Rabies virus infection of cultured rat sensory neurons.

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for Microbiology

Rabies Virus Infection of Cultured

Rat

Sensory Neurons

ERIKLYCKEt AND HENRITSIANG* Unite Rage, Institut Pasteur, Paris, France Received 10 February 1987/Accepted1 June 1987

The axonaltransportof rabies virus (challenge virus strain of fixed virus)was studied indifferentiatedrat embryonic dorsal root ganglion cells. In addition, we observed the attachment ofrabies virus to neuronal extensions and virus production by infected neurons. A compartmentalized cell culture system was used, allowing infectionandmanipulationofneuronalextensionswithoutexposing theneuralsomatothevirus.The cultures consisted of60% large neuronal cells whose extensions exhibited neurofilament structures. Rabies virus demonstrated highbinding affinitytounmyelinated neurites, assuggested byassaysof virus adsorption

andimmunofluorescence studies. Therateof axoplasmictransportofviruswas12to24mm/day,includingthe time required for internalization of the virus into neurites. The virus transport could be blocked by cytochalasin B, vinblastine, andcolchicine, noneof which negatively affected the production of virusin cells

oncetheinfectionwasestablished.Itwasconcluded that,for the retrograde transfer of rabies virusbyneurites from the peripherytothe neuronalsoma,the integrityof tubulin-andactin-containingstructuresis essential. Theratsensory neuronswerecharacterizedaspermissive, moderately susceptible, but low producers of rabies

virus. Theseneuronswerecapable of harboring rabiesvirusforlongperiodsof time and abletoreleasevirus intotheculturemedium without showingany morphological alterations. The involvement ofsensoryneurons

in rabies virus pathogenesis, bothinviral transportandas asiteforpersistent viral infection, is discussed.

The spread of rabies infection through theaxoplasmic flow in peripheral nerves and neurons in the central nervous

system(CNS)representsanimportant and significant patho-genetic characteristic. Interruption of the peripheral axoplasmic flow by ligature or sectioning (4, 15, 26) or by pharmacological impairment of the microtubulin function prevents rabies virus transport from the peripheral virus inoculation sitetothe CNS (8, 43). Peripheralnervesof both

motor and sensory neurons canbe used forpropagation of rabies virusinfection (10, 20), although sensory neurons are

mostprobably involved in the virus transfer from the site of the primary infection to the CNS (35). It is generally ob-served, by electron microscopy, that rabies virus buds from infected neurons and subsequently enters into uninfected cells(23, 24, 34, 39). However, the hypothesis ofanaxonal

transportof virus inthe CNS hasneverbeendiscarded (35). Virusinoculation into theeyehasresulted in virustransport into the CNS(31). Recently,experimental evidence has been provided foran axonal transport of rabies virus within the CNS. Stereotaxic inoculation of the virus into rat brains

gavepatterns ofvirus spread which areconsistentwith the hypothesis ofan axoplasmic flow-mediated transport(19).

Infection ofcultured nerve cells (44, 47) suggested that susceptibility to rabies virus is likely to vary markedly

depending on the types of neuronal cells. In the present study, we used rat dorsal root ganglia (DRG) neurons for evaluating axonal transport of rabies virus in vitro. In addition, westudiedvirusbindingtoneuritic extensions and itsreplication insensory neurons. Forthispurpose,wetook advantage ofa compartmentalized cell culture system (11) which allowed usto infect and manipulate neuronal exten-sions without exposingtheneuronal cell somatothe virus.

* Correspondingauthor.

t On leave of absencefrom theDepartmentofVirology,Institute

of MedicalMicrobiology, UniversityofGoteborg, Goteborg,

Swe-den.

MATERIALS AND METHODS

Virus.Challenge virus strain (CVS) fixed rabies viruswas obtained from supernatants of BHK-21 (baby hamster kid-ney)-infected cells. Cell monolayers were infected with a multiplicity of infection of 5 PFU/ml and incubated for 3 days at 37°C in Eagle minimal essential medium containing 2% fetal calf serum (Flow Laboratories Ltd., Ayrshire, United Kingdom), 2 mM glutamine, penicillin (100 U/ml), and streptomycin (100

[Lg/ml).

