virion purification. Virions released from cells were purified as de-scribed previously (36) with minor modifications. The culture media ofMDCK/EGFP-Rab11WT and -Rab11S25N cells infected with MuV wereharvested at 24 h postinfection (p.i.) and centrifuged at 7,500 � g for 2min to remove cell debris. The supernatants were layered on 20% sucrosein TNE (0.1 M NaCl; 0.01 M Tris-HCl, pH 7.5; 0.001 M EDTA) (wt/vol)and centrifuged in an SW41 rotor (Beckman Coulter, Inc., Brea, CA) at140,000 � g for 1.5 h. Pellets were then resuspended in 0.5 ml of TNE andmixed with 1.3 ml of 80% sucrose in TNE, making 1.8 ml of �60% sucrosesolution with samples. Layers containing 50% sucrose (1.8 ml) and 10%sucrose (0.6 ml) in TNE were applied to the samples in the �60% sucrosesolution. The sucrose gradient solutions were then centrifuged in anSW55Ti rotor (Beckman) at 110,000 � g for 3 h. The fractions (1 ml each)were collected from the top of the gradient. MuV virions, which werefloated in the second fraction from the top, were precipitated with trichlo-roacetic acid (TCA) and analyzed by immunoblotting.

RESULTSMuV entry is bipolar, but release is restricted to the apical sur-face in polarized epithelial cells. To assess restriction effects of thepore size for migration of MuV through membrane filters, non-polarized Vero cells were infected with MuV and grown on0.4-�m or 3.0-�m Transwell filters, and at 24 h p.i., the virus titersin the apical and basolateral chambers were determined. Virustiters in the basolateral chamber were �10 times lower than thosein the apical chamber, when 0.4-�m filters were used (Fig. 1A).On the other hand, the difference was less than 3 when 3.0-�mfilters were used (Fig. 1A). Thus, 3.0-�m filters were used for thiswork, unless otherwise noted. To analyze the directional entry andrelease of MuV in epithelial cells, polarized MDCK cells were in-fected with MuV at either the apical or basolateral surface, and

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virus titers in the apical and basolateral media were determined,respectively. As shown in Fig. 1B and C, MuV was predominantlydetected in the apical chamber, regardless of the virus entry route.The basolaterally infected cells produced �3-fold-lower virus ti-ters than the apically infected cells (Fig. 1C). However, this reduc-tion was likely due to the small restriction of virus migrationthrough the 3.0-�m filters, as shown in Fig. 1A. Therefore, theefficiency of virus entry was comparable between the apical andbasolateral infection. MuV infection did not cause significant cy-topathic effects in MDCK cells or disrupt the integrity of the po-larized cell layer displaying a high TER (�180 �/cm2) until 96 hp.i. As in MDCK cells, MuV showed the bipolar entry, the apicalrelease, and little cytopathic effect in another polarized epithelialcell line, Calu-3 (Fig. 1D and E). Analyses by confocal microscopyshowed that each viral particle component, i.e., the N (vRNP), M(matrix), and HN (membrane) proteins, was predominantlytransported to the apical surface in both polarized MDCK andCalu-3 cells (Fig. 1F and G). Collectively, these data indicate thatMuV entry is bipolar, while viral release is restricted to the apicalsurface in polarized epithelial cells.

Rab11 plays key roles in apical transport of vRNP and effi-cient virus production in polarized epithelial cells. Rab11-de-pendent apical transport has been reported to function in traffick-ing of the vRNP complex and efficient virus production of manyRNA viruses, such as IAV, RSV, SeV, and MV (26-30, 37). Toexamine the roles of Rab11 in the apical transport of MuV vRNP,the intracellular localizations of MuV proteins in MDCK cells ex-pressing the EGFP-Rab11 wild-type (Rab11WT) or its dominantnegative form (Rab11S25N) were used (29). As shown in Fig. 2A,the MuV N protein was colocalized with EGFP-Rab11WT andaccumulated at the apical surface, whereas it was concentratedin the cytoplasm of polarized MDCK cells expressing EGFP-Rab11S25N. In EGFP-Rab11WT-expressing MDCK cells, the Mprotein was accumulated at the apical surface but poorly colocal-ized with EGFP-Rab11WT (Fig. 2A). On the other hand, the Mprotein mostly showed a diffuse distribution pattern in the cyto-plasm in EGFP-Rab11S25N-expressing MDCK cells (Fig. 2A).Similar distribution patterns of the N and M proteins were alsoobserved in polarized Calu-3 cells expressing either EGFP-Rab11WT or -Rab11S25N (Fig. 2B). In contrast, the N and M

