JOURNAL OF VIROLOGY, Dec. 1994, p. 8428-8432 Vol. 68, No. 120022-538X/94/$04.00+0Copyright X 1994, American Society for Microbiology

Proteolytic Processing of Reovirus Is Required forAdherence to Intestinal M Cells

HELEN M. AMERONGEN,1t GREAME A. R. WILSON,2t BERNARD N. FIELDS,2AND MARIAN R. NEUTRAl*

GI Cell Biology Laboratory, Children's Hospital, and Departments of Pediatrics1 andMicrobiology and Molecular Genetics, 2 Harvard Medical School,

Boston, Massachusetts 02115

Received 13 June 1994/Accepted 6 September 1994

Reovirus adheres specifically to apical membranes of mouse intestinal M cells and exploits M-celltransepithelial transport activity to enter Peyer's patch mucosa, where replication occurs. Proteolyticconversion of native reovirus to intermediate subviral particles (ISVPs) occurs in the intestine, but it is notknown whether conversion is essential for interaction of virus with M cells. We tested the capacity of nativevirions, ISVPs, and cores (that lack outer capsid proteins) to bind to intestinal epithelial cells in vivo and foundthat only ISVPs adhered to M cells. Thus, intraluminal conversion of native reovirus to ISVPs is a prerequisitefor M-cell adherence, and outer capsid proteins unique to ISVPs (either or1 or products of j,1) mediateinteraction of virus with M-cell apical membranes.

Many viruses gain entry to their hosts by crossing anepithelial barrier. In mammalian intestines, a large number ofpathogens enter regions of the intestinal mucosa containingorganized mucosal lymphoid tissues, which are composed oflymphoid follicles covered by a specialized follicle-associatedepithelium (4, 8, 17). The follicle-associated epithelium con-tains M cells, a unique epithelial cell type whose function is totransport samples of luminal material, including antigens andmicroorganisms, to the underlying mucosa for the purpose ofinducing a protective mucosal immune response (12). In mice,reovirus types 1 and 3 ingested orally exploit the vesiculartransepithelial pathway of M cells to invade the mucosa,proliferate locally, and spread systemically (15, 24). By adher-ing specifically to the apical membranes of M cells, reovirusesensure their own transepithelial transport and efficient deliveryto mononuclear cells (most likely macrophages) within thePeyer's patch mucosa, where replication takes place. Thus,M-cell adherence is the first direct encounter of reovirus withthe cells of the host and an essential first step in viralpathogenesis.The biochemical features that allow reoviruses to recognize

and adhere to M cells are not known. Interaction with M cellsis a general strategy used by a variety of microorganisms whoselife cycle involves crossing mucosal barriers (11). For example,poliovirus (19), human immunodeficiency virus (1), and severalgram-negative bacteria (11) also selectively bind to M cells andinvade via this route. Neither the M-cell receptors nor themicrobial ligands involved for any infectious agent have beenidentified. The molecular structure of reovirus and the se-quence of biochemical alterations that occur in reovirus capsidproteins during the early stages of host cell infection have beendefined in detail (5, 15). Thus, reovirus provides a valuable

* Corresponding author. Mailing address: GI Cell Biology, Enders461, Children's Hospital, 300 Longwood Ave., Boston, MA 02115.Phone: (617) 735-6229. Fax: (617) 730-0404.

t Present address: Department of Anatomy, University of ArizonaCollege of Medicine, Tucson, AZ 85724.

t Present address: Virus Laboratories, Laboratory Centre for Dis-ease Control, Tunney's Pasture, Ottawa, Ontario KlA OL2, Canada.

model system with which to explore the basis of selectivemicrobe-M-cell interactions.The reovirus outer capsid is composed of 600 copies of each

of two major proteins: Rl (1i1C/I1N), which forms the basicshell of the external capsid, and cr3, which decorates the nativevirus surface (5). In an intact virion, 5% of the ,u protein ispresent in an uncleaved form while 95% has been cleaved togenerate a small myristoylated piN fragment and a large pulCprotein; both fragments are found in the outer capsid (16). Inaddition, about 36 to 48 copies of viral hemagglutinin proteina1 are located (probably as trimers or tetramers) at theicosahedral vertices along with an additional protein, X2, thatspans the capsid (9, 13). Treatment of virus with trypsin orchymotrypsin under controlled conditions in vitro results in anintermediate subviral particle (ISVP) generated by proteolyticremoval of a3 and cleavage of ,ulC into a large, amino-terminal subfragment called 8 and a smaller carboxy-terminalsubfragment termed 0. 8, 0, and pulN all remain associated withthe ISVP. In addition, there is a transition of crl from a foldedto an extended conformation (6). ISVPs are stable and highlyinfectious. Further protease treatment removes the remainingouter capsid proteins, yielding stable viral cores that cannotinfect cells in vitro but are transcriptionally active (7).

In cultured cells, proteolytic cleavage to ISVPs occurs inendosomes or lysosomes (20), but in mice, native virions firstencounter proteases in the intestinal lumen. Although ISVPsare generated in the intestine (3) and inhibitors of luminalserine proteases reduce viral infectivity in mice (2), it is notknown at what step the conversion of virus to ISVPs is essentialfor infectivity, i.e., for interaction of reovirus with M cells, orfor the subsequent infection of mucosal macrophages. In thisstudy, we directly tested the capacity of three forms of reovi-rus-native virions, ISVPs, and cores-to bind to the apicalmembranes of mouse intestinal epithelial cells.We first tested whether ISVPs generated by protease treat-

ment in vitro are able to selectively recognize and adhere toM-cell apical membranes in vivo. Reovirus serotype 1 (Lang)from a standard laboratory stock was grown in mouse L cells insuspension culture, purified, and concentrated as describedpreviously (14, 22). Numbers of viral particles were determinedspectrophotometrically, and infectivity was measured by

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I77FIG. 1. Adherence of reovirus ISVPs to M cells in mouse intestines. ISVPs were generated by chymotrypsin digestion in vitro and inoculated

into ligated loops of mouse ileum containing a Peyer's patch. Epithelial cells of tissues collected after 30 min were examined by electronmicroscopy. ISVPs were consistently observed on apical membranes and in endocytic vesicles ofM cells but were generally absent from neighboringabsorptive cells. Thus, ISVPs can selectively bind to M cells. Bar, 1 pm.

plaque assay using L-cell monolayers. ISVPs were prepared bychymotrypsin digestion of purified reovirus (10) and injectedinto ligated loops of distal mouse ileum containing a Peyer'spatch. Two 6-week-old female BALB/c mice (Charles RiverLaboratories, Wilmington, Mass.) were anesthetized with2,2,2-tribromoethanol (Avertin; Aldrich Chemical Co., Mil-waukee, Wis.). About 5 cm of the distal ileum was exposedthrough a midline incision and flushed with phosphate-buff-ered saline. A short segment (approximately 1 cm long) wasligated and inoculated with 10 2 ISVPs, and the intestine wasreturned to the abdominal cavity. After 30 min, mice werekilled by cervical dislocation and the loops were excised, rinsedwith cold phosphate-buffered saline, and immersed in a fixativesolution containing 2.5% glutaraldehyde, 2% formaldehyde, 4mM CaC12, and 2 mM MgCl2 in 0.1 M Na cacodylate buffer.Mucosal samples were postfixed in 1% OS04 and 0.5% uranylacetate and processed for electron microscopy as previouslydescribed (23).

Binding and endocytosis of ISVPs were evaluated by directexamination of the epithelium by electron microscopy. ISVPswere consistently observed on M-cell apical membranes (Fig.1) and within endocytic vesicles in the apical cytoplasm of Mcells, whereas few viral particles were associated with neigh-boring absorptive cells. Occasional absorptive cells both on thefollicle-associated epithelium and on adjacent vili showedadherence of large numbers of ISVPs to the glycocalyx cover-ing the apical microvillous border, but there was no evidence ofendocytosis by these cells.Having confirmed that ISVPs can selectively bind to M cells,

we then sought to determine whether conversion of nativevirions to ISVPs is a prerequisite for M-cell binding. M-cellbinding and endocytosis of native virions were evaluated in thepresence and absence of protease inhibitors administeredintraluminally at concentrations previously shown to inhibitviral infectivity in vivo (2, 3). It was important first to confirmthat native virions were indeed converted to ISVPs in theintestinal lumens of our untreated mice and that conversionwas effectively blocked by the intraluminal infusion of protease

inhibitors that we used in vivo. This was done by inoculatingintestinal segments with 1012 virus particles that had beenmetabolically labeled with [35S]methionine (20), recoveringluminal contents after 30 min, and analyzing viral proteins bysodium dodecyl sulfate (SDS)-polyacrylamide gel electro-phoresis (PAGE) and fluorography (3). Reovirus ISVPs arereadily distinguished from native virions by this method be-cause in ISVPs, the a3 band, prominent in lysates of nativevirus, is absent or reduced and the native viral 72-kDa bandrepresenting the intact ,ulC protein is shifted downward to a59-kDa band representing delta (16). It should be noted thatthe ISVPs also contain ,uN and 0, but these smaller fragmentsare not detected under the PAGE conditions of this experi-ment (14, 16). Luminal material recovered from control mice30 min after inoculation with native virions showed a viralprotein profile typical of ISVPs, indicating that endogenousproteases in the normal mouse intestine had effectively con-verted virus to ISVPs (Fig. 2).

Pilot experiments designed to confirm the ability of proteaseinhibitors to block reovirus conversion showed that when 7 ,ugof aprotinin per ml alone was added to the viral suspension justbefore infusion into the intestinal lumen, conversion to ISVPswas substantially but not completely inhibited (data notshown). A mixture of 7 ,ug of aprotinin per ml and 10% fetalbovine serum, however, completely prevented the conversionof native virus to ISVPs (Fig. 2), and thus, both aprotinin andserum were used in subsequent experiments. To rule out apossible direct effect of aprotinin or serum on virus bindingunrelated to protease inhibition, ISVPs were generated bychymotrypsin treatment of virus in vitro and inoculated intoligated intestinal loops of two mice, with or without aprotininand serum. Adherence of reovirus particles to M cells is usuallyfollowed by endocytosis, and in vivo, both events are occurringat any one time; therefore, estimates of total viral bindingshould take into account virus particles already taken up inendocytic vesicles, as well as those on apical cell surfaces. Inthis and subsequent experiments, viral adherence to M cellswas visualized by electron microscopy and analyzed quantita-

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FIG. 2. Protease inhibitors prevent conversion of native reovirus toISVPs in the intestinal lumen in vivo. Intestinal segments wereinoculated with 1012 native reovirus particles (type 1/Lang) that hadbeen metabolically labeled with ["S]methionine. Luminal contentswere recovered after 30 min and analyzed by SDS-PAGE and fluorog-raphy, along with control preparations of native reovirus and ISVPsgenerated in vitro. In lane 1, native reovirus shows three prominentbands representing viral proteins X, RIC, and (X3. In lane 2, ISVPsdiffer from native virions in that the cr3 band is lost and the pLlCprotein band is shifted downward to a band representing the ,ulCproduct, B. Smaller fragments of ,ulC (plN and 0) were not retainedin this gel. In lane 3, viral proteins recovered from mice inoculated withnative virus along with aprotinin and serum show a protein profiletypical of native virus. Thus, protease inhibitors effectively preventedthe conversion of native virus to ISVPs. In lane 4, viral proteinsrecovered from the intestines of control mice show a profile typical ofISVPs, indicating that endogenous intestinal proteases have convertedvirus to ISVPs.

tively by counting the viral particles associated with the surfaceor apical endocytic vesicles of each M cell observed in ultrathinsections cut perpendicular to the surface of the follicle-associated epithelium (Fig. 3). The counts obtained indicatednot the total number of virus particles associated with individ-ual M-cell surfaces but rather a representative fraction thereofwhich could be used to compare M cells of one animal to thoseof another. The statistical significance of differences in thesecounts was evaluated by a two-tailed t test using Statviewsoftware on an Apple Macintosh computer. This analysisshowed that binding of ISVPs to M cells was unaffected byaprotinin or serum (Fig. 3A).To directly assess the role of proteolytic processing on viral

adherence to M cells, 1012 native virions, either with or withoutaprotinin and serum, were inoculated into ligated ileal loops ofsix mice in three separate experiments. Peyer's patch tissueswere collected after 30 min and processed and analyzed as

described above. Native reovirus administered without inhibi-tors adhered efficiently to M cells (Fig. 3B and 4A), as

previously observed (24). When endogenous luminal proteaseactivity was inhibited, however, M-cell binding and endocytosisof virus were significantly decreased (Fig. 3B and 4B). Thus,intraluminal conversion of native reovirus to ISVPs is a

prerequisite for M-cell adherence.We then sought to determine which viral proteins might be

involved in interaction of ISVPs with M cells. Prolongedprotease treatment of ISVPs in vitro completely removes theor1 protein, as well as p1N, 8, and 0 (the three subfragmentsderived from ,ul and Llc). The resultant viral cores are devoid

B

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FIG. 3. Effect of proteolytic processing on the binding of nativereovirus and ISVPs to M cells in vivo. Results of three separateexperiments are shown. In all cases, viral particles were inoculated intoligated ileal loops of mice, Peyer's patch tissues were collected after 30min, and adherence to M cells was analyzed by electron microscopy asdescribed in the text. (A) Reovirus ISVPs were produced in vitro andinoculated either with or without protease inhibitors (PI). Binding ofISVPs to M cells was unaffected by inhibitors, indicating that ISVPscan adhere to M cells without further protease processing. (B) Nativereovirus particles were inoculated either with or without proteaseinhibitors. Virus administered without inhibitors adhered efficiently toM cells. In the presence of inhibitors, however, M-cell binding andendocytosis of virus were significantly decreased (P = 0.0052). Thus,intraluminal conversion of native reovirus to ISVPs is a prerequisitefor M-cell adherence. (C) ISVPs and viral cores were produced bycontrolled digestion of reovirus in vitro, and conversion of ISVPs tocores was confirmed by SDS-PAGE (data not shown). Equalnumbers of cores or ISVPs (1012 particles) were injected intoligated loops of mouse ileum. Electron microscopic analysis showedthat ISVPs were consistently present on M-cell surfaces but coreswere not (P = 0.0002).

of outer capsid components, with the exception of the X2spikes at the vertices of the particle (5, 15). If X2 is involved inISVP binding to M cells, then both ISVPs and cores would beexpected to bind. If binding is mediated by any of the subfrag-ments of ,ul/,ulC or the extended cr1 protein, however, coresshould be unable to bind.

Cores were produced by digestion of native reovirus parti-cles in vitro with chymotrypsin (14). SDS-PAGE of the di-gested particles showed loss of the crl and delta bands (datanot shown), confirming that conversion to cores was complete.In three separate experiments, equal numbers of cores orISVPs (10' particles) were injected into ligated loops ofmouse ileum (two mice per experiment and one or two ligatedloops per mouse) and association of viral particles with M cellswas assessed by electron microscopy as described before.ISVPs were present on M-cell surfaces and in M-cell endo-somes, as expected, but adherence or endocytosis of cores wasnot observed (Fig. 3C), although clusters of viral cores wereseen associated with luminal debris. To test the possibility thatsuch aggregation reduced the numbers of viral cores that werefree to diffuse to the epithelial surface, we inoculated a largenumber of cores (1013 particles) but still did not observe M-cellbinding or endocytosis. Thus, removal of the outer capsidprotein crl and ul1/p.lC fragments abolished M-cell adherence.These observations provide an important clue as to the

nature of this virus-cell interaction. The fact that ISVPs, theactive, infectious form of reovirus, adhere selectively to M cellsof Peyer's patches whereas native virions and viral cores do notindicates that outer capsid proteins unique to ISVPs mediateinteraction of virus with apical membrane components of Mcells. The intact viral particle is a compact, highly stable

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FIG. 4. Adherence of reovirus to M cells in the absence andpresence of protease inhibitors. Native reovirus was inoculated intoligated intestinal loops as described in the legend to Fig. 3B. (A) In theabsence of inhibitors, viral particles were observed adhering to apicalsurfaces and in endocytic compartments of M cells. (B) In the presenceof protease inhibitors, little or no virus adhered to M-cell surfaces. Bar,1 ,um.

structure whose function is thought to be related to survivaloutside the host and passage through the acidic environment ofthe stomach (15). The ISVP is generated in the small intestineand is dramatically altered: the or3 protein is removed, the plprotein and its product ,ulC are further cleaved, and attach-ment protein al is extended (5, 15). In this activated form, thevirus is primed to interact with cells. The two most prominentsurface components of ISVPs, cl and ,ul products (includingfragments pulN, 8 and 0), are thought to have differentfunctions during infection of target mononuclear cells (5). Thecrl protein binds to host cell surface receptors that appear tobe sialylated glycoproteins or possibly glycolipids (8). The ,ulprotein and its products have recently been shown to interact

with cell membranes, as evidenced by the capacity of ISVPs tocause release of 51Cr from prelabeled cells (10) and togenerate ion channels in planar lipid bilayers (21). It is thuspossible that either al or products of ,l are involved in M-cellrecognition and entry. The X2 pentamers, proteins that formthe spike to which the al protein is attached, are also found atthe surface of the ISVP, but the presence of X2 pentamers oncores (which do not bind to M cells) argues against a direct rolein M-cell recognition. Further studies are required to deter-mine which of these proteins is critical for M-cell attachment.We have established that proteolytic processing of native

reovirus by enzymes in the intestinal lumen of a host is aprerequisite for M-cell transport of virus into Peyer's patchmucosa. These studies confirm that digestive proteases are thekey signal that triggers conversion of intact viral particles toinfectious ISVPs (2, 3). Reovirus thus serves as a strikingexample of how viruses use specific internal environments ofthe host as part of their own life cycle. The fact that otherviruses and microorganisms use M cells of the gastrointestinaltract as a portal of entry (12) raises the possibility that otherpathogens also exploit this enzyme-rich environment to changefrom an inactive, nonadherent form to an active form that canbind to M cells. Although there is no evidence for this, there isabundant evidence that gut proteases convert viruses from oneform to another (18). It is possible that intraluminal alterationsenable certain bacterial, as well as viral, pathogens to bind toM cells.The M-cell transepithelial transport pathway used by reovi-

rus leads directly to mucosal sites that are specialized forgeneration of mucosal immune responses (8). This suggeststhat information about the mechanism whereby reovirus inter-acts with M cells could be exploited to deliver other antigens tomucosal inductive sites. Although engineering of foreign pro-teins into reovirus is not feasible, reovirus outer capsid pro-teins might serve as targeting molecules to enhance theeffectiveness of oral vaccines.

This work was supported by National Institutes of Health researchgrants R01 DK21505, R37 HD17557 (to M.R.N.), and P50 NS16998(to B.N.F.). Additional support was provided by NIH grant DK34854to the Harvard Digestive Diseases Center.

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