Infected supernatants were centrifugedtoremovecelldebris, divided into aliquots, and storedat -70° until used.

Titration of infectious virus. The concentration of infec-tious viruswas determined on cultures of chicken embryo-relatedcellsasdescribedby Smithet al. (38). Briefly, these cellswere grownin amedium containing 1.2g ofNaHCO3

perliterandsupplementedwith10% fetalcalfserum,2 mM glutamine, and antibiotics.After6days of incubationina5% C02, humidity-saturated incubator at 37°C, the cells were

fixed withformaldehyde and stained withcrystal violet. Rabies virus antiserum. Anti-rabies virus antiserum was prepared by immunizing rabbits with beta-propiolactone-inactivated, purified rabies virus emulsified in Freund adju-vant, as described earlier (3). Rabbits were given four immunizing injections at 1-week intervals. One week after the last injection,ablood samplewascollectedfor titration

of the antibodies, using the plaque inhibition test. The antibody titerwasexpressed asinternationalunits, usingas a reference an international rabies antibody standard. The titerof the serumusedwas30 international units.

Nervecell cultures. DRGcellswereobtainedby dissecting

10 to 12 embryos of 15- to 17-day-pregnant Wistar rats. Ganglia were collected using forceps under a dissection microscope, and cells were dissociated by treatment with 0.25%trypsininCa2+-andMg2+-freeHanks bufferatpH7.2 for 30 min at 37°C. The cells were then separated from trypsin by low-speed centrifugationandsuspendedin culture 2733

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2734 LYCKE AND TSIANG

FIG. 1. The two-chamber cell culturesystem, acollagen-coated

petri dish with scratches made in the coat to direct neurites extending from differentiating neurons. In the centerof the dish, a

cloning cylinder is attached to its surface by silicon high-vacuum greaseandmethylcellulose. Neuronal cellsareseededinthecloning

cylinder (inner culturecompartment); neuritic extensionswillgrow

across the grease-cellulose seal and invade the outside of the cylinder (outerculture compartment).

medium. DRG cellswereseeded into theinnercompartment

ofatwo-chambercell culturesystemoriginally developed by Campenot (11). The system, which was adaptedfor studies

on nerve cell-virus interactions (32, 49), allows neuronal extensions which have outgrown in theouterculture cham-berto be infected with the virus, without exposure of the neuralcell somainthe innercompartment. I'hesystem(Fig. 1) consisted of a 35-mm collagen-coated (Vitrogen 100, Collagen Corp., Palo Alto, Calif.) plastic plate (Falcon; Becton Dickinson, Grenoble, France), scratched across its

centerwithstainless-steel needles placed inabrush 0.5mm

apart. An8-mm-wide glass cylinderwas putinthecenterof the plate and sealed with high-vacuum silicongrease. Inthe lower partof the glass cylinder, the trenches formed by the scratches in the collagen coat were sealed with 1%

methylcellulose (Methocel 400, Fluka AG, Buchs, Switzer-land) in Hanks buffer (pH 7.2). The glass cylinderserves as atight diffusion barrierinthe culture. After1 day of culture,

the cells were treated with 2.8 jig of cytosine arabinoside (SigmaChemical Co.,St. Louis, Mo.)permlofmediumfor

2 days to reduce the growth of cells exhibiting mitotic activity. On day 4, the cultures were rinsed and fresh

medium was added.

Wefurthermodified thetechniquedescribedabove forthe

present study.Theposition oftheglass cloning cylinderwas

identified on the external side of the plate to facilitate the microscope recognition of the areas observed, which are

eitherinsideoroutside the barrierevenafterremoval ofthe

cloning cylinder. We also used FIO cell culture medium (Boehringer, Mannheim, Federal Republic ofGermany)

sup-plemented with 10%, fetal calfserum because this medium

appeared torestrictgrowth ofcontaminatingfibroblasts. To stimulate differentiation of neuronal cells and outgrowth of neurites, UV light-sterilized GTlb ganglioside (thegenerous

gift of B. Hauttecoeur, Institut Pasteur) and nerve growth factor (Collaborative ResearchInc.,Lexington, Mass.)were added as suggested by Hauw et al. (21) to final concentra-tions of10 and 25 ng/ml, respectively.

It wasrepeatedlyobserved that the wall between the inner and outer culture compartments seemed a tight diffusion barrier, as (i) the fluid level inside the glass cylinder re-mained at a relatively higher level than outside fluid; (ii) radiolabeled low-molecular-weight substances did not dif-fuseacrossthe barrier; and (iii) the addition ofconcentrated

H2SO4 outside the glass cylinder to destroy peripheral neurites did not result in any morphological cell changes inside the barrier.

Destruction of neuritic extensions. Neuritic extensions pre-sent in the outer culture chamber were destroyed by the addition of200 to300,ul of concentrated H2S04. Aftera few secondsofcontactwith theacid, the culturewasrinsedwith medium until neutrality was restored.

Immunofluorescence. Rabies virus-infected cultures were stained witharabbit anti-rabies virus nucleocapsid immuno-globulin G conjugated with fluorescein isothiocyanate (FITC; kindly prepared byP.Versmisse)asdescribedearlier (46). Stainedcultureswerewashed with phosphate-buffered saline, mounted in Elvanol, and examined with an inverted Zeiss 1M32 microscope (Oberkochen, Federal Republic of Germany).

Staining of neurites with monoclonal antibodies against neurofilaments was performed with a mouse immunoglobu-linGl antibody (clone 2F11; Monosan, Uden, The Nether-lands).

RESULTS

Uninfected DRG cell culture. The DRG cells seeded into the inner culture compartment extended neurites which within 8 to 12 days grew across the seal into the areaoutside the barrier. About 2 x 105 to 3 x 105 cells were present in each cylinderascalculated from six cultures, of which 60% consisted of large neuronal cells. Neurons extending neurites outside the barrier hadno particular locationin the culture; they wereobserved with the samefrequency inthe centerof the culture as in the periphery. However, only a minor proportion of the neurons extended neurites capable

of reaching the outer culture compartment. No more than 236 neurites were counted in the outer compartment ofa culture(themeanvaluewasintherangeof68 to96;n = 18). However,since theneuronalextensions seemed mostoften to be able topenetrate thediffusion barrier onlyasfascicles of extensions twistedtogether, thetruenumberof neuritesin a culture might be greater. Most frequently, the neuritic outgrowth wasrestricted tothe areabetween twotrenches. However, someneurites wereable to traverse the trenches and formaneuritic network. Dependingon theefficiency of antimitotic treatment, neuritic extensions werefound to be associated or not with Schwann cells (Fig. 2). Staining the neuriteswith an FITC-labeled antineurofilament monoclonal antibody allowed us to demonstrate the presence of neuro-filaments in the extensions (Fig. 3). The neurites as well as the neurons maintained their morphological and physiologi-cal characteristics during the 3 or6 weeks of the culture.

Neuronal replication of CVS. Infection of DRG cultures directly in the cloning cylinder showed that the main target cells ofthe CVS virus werethe large sensory neurons (Fig. 4). However, production of virus and its release into the culture fluid over a24- to30-h postinfection period required amultiplicity of infectionintherangeof20PFU/ml. Kinetics J. VIROL.

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FIG. 2. Neuritic extensions. Neurites in the outer culture chamber 10 to 12 days after DRG cells were seeded into the inner culture compartment. Dependinguponrelative efficiencyoftreatmentwithcytosinearabinoside,the drug used to reducegrowthofcells exhibiting mitotic activity, theoutgrowing neurites were or were notassociatedwith Schwanncells. Magnification, x2,000.

ofreplication atthismultiplicity ofinfection, asdetermined by assaying the PFU in the culture fluid at various time intervals, isillustrated in Fig. 5. At48 hpostinfection (p.i.),

104-5

PFU/ml (n = 5) was determined in the culture fluid, whereas 104-0 PFU/ml was detectable as cell-bound virus.

FIG. 3. Neurofilaments in neuritic extensions. Immunofluores-cencestaining usinganFITC-labeledantineurofilament monoclonal antibody.Magnification, x3,200.

The intracellular virus was set free by freeze-thawing the infectedcells in suspension. In this way 1 log PFU was lost (notshown),and it was estimated that approximately75%of thetotal amountof detectable infectious virusremainedcell associated. The CVS-infected DRG neurons seemed to be low producers of virus since only 1 PFU of virus was produced per cell over a 48-h period. On the other hand, the gross morphology of most neurons in infected cultures

appearedtobe rather wellpreserved over 2 to 4 weeks p.i.

One week after infection, the neurites became slender and

appearedto "float" in theculturemedium, sometimesbeing attached atbothendsonly. Infectedneurons in the cultures

survivedforat least4weeks,overwhich interval they were

continuouslyproducing infectious viralparticles.

Rabies virus

attachment

to neuronal extensions. CVS was inoculated into the outer compartment of a number of

cultures to a final concentration of

107.85

PFU/ml. The

cultures were incubated at 37°C, samples were drawn at

various times between 1 and 4 h p.i., and residual virus

activity was assayed. The virus concentration obtained

under these conditions was compared with the concentra-tions of virus remaining on plates containing no cells but

otherwisetreated similarlyand in parallelwith thecultures. Within 1 h after inoculation, 90% ofthe virus had disap-pearedfrom the culturefluid and apparentlybeenadsorbed

to neurites (Fig. 6). To visualize the virus attachment, neurites exposedto virusfor4h at

37°C

were examined

by

immunofluorescence. CVS virus showed a high adsorption affinity for the neurites of DRG cells, since viral

antigens

were found to be adsorbed all along the neuritic plasma

membrane (Fig. 7).

Finally,although themajority of infected cells were neu-rons, viral antigens were also found to be associated with cells which by their

morphology

and appearance in the cultures were classified as Schwann cells. This

finding,

whichpointstothepossible infection ofSchwann cellswith

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.I._1Es.--<.:.n .. i...:

I.~~~~~~~~~~W

FI oio luoecn DR cll at172 hp withrabies viu (CS Imnfursecsting (A.a wth an FITlaele

anti-rabies virus nucleoproteinconjugate. (B) Phase-contrast microscope picture. Magnification, x3,200.

CVS virus, will not be discussed in the present paperbutwill be dealt with elsewhere.

Transportofrabies virus in neuronal extensions. Neurites were infected by adding the virus (1066 PFU in a 0.5-ml volume) to the outer compartment of DRG cell cultures. At 1 h p.i., nonadsorbed virus was removed and the medium was replaced with fresh medium. The cloning cylinder was

filled with medium containing 8 international units of anti-rabies virus serum per mltoexcludenonspecific extraneural spread ofvirus. Incubation of the culturesat37°Cresultedin

propagation of the infection from the neurites to the cell soma. The presence of neutralizing antibodies in the cell cultures did not significantly modify the infection; most of the neuronsin thecylinderwerefoundtobeinfectedasthey were inuntreated control cultures. At 24to 30 hp.i., afew

5r

41 E

U. IL

0)

0

-i

single neurons in the culture inside the diffusion barrier

showed the presenceof rabies antigenasrevealedby immu-nofluorescence (Fig. 8). The infection spread from these primarily infected neurons to the neuritic network. Foci of infectednervecellswereobserved whichinvadedthewhole culture (Fig. 9).

The rateofuptake and transport of viruswassubsequently

estimated from a series of experiments in which the time necessaryforestablishing infection inside thecloning

cylin-der was determined after the virus

(107-85

PFU/ml)

was

inoculated outside the diffusion barrier. The cultures were incubated at 37°C for 1 h, and after various times

ranging

from 1 to 24 h, neurites outside the barrierwere destroyed.

8.or

7.51

ILA U.

6

7.0

0

6.5 3

F

1 24 48 72 96

HOURS FIG. 5. Rabies virus(CVS)replication in rat sensory neurons of dorsalrootganglia. Amounts of virus recovered from cultureliquids

at various timesp.i.

---'A

I

I@

I ***

. . . . .

2 4 6

HOURS

FIG. 6. Attachment rate of CVS to to neurites of the outer culturechamber. Dotted line, Concentration ofresidual infectious virus,plotted against time. Solidline. Controldisplaying reduction of virus ofacell-fiee culture system.

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FIG. 7. Rabies virusantigenattached to neurites. Immunofluo-rescence using FITC-labeled rabbit anti-rabies virus serum for demonstration of viral antigens on neuriticextensions.Theneurites wereexposed to virus for 4 h.Magnification. x4.O00.

Thecultures were furtherincubated at 37°C in the presence of anti-rabies virus serum for a total period of 72 h, after which time the number of fluorescent foci was recorded. About 3.5 h was required for internalization and neuritic transport of the virus across the diffusion barrier (Fig. 10). Therefore a somatopetal transport rate of approximately 12 mm/day wasestimated. However, it cannot be excluded that the virus may also be transported at a higher rate. In

particular,single fluorescent neuronswereobservedassoon as 2or 3 h after neurite exposure to virus.

Drug-induced blockade of virus uptake and transport by neurites. The uptake, internalization, and neuritic transport of CVS virus wereexamined in the presence of two drugs,

cytochalasin B and vinblastine, which are known to affect plasma membrane andcytoskeleton functions. Rabies virus was alsoconsidered as a biological tracer for the inhibitory effectof colchicineonaxonal transport in cultured neurons.

Cytochalasin B and vinblastine were added to the outer chamber of cultures either simultaneously with or4 or24 h before CVS inoculation (0.5 ml ofa suspension containing

107.85

PFU/ml). The drugs were tested either separately at 0.1 mM and 0.01 mM or in combination at the same

concentrations. Infected and treated cultureswereincubated at 37°C for a total period of 72 h. Fluorescent foci were

counted,andproduction ofinfectious viruswasdetermined. Atthe concentrations used, the drugs exhibited no cyto-toxic effects, since morphological changes in the neurites were not seen and CVS replication in DRG cells was not

inhibited. On theotherhand, bothdrugs suppressed uptake

andneuritic transport ofvirus,asindicatedbythereduction of the number of fluorescent foci and of the quantities of virus produced in the presence of the drugs (Table 1). The drugs were still effective in blocking virus transport by the neurites when added 4h p.i.

Colchicineataconcentrationof0.1 mM wasadded(0.5ml perculture) to the outer compartments of cultures showing many neuronal extensions. Colchicine treatment was per-formed either1 to 4hbefore, or atthe same timeas. or 1 to 4 hafter virusinoculation (0.5ml ofasuspensionwith

107.54

PFU/ml). It should be mentioned that colchicine did not inhibit replication of CVS virus in DRG cultures at the concentration used and didnotcauseanygrossmorhological

changes inexposed neurites throughoutthe

experiments.

Figure 11 shows the colchicine added 1 h p.i. or before

inoculation blocked infection spread across the culture bar-rier. In addition. at 2 h p.i. only about

50%

of the final number offluorescent foci, as related to untreated cultures, wasdetected, suggesting a partial impairment of the neuritic transport. Virus transport could not becompletely inhibited

by colchicine treatment, and residual transport was ob-served in different experiments.

DISCUSSION

In the present study, we have investigated on cultured neurons of rat DRG the involvement of sensory neurons in the pathogenesisof rabies virus. Immature neuronal cellsin culture are induced by nerve growth factor to differentiate

with theformation of neurite extensions. Within a few days, youngneurites develop into what appears to be axons (16), and theirunordered somallike cytoplasmgraduallyacquires the highly ordered, adult neuritic cytoskeleton structure (25). Taking advantage of the nerve growth factor-induced

differentiation of neuronal cells, we have studied the inter-actionofrat sensory neurons withrabies virus (CVS strain), using a compartmentalized cell culture system. We have focusedourinterestonthe axonal transport of rabies virus. Fixed rabies virus (CVS strain) was capable of readily infecting DRG neurons withoutrequiring prioradaptationby serial passages, which points to the natural susceptibility of these cells to the virus. Rabies virus exhibited a very high binding affinity for theunmyelinated neurites of rat sensory neurons. Some

90%

of the added virus was taken up from the culture fluid within1 h, and viralantigens were presentinthe neuronal extensions as shown by immