Proteolytic processing of reovirus is required for adherence to intestinal M cells.

JOURNALOFVIROLOGY,Dec. 1994,p.8428-8432 Vol. 68, No. 12

0022-538X/94/$04.00+0

CopyrightX 1994, AmericanSocietyforMicrobiology

Proteolytic Processing

of Reovirus Is

Required

for

Adherence

to

Intestinal M Cells

HELEN M. AMERONGEN,1t GREAME A. R.WILSON,2t BERNARD N. FIELDS,2

ANDMARIAN R. NEUTRAl*

GI CellBiology Laboratory, Children's Hospital, and Departments of Pediatrics1 and Microbiology and Molecular Genetics, 2 Harvard Medical School,

Boston, Massachusetts 02115

Received13June1994/Accepted 6 September1994

Reovirus adheres specifically to apical membranes of mouse intestinal M cells and exploits M-cell

transepithelial transport activity to enter Peyer's patch mucosa, where replication occurs. Proteolytic conversion of native reovirus to intermediate subviralparticles (ISVPs) occursin theintestine, but it isnot known whether conversionis essential for interaction of virus with M cells. We tested thecapacityof native

virions,ISVPs, andcores(thatlack outercapsid proteins)tobind to intestinalepithelialcells invivoandfound thatonlyISVPs adheredtoM cells.Thus, intraluminal conversionof native reovirustoISVPs isaprerequisite for M-cell adherence, and outer capsid proteins unique to ISVPs (either or1 or products of j,1) mediate interaction of virus withM-cell apicalmembranes.

Many viruses gain entry to their hosts by

crossing

an

epithelialbarrier. Inmammalianintestines, alargenumber of pathogens enter regions of the intestinal mucosa containing organized mucosal lymphoid tissues, which arecomposed of lymphoid follicles coveredby a specialized follicle-associated

epithelium (4, 8, 17). The follicle-associated epithelium

con-tains Mcells,auniqueepithelialcell type whose function isto

transportsamplesof luminalmaterial, including antigens and

microorganisms,to theunderlyingmucosafor the purposeof inducingaprotective mucosal immune response(12).Inmice,

reovirus types 1 and 3 ingested orally exploit the vesicular

transepithelial pathway of M cells to invade the mucosa, proliferatelocally,andspreadsystemically (15, 24).By

adher-ingspecificallytotheapical membranes ofMcells, reoviruses

ensuretheirowntransepithelial transport and efficient delivery

to mononuclear cells (most likely macrophages) within the Peyer's patch mucosa, where replication takes place. Thus, M-cell adherence is the first directencounterof reovirus with the cells of the host and an essential first step in viral

pathogenesis.

The biochemical features that allow reoviruses to recognize andadheretoMcellsare notknown. Interaction withMcells isageneral strategy used by a variety of microorganisms whose

lifecycle involves crossing mucosal barriers (11). For example,

poliovirus(19),humanimmunodeficiencyvirus(1), and several gram-negativebacteria (11)alsoselectively bind to M cells and invade via this route. Neither the M-cell receptors nor the microbialligands involved for any infectious agent have been identified. The molecular structure of reovirus and the se-quence ofbiochemical alterations that occur in reovirus capsid proteins during the early stages of host cell infection have been defined in detail (5, 15). Thus, reovirus provides a valuable

*Correspondingauthor. Mailing address: GI Cell Biology, Enders 461, Children's Hospital, 300 Longwood Ave., Boston, MA 02115. Phone: (617)735-6229. Fax: (617) 730-0404.

tPresent address: DepartmentofAnatomy, UniversityofArizona CollegeofMedicine, Tucson, AZ 85724.

tPresentaddress: VirusLaboratories, LaboratoryCentre for Dis-easeControl,Tunney's Pasture, Ottawa, OntarioKlAOL2, Canada.

model system with which to explore the basis of selective microbe-M-cell interactions.

The reovirus outercapsid is composed of 600copiesof each oftwo major proteins:

Rl

(1i1C/I1N), which forms the basic shell of the externalcapsid,andcr3,which decorates the native virus surface (5). In an intactvirion, 5% of the ,u protein is presentin an uncleaved formwhile 95% has been cleaved to generateasmallmyristoylated piNfragment andalargepulC

protein;bothfragmentsarefound inthe outercapsid(16).In

addition,about 36 to48copies of viralhemagglutinin protein

a1 are located (probably as trimers or tetramers) at the icosahedral verticesalongwithan additionalprotein, X2, that spans the capsid (9, 13). Treatment of virus with trypsin or chymotrypsinunder controlled conditions in vitroresults in an intermediate subviralparticle(ISVP)generatedbyproteolytic removal of a3 and cleavage of ,ulC into a large, amino-terminalsubfragment called 8 and asmallercarboxy-terminal subfragment termed 0. 8, 0, and

pulN

allremainassociated with the ISVP.Inaddition, there isatransition ofcrl from a folded

to anextended conformation (6).ISVPs are stable and highly infectious. Further protease treatment removes the remaining

outer capsid proteins, yielding stable viral cores that cannot infectcells in vitro but are transcriptionally active (7).

In cultured cells, proteolytic cleavage to ISVPs occurs in endosomesor lysosomes(20), but in mice, native virions first

encounterproteases in the intestinal lumen. Although ISVPs

are generated in the intestine (3) and inhibitors of luminal serine proteases reduce viral infectivity in mice (2), it is not knownatwhat stepthe conversion of virus to ISVPs is essential forinfectivity, i.e.,forinteraction of reovirus with M cells, or forthesubsequentinfection of mucosal macrophages. In this

study, we directly tested the capacity of three forms of reovi-rus-native virions, ISVPs, and cores-to bind to the apical membranes of mouse intestinal epithelial cells.

Wefirst tested whether ISVPs generated by protease

treat-ment invitro are able to selectively recognize and adhere to M-cell apicalmembranes in vivo. Reovirus serotype 1 (Lang) fromastandardlaboratory stock was grown in mouse L cells in suspension culture, purified, and concentrated as described

previously (14,22). Numbers of viral particles were determined spectrophotometrically, and infectivity was measured by

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I77

FIG. 1. Adherence of reovirus ISVPs toMcells in mouseintestines. ISVPs weregenerated 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 electron microscopy. ISVPs wereconsistently observed on apical membranes and inendocyticvesicles ofMcells but were

generally

absent fromneighboring absorptive cells. Thus, ISVPs canselectively bind to M cells. Bar, 1 pm.

plaqueassayusingL-cellmonolayers. ISVPs were prepared by

chymotrypsin digestion of purifiedreovirus (10) and injected

into ligatedloops ofdistalmouse ileum containing a Peyer's

patch. Two 6-week-old female BALB/c mice (Charles River

Laboratories, Wilmington, Mass.) were anesthetized with

2,2,2-tribromoethanol (Avertin; Aldrich Chemical Co., Mil-waukee, Wis.). About5 cm of the distal ileum was exposed through a midline incision and flushed with

phosphate-buff-eredsaline. Ashort segment

(approximately

1 cmlong)was

ligated and inoculated with 10 2 ISVPs, andtheintestine was

returned to the abdominal cavity. After 30 min, mice were

killedby cervical dislocation andtheloopswereexcised,rinsed withcoldphosphate-buffered saline, and immersedin afixative solutioncontaining2.5%glutaraldehyde, 2% formaldehyde,4

mMCaC12, and2 mMMgCl2in 0.1 M Nacacodylatebuffer.

Mucosalsampleswere

postfixed

in1%

OS04

and0.5%

uranyl

acetate and processed for electron microscopy as

previously

described(23).

Bindingandendocytosis ofISVPs wereevaluatedbydirect

examination ofthe epithelium byelectronmicroscopy. ISVPs

were consistently observedon M-cellapicalmembranes

(Fig.

1) andwithinendocytic vesicles inthe

apical cytoplasm

of M

cells, whereas few viral

particles

were associatedwith

neigh-boringabsorptive cells. Occasional

absorptive

cellsbothonthe

follicle-associated

epithelium

and on

adjacent

vili

showed adherence oflarge numbersofISVPstothe

glycocalyx

cover-ingtheapical microvillousborder,buttherewas noevidenceof

endocytosis bythesecells.

Having confirmed thatISVPscan

selectively

bindtoM

cells,

we then sought to determine whether conversion of native virions to ISVPs is a prerequisite for M-cell

binding.

M-cell

bindingandendocytosisof native virionswereevaluated in the presence and absence of protease inhibitors administered intraluminally at concentrations

previously

shown to inhibit viralinfectivity in vivo(2,

3).

Itwasimportantfirsttoconfirm

that native virions were indeed converted to ISVPs in the intestinal lumens of our untreated mice and that conversion waseffectively blockedbytheintraluminalinfusion ofprotease

inhibitors thatweusedin vivo. This wasdone byinoculating intestinal segments with 1012 virus particles that had been

metabolically labeled with

[35S]methionine

(20), recovering

luminalcontents after 30 min, and analyzingviralproteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel

electro-phoresis (PAGE) and fluorography (3). Reovirus ISVPs are

readily

distinguished from native virions by this method

be-causein ISVPs, the

a3

band, prominentin lysates of native

virus, is absent or reduced and the nativeviral 72-kDa band

representing

the intact ,ulCproteinis shifted downwardto a

59-kDa bandrepresenting delta(16). It should be notedthat theISVPs also contain,uNand 0,butthesesmallerfragments

are not detected under the PAGE

conditions

of this

experi-ment (14, 16).Luminalmaterial recoveredfromcontrolmice 30 min after inoculation with native virions

showed

a viral

protein profile

typical

of ISVPs, indicating that endogenous

proteases in the normal mouse intestine had effectively con-vertedvirus toISVPs(Fig. 2).

Pilotexperimentsdesignedtoconfirmtheability ofprotease

inhibitorstoblockreovirus conversionshowedthat when 7 ,ug ofaprotininper ml alone wasaddedtotheviralsuspension

just

beforeinfusionintothe intestinal

lumen,

conversiontoISVPs was substantially but not

completely

inhibited

(data

not

shown).

A mixtureof 7 ,ugof

aprotinin

per ml and 10% fetal bovine serum, however,

completely

prevented

the conversion

ofnativevirus toISVPs

(Fig.

2),

and

thus,

both

aprotinin

and serum were used in

subsequent

experiments.

To rule out a

possible

direct effect of

aprotinin

or serum on virus

binding

unrelated to protease

inhibition,

ISVPs were

generated by

chymotrypsin

treatment ofvirus in vitro and inoculated into

ligated intestinal loops oftwo

mice,

withorwithout

aprotinin

and serum. Adherence ofreovirus

particles

toMcellsis

usually

followedby

endocytosis,

andinvivo, bothevents are

occurring

at any one time;

therefore,

estimates of total viral

binding

should take into account virus

particles

already

taken up in

endocytic

vesicles, aswellasthose on

apical

cellsurfaces. In this andsubsequent

experiments,

viral adherence to M cells

wasvisualized

by

electron

microscopy and

analyzed

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8430 NOTES

1 2

34

:.

_ ^_~~~~~~40 . _

Alc_

.

5-ar2-

_a3'

CT3

=

A

CD 30

V) 20

-4-,

X 10

-.> O0

__

FIG. 2. Proteaseinhibitors prevent conversion of native reovirusto

ISVPs in the intestinal lumen in vivo. Intestinal segments were

inoculated with 1012 native reovirus particles (type 1/Lang) that had been metabolically labeled with ["S]methionine. Luminal contents

wererecovered after 30minandanalyzed bySDS-PAGE and fluorog-raphy, along with control preparations of native reovirus and ISVPs generated in vitro. In lane 1,native reovirus shows threeprominent bands representingviral proteins X,

RIC,

and (X3. In lane 2, ISVPs

differ from nativevirions in that the cr3 band is lost and the pLlC protein band is shifted downward to a band representing the ,ulC product, B. Smaller fragmentsof,ulC(plNand0)were notretained in thisgel.Inlane3,viralproteinsrecovered from mice inoculated with native virus alongwith aprotinin and serum show a protein profile typicalof native virus.Thus,protease inhibitorseffectively prevented the conversion of native virus to ISVPs. In lane 4, viral proteins recovered from the intestines of control mice showaprofile typicalof ISVPs, indicating that endogenous intestinalproteaseshave converted virustoISVPs.

tively by countingtheviralparticlesassociated with the surface

orapical endocyticvesicles of each M cell observed in ultrathin

sections cut perpendicular to the surface of the follicle-associatedepithelium (Fig. 3).The countsobtained indicated notthe totalnumberof virus particlesassociated with

individ-ual M-cell surfaces butratherarepresentativefractionthereof

which could be usedtocompareMcells ofoneanimaltothose

ofanother. The statistical significance of differences in these counts was evaluated by a two-tailed t test using Statview

software on an Apple Macintosh computer. This analysis

showed that binding of ISVPs to M cells was unaffected by

aprotininor serum(Fig. 3A).

To directly assessthe role of proteolytic processingonviral

adherencetoM

cells,

1012

native

virions,

eitherwithorwithout

aprotininandserum, wereinoculated into ligated ilealloops of

six mice in three separate experiments. Peyer's patch tissues

were collected after 30 min and processed and analyzed as

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

previously observed (24).When endogenous luminalprotease

activitywasinhibited, however,M-cell binding and endocytosis

ofviruswere significantly decreased (Fig. 3B and 4B). Thus,

intraluminal conversion of native reovirus to ISVPs is a

prerequisite for M-celladherence.

We thensought todetermine which viralproteins might be

involved in interaction of ISVPs with M cells. Prolonged protease treatmentofISVPs in vitro completelyremoves the or1 protein, aswellas p1N, 8, and 0 (the three subfragments

derived from,ul and Llc). Theresultant viralcores aredevoid

B

ISVPs ISVPs

+PI

12 10 8 6 4 2 0

C

12-10

6 4-2

-VRUS VlRUS

-VIUS VIRUS +PI

L

CORES ISVPs

FIG. 3. Effectofproteolytic processing on thebinding of native reovirus and ISVPs to M cells in vivo. Results of three separate experimentsareshown. Inallcases,viralparticleswereinoculated into ligated ileal loops ofmice, Peyer'spatchtissueswerecollected after 30 min,and adherencetoMcellswasanalyzed by electronmicroscopyas describedin thetext. (A) Reovirus ISVPswereproduced in vitro and inoculated either withorwithoutproteaseinhibitors(PI). Binding of ISVPstoM cellswasunaffectedbyinhibitors, indicating that ISVPs canadheretoMcells without furtherproteaseprocessing. (B)Native reovirus particles were inoculated either with or without protease inhibitors. Virusadministeredwithout inhibitorsadheredefficientlyto M cells. In thepresence ofinhibitors, however, M-cell binding and endocytosis ofvirusweresignificantly decreased (P= 0.0052). Thus, intraluminalconversion of nativereovirusto ISVPs isaprerequisite forM-cell adherence. (C) ISVPs andviral cores were produced by controlleddigestionof reovirusinvitro, and conversion of ISVPsto cores was confirmed by SDS-PAGE (data not shown). Equal numbers of cores or ISVPs (1012 particles) were injected into ligated loopsof mouse ileum. Electronmicroscopic analysis showed that ISVPs wereconsistently presentonM-cell surfacesbutcores werenot (P= 0.0002).

of outer capsid components, with the exception of the X2 spikesatthe verticesof theparticle(5, 15).If X2 is involved in ISVPbindingtoMcells, then both ISVPs andcoreswould be

expectedtobind. Ifbindingismediatedby any of the subfrag-ments of,ul/,ulCor the extended cr1 protein, however,cores should beunable tobind.

Coreswere produced by digestion of native reovirus parti-cles in vitro with chymotrypsin (14). SDS-PAGE of the

di-gested particles showed loss of thecrl anddelta bands (data

notshown), confirming that conversion to cores was complete.

In three

separate

experiments, equal numbers of cores or ISVPs

(10'

particles) were injected into ligated loops of mouseileum(twomice per experiment and one or two ligated loops permouse) and association of viral particles with M cells

was assessed by electron microscopy as described before. ISVPs were present on M-cell surfaces and in M-cell endo-somes,asexpected, but adherence or endocytosis of cores was

not observed (Fig. 3C), although clusters of viral cores were

seenassociated with luminaldebris. To test the possibility that suchaggregation reduced the numbers of viral cores that were free todiffuse to the epithelial surface, we inoculated a large numberof cores(1013particles) but still did not observe M-cell binding or endocytosis. Thus, removal of the outer capsid protein

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, the

active,infectious form of reovirus, adhere selectively to M cells ofPeyer's patches whereas native virions and viral cores do not indicates thatouter capsid proteins unique to ISVPs mediate interaction of virus with apical membrane components of M cells. The intact viral particle is a compact, highly stable J. VIROL.

.*..

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A

It.

)"I"r. 00 v.

.1

B

FIG. 4. Adherence of reovirus to M cells in the absence and presence ofprotease inhibitors. Native reoviruswasinoculated into ligatedintestinalloopsasdescribedinthelegendtoFig.3B.(A)Inthe

absence ofinhibitors,viralparticleswereobservedadheringtoapical

surfaces and inendocytic compartmentsof Mcells.(B)Inthe presence

ofprotease inhibitors,littleor novirusadhered toM-cellsurfaces.Bar,

1 ,um.

structure whose function is thought tobe related to survival

outside the host andpassagethroughtheacidicenvironment of

thestomach(15).The ISVP isgeneratedinthe smallintestine

and isdramaticallyaltered: the or3 proteinisremoved,thepl protein and its product ,ulC are further cleaved, and

attach-mentproteinal isextended(5, 15).Inthis activatedform,the

virus isprimedtointeract with cells. The two mostprominent

surfacecomponentsofISVPs, cl and ,ulproducts (including fragments pulN, 8 and 0), are thought to have different

functionsduringinfection oftargetmononuclear cells(5).The crlproteinbinds to host cell surface receptorsthatappear to

be sialylated glycoproteinsor possibly glycolipids (8).The ,ul proteinand itsproducts haverecentlybeen shown tointeract

with cellmembranes, as evidencedby the capacity of ISVPs to

cause release of 51Cr from prelabeled cells (10) and to

generate ion channels in planar lipid bilayers (21). It is thus

possiblethat either alorproducts of ,l are involved in M-cell

recognition and entry. The X2 pentamers, proteins that form thespike to which the al protein is attached, are also foundat

the surface of the ISVP, but the presence ofX2 pentamerson cores(which do not bind to M cells) argues againstadirect role in M-cell recognition. Further studies are required to deter-mine which of theseproteins is critical for M-cellattachment.

We have established that proteolytic processing of native reovirus by enzymes in the intestinal lumen of a host is a prerequisite for M-cell transport ofvirus into Peyer's patch

mucosa.These studies confirm thatdigestive proteases arethe key signal that triggers conversion of intact viral particlesto infectious ISVPs (2, 3). Reovirus thus serves as a striking exampleof how virusesuse specific internal environments of the host as part of their own life cycle. The fact that other viruses andmicroorganisms use M cells of thegastrointestinal

tract as aportal of entry(12) raises thepossibility that other

pathogensalsoexploit this enzyme-rich environment tochange

fromaninactive, nonadherent form to an active form thatcan bind to M cells.Althoughthere isnoevidence forthis, there is abundant evidence that gut proteasesconvertvirusesfromone

form to another(18).It ispossiblethat intraluminal alterations enable certainbacterial, aswell asviral, pathogens to bind to M cells.

The M-celltransepithelial transportpathwayusedby reovi-rus leads directly to mucosal sites that are specialized for

generation of mucosal immune responses (8). This suggests that information about the mechanismwherebyreovirus

inter-actswithM cells could beexploitedtodeliver otherantigensto

mucosal inductive sites.Although engineeringofforeign pro-teins into reovirus is not feasible, reovirus outer capsid pro-teins might serve as targeting molecules to enhance the effectiveness of oral vaccines.

This workwassupported byNationalInstitutesof Healthresearch grants R01DK21505, R37 HD17557(to M.R.N.), andP50NS16998

(to

B.N.F.).Additionalsupportwasprovided byNIH grantDK34854 tothe HarvardDigestiveDiseasesCenter.

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