FIG 1 Directional entry and release of MuV from polarized epithelial cells. (A) Vero cells on 0.4-�m or 3.0-�m polycarbonate Transwell filters were infectedwith MuV at a multiplicity of infection (MOI) of 5.0. Apical and basolateral culture supernatants were collected separately at 24 h p.i., and the infectious titerswere determined by plaque assay. (B to E) Polarized MDCK (B and C) and Calu-3 (D and E) cells on 3.0-�m Transwell filters were infected with MuV from theapical or basolateral surface at an MOI of 5.0. Apical and basolateral culture supernatants were collected separately at 24 h p.i., and the infectious titers weredetermined by plaque assay (C and E). The percentages of total release in the apical and basolateral media are shown in panels B and D. (F and G) PolarizedMDCK (F) and Calu-3 (G) cells infected with MuV were immunostained at 24 h p.i. with mouse anti-N, -M, or -HN MAb and AF488-conjugated anti-mouseIgG. Cortical actin and cell nuclei were visualized by AF594-phalloidin (red) and DAPI (blue), respectively. The significance of differences was determined byStudent’s t test.

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FIG 2 Rab11 plays a role in apical transport of vRNP in polarized cells. Polarized MDCK (A), Calu-3 (B), or nonpolarized Vero (C) cells expressing EGFP-Rab11WT or -Rab11S25N were infected with MuV at an MOI of 1.0. At 24 h postinfection, the cells were immunostained with mouse anti-MuV N, M or HNMAb and AF633-conjugated anti-mouse IgG (pseudocolored red). Cortical actin was visualized by AF594-phalloidin (pseudocolored blue).

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proteins were barely localized at the plasma membrane in bothEGFP-Rab11WT- and EGFP-Rab11S25N-expressing Vero cells(Fig. 2C). Expression of Rab11S25N did not influence the local-ization pattern of the HN protein any of the three cell lines (Fig. 2Ato C). These results suggested that the vRNP, M, and HN proteinsare separately transported to the apical surface and that Rab11contributes differently to the intracellular transport of the vRNPand M and HN proteins in polarized epithelial cells.

Next, to investigate the effect of altered Rab11 function onMuV propagation, the virus titers released from the cells express-ing either EGFP-Rab11WT or -Rab11S25N were determined (Fig.3A). In a control infection, expression of EGFP-Rab11S25N didnot inhibit vaccinia virus replication in polarized MDCK cells(Fig. 3B). In contrast, virus titers in polarized MDCK and Calu-3

cells expressing EGFP-Rab11S25N were �20-fold and �3-foldlower, respectively, than those in the EGFP-Rab11WT-expressingcells. In the case of nonpolarized cells, the virus titer in the cellsexpressing EGFP-Rab11S25N was unaffected (293T cells) or wasslightly higher than that in the cells expressing EGFP-Rab11WT(Vero cells), as seen in MV infection (29).

To further characterize the role of Rab11 in MuV propagation,the viral protein synthesis and the physical particle production inEGFP-Rab11WT- and EGFP-Rab11S25N-expressing cells wereanalyzed. Although viral protein synthesis was slightly decreasedin MDCK cells expressing EGFP-Rab11S25N (Fig. 3C), this re-duction was not sufficient to explain the inhibition of virus pro-duction shown in Fig. 3A. In contrast, physical viral particles pu-rified and detected by immunoblotting of the N and M proteins

FIG 3 Rab11-mediated transport of vRNP is important for efficient MuV release from the apical surface of polarized cells. (A) Polarized MDCK or Calu-3 ornonpolarized Vero or 293T cells expressing either of the EGFP-Rab11s were infected with MuV at an MOI of 5.0. At 24 h p.i., the supernatants were collected, andthe infectious titers were determined. The results shown are from three independent experiments, with the error bars representing the standard deviations. (B)Polarized MDCK cells expressing either of the EGFP-Rab11s were infected with vaccinia virus at an MOI of 10. At 24 h p.i., both the supernatants and cells werecollected and sonicated, and then the infectious titers were determined. The results shown are from three independent experiments, with the error barsrepresenting the standard deviations. (C) Polarized MDCK cells expressing either of the EGFP-Rab11s were infected with MuV. At 24 h p.i., the cell lysates weresubjected to immunoblotting with the indicated antibodies. The relative band intensities of MuV N were normalized by the �-actin level, and the relativeexpression of MuV N based on the levels of cells expressing EGFP-Rab11WT is shown, with the error bars representing the standard deviations. (D) PolarizedMDCK cells expressing either of the EGFP-Rab11s were infected with MuV. At 24 h p.i., the proteins in the cell lysates and the floated fraction were detected byimmunoblotting with the indicated antibodies. The relative band intensities of MuV N and M in the floated fraction were normalized by those in the cell lysate,and the relative expressions of MuV N and M were determined based on the levels of cells expressing EGFP-Rab11WT. (E and F) Polarized MDCK expressingeither of the EGFP-Rab11s on 3.0-�m Transwell filters were infected with MuV from the apical surface at an MOI of 5.0. Apical and basolateral culturesupernatants were collected separately at 24 h p.i., and the infectious titers were determined by plaque assay. The percentages of total release in the apical andbasolateral media are shown in panel E. The significance of differences was determined by Student’s t test.

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were barely detectable in the culture media of EGFP-Rab11S25N-expressing cells, indicating the impairment of particle formationin these cells (Fig. 3D). In order to analyze the effect of interrup-tion of Rab11-mediated protein sorting on directional MuV bud-ding, virus titers in both the apical and basolateral media ofMDCK cells expressing either EGFP-Rab11WT or -Rab11S25Nwere determined. In both MDCK/EGFP-Rab11WT and -Rab11S25Ncells, the progeny virus was predominantly detected in the api-cal-chamber media (Fig. 3E and F), indicating that the disrup-tion of Rab11 function did not alter the budding side. Takentogether, these findings indicate that Rab11-mediated apicaltransport is required for efficient virus production in polarizedepithelial cells.

Rab11-mediated transport of vRNP is dependent on cell po-larity. Next, we assessed the apical transport of vRNP mediated byRab11 in the context of polarity. At all measurement time pointsbefore 4 days postseeding (p.s.), MDCK cells were not yet polar-ized, as indicated by the low TER (180 �/cm2) and the lack ofdistinct ZO-1 staining, which is a marker of the tight junction, atthe cell junction (Fig. 4A). At 4 days p.s., MDCK cells had estab-lished a polarized monolayer displaying a high TER (�180�/cm2) and clear ZO-1 staining. Both nonpolarized (1 days p.s.)and polarized (5 days p.s.) MDCK cells were infected with MuV,and virus production and vRNP transport were analyzed. Expres-sion of EGFP-Rab11S25N caused a 3-fold reduction in the re-leased progeny virus in nonpolarized MDCK cells but had a muchmore pronounced effect in polarized MDCK cells, where a �30-fold reduction was observed (Fig. 4B). As shown above, the apicaltransport of vRNP was not observed in polarized MDCK cellsexpressing EGFP-Rab11S25N (Fig. 4C). On the other hand, theexpression of EGFP-Rab11S25N had a small effect on the surfacetransport of vRNP in nonpolarized MDCK cells. The data showedthat Rab11 is involved in the trafficking of vRNP, especially incompletely polarized epithelial cells.

Apical transport of vRNP is dependent on the MT network.Since it has been reported that the vRNP complexes of other RNAviruses are transported along MTs (28–30, 38), we analyzed theeffects of nocodazole-induced MT disruption on the vRNP traf-ficking and virus release in polarized epithelial cells. Nocodazoletreatment disrupted the structures of MTs (Fig. 5A) and reducedMuV release from the cells (Fig. 5B). Since viral protein synthesiswas not affected by nocodazole treatment (Fig. 5C), the MTs weresuggested to play roles in the viral assembly and/or budding steps.Immunofluorescence analysis revealed that the N protein wasrarely observed at the apical surface of nocodazole-treated cells(Fig. 5D). Instead, we observed inclusion bodies (IBs) that werelarger than those in the absence of the drug. Furthermore, fewersmall N-positive dot-like structures, which were thought to be thevRNP en route to the assembly site, were observed in the nocoda-zole-treated cells. These findings suggest that MuV vRNPs aretransported in an MT-dependent manner from IBs to the apicalmembrane, where viral assembly and budding occur.

DISCUSSION

In general, virus entry in polarized cells is correlated with theapical and basolateral distribution of the entry receptor (17–21).Because the MuV receptor, sialic acid, is expressed on both theapical and basolateral surfaces, MuV enters the polarized epithe-lial cells from both sides. The infection from the apical surface hasthe advantage that it facilitates the transmission of virus to neigh-

boring cells, because virus release occurs via the apical (luminal)side, leading to an efficient regional spread. On the other hand, thebasolateral entry is likely used for secondary infection via thebloodstream during the viremic phase. Therefore, the bipolar en-try of MuV is thought to be an essential viral strategy for theestablishment of local and systemic infections.

In contrast to the bipolar entry, the release of MuV occurspredominantly from the apical surface. The direction of virus re-lease is assumed to determine the spread of infection (39). Baso-lateral virus release is thought to contribute to systemic spread,

FIG 4 Rab11-mediated transport of vRNP is dependent on cell polarity. (A)Polarized and nonpolarized MDCK cells were immunostained with rabbitanti-ZO-1 PAb. (B) Polarized or nonpolarized MDCK cells expressing eitherof the EGFP-Rab11s were infected with MuV at an MOI of 5.0. At 24 h p.i., thesupernatants were collected, and the infectious titers were determined. Theresults shown are from three independent experiments, with the error barsrepresenting the standard deviations. The significance of differences was de-termined by Student’s t test. (C) Polarized or nonpolarized MDCK cells ex-pressing either of the EGFP-Rab11s were infected with MuV. At 24 h p.i., thecells were immunostained with mouse anti-MuV N MAb and AF633-conju-gated anti-mouse IgG (pseudocolored red). Cortical actin was visualized byAF594-phalloidin (pseudocolored blue).

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while apical virus shedding from the epithelia causes primarilylocal infections restricted to mucosal surfaces. In keeping with thismodel, the budding of wild-type SeV is restricted to the apicalsurface and thus causes a local respiratory infection, while muta-tion in the M protein has been shown to disrupt the cellular po-larity and cause bipolar budding of the virus, resulting in a sys-temic infection in mice (40, 41). In the case of the MuV infection,apical release supports efficient virus replication in the glandularepithelium, especially the parotid gland, and virus shedding insaliva, leading to person-to-person transmission. Selective apicalrelease from epithelial cells is a common strategy of paramyxovi-ruses, even though the virus causes either a systemic infection or alocal respiratory infection, indicating that the direction of virusrelease may not be a determinant of paramyxoviral tissue tro-pisms.

So far, the intracellular trafficking of MuV structural compo-nents has not been addressed. In this study, we found that apicaltransport of the N protein was mediated by a Rab11- and MT-dependent mechanism in polarized epithelial cells. A number ofstudies have reported that the N protein of paramyxoviruses con-stitutes vRNP in concert with the P and L proteins and viralgenomic RNA (2). Since we previously observed colocalization ofthe N protein with other vRNP components in infected cells (35),it would appear that the intracellular dynamics of the MuV Nprotein in the infected cells represents the vRNP trafficking. In-tracellular sorting and trafficking of the apical and basolateralmembrane components are important to establish and maintainepithelial cell polarity (12). Apical recycling endosomes (ARE) areinvolved in regulated recycling of specialized apical proteins (42),

and Rab11 is a key regulator of ARE and plays roles in the apicalvRNP trafficking and particle formation of many RNA viruses inpolarized epithelial cells (26–30). However, the disruption ofRab11 function did not affect the apical expression of the HNprotein. As the surface glycoproteins are indispensable for theproduction of infectious MuV, it must be expected that the apicalbudding is not altered, even though the vRNP and M protein aredifferently targeted. Collectively, our data suggest that Rab11 actsas an activator of MuV release from polarized epithelial cells butthat it is not a regulator of selective apical release.

Rab11 interacts with many effector proteins and regulates var-ious cellular functions, including plasma membrane recycling, en-dosomal membrane organization, and cytokinesis, and its func-tions are dependent on the cell type, such as polarized ornonpolarized (43–46). Our data suggested that the Rab11-depen-dent vRNP transport would be important for efficient virus releasefrom polarized cells, because the number of virus particles re-leased from polarized cells, in which vRNP was transported in aRab11-mediated manner, was much higher than that from non-polarized cells, and the efficient release from polarized cells wascancelled by disrupting the Rab11 function (Fig. 4C). Therefore,Rab11 might have additional roles in the late stages, such as as-sembly and/or budding, at the apical surface of polarized epithelialcells.

The transport of MuV HN protein was not regulated by Rab11.Like other envelope glycoproteins, the HN protein is synthesizedin the endoplasmic reticulum (ER) and transported by the secre-tory pathway. In contrast, the apical accumulation of the M pro-tein was impaired by disrupting the Rab11 functions. Data from

FIG 5 Roles of microtubules in the apical transport of vRNP and viral production. (A) Polarized MDCK cells treated with nocodazole (5 �M) or dimethylsulfoxide (DMSO) were immunostained with mouse anti-�-tubulin MAb. (B to D) Polarized MDCK cells infected with MuV at an MOI of 5.0 were treated withnocodazole (5 �M) or DMSO. (B) The supernatants were collected at 24 h p.i., and the infectious titers were determined. The results shown are from threeindependent experiments, with the error bars representing the standard deviations. The significance of differences was determined by Student’s t test. (C) At 24h p.i., cell lysates were collected and subjected to immunoblotting with mouse anti-�-actin MAb and rabbit anti-MuV N PAb. The relative band intensities ofMuV N were normalized to the �-actin level, and the relative expression of MuV N is shown based on the levels of DMSO-treated cells. (D) At 24 h p.i., the cellswere immunostained with mouse anti-MuV N MAb (green). Cell nuclei were stained with DAPI (blue).

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various viruses showed that viral matrix proteins themselves maybe soluble in the cytoplasm or associate with the cellular mem-brane via electrostatic forces or specific fatty acid modification (6).The mechanism of intracellular transport of MuV M protein ispoorly understood, but our data suggested that the M protein mayuse a particular transport machinery regulated directly or indi-rectly by Rab11. Alternatively, the apical accumulation of the Mprotein may be due to association with a highly stable apical bind-ing partner, such as the N protein, because the interaction of the Mprotein with the N protein of parainfluenza virus 5, which belongsto the genus Rubulavirus, is important for viral particle assembly(47). The Rab11-mediated vRNP transport could facilitate the in-teraction between the N and M proteins and the efficient virusbudding at the apical surface.

Because epithelial tissues of the respiratory, digestive, and re-productive tracts act as barriers between the body cavities andunderlying tissues, viruses that cause systemic infections mustfind ways to penetrate the barriers. Some viruses, such as HIV(48), Epstein-Barr virus (49), and hepatitis C virus (50), targetimmune cells and use their innate capacities to cross the epithelia.As another example, NiV has been reported to disrupt the integ-rity of the epithelial cell layer by syncytium formation, allowingsystemic spread into the body (21). In the present study, weshowed that MuV is released predominantly from the apical sur-face. This finding indicates that MuV does not escape the epithe-lial barrier simply by passing through the epithelial cells from theapical to basolateral side. Furthermore, unlike NiV infection,MuV infection did not cause significant cytopathic effects in eitherpolarized MDCK or Calu-3 cells and did not disrupt the integrityof the polarized cell layer, suggesting that transmigration of api-cally released virus to the basolateral side through the partly dis-rupted epithelial barrier does not occur. In the case of MV, whichis also transmitted via the respiratory route and causes systemicinfections, macrophages and dendritic cells in the lung tissue areconsidered the main cells targeted for the epithelial crossing (51).Further studies will be needed to clarify how MuV breaks throughthe epithelial barrier during initial infection.

In conclusion, the present study showed that MuV enters po-larized epithelial cells from both the apical and basolateral sidesand is predominantly released from the apical surface. We havealso demonstrated that the Rab11-positive ARE is an importantcellular factor in the process of apical vRNP transport and virusproduction. These data should provide insights into the mecha-nism of MuV transmission and pathogenesis.

ACKNOWLEDGMENTS

We thank Tadaki Suzuki of the Department of Pathology, National Insti-tute of Infectious Diseases, Tokyo, Japan (NIID), for the plasmids. Wealso thank all the members of the Department of Virology III, NIID, fortheir technical advice and critical input.

This work was supported in part by a grant from the Ministry ofHealth, Labour and Welfare of Japan to Hiroshi Katoh.

We declare no conflict of interest.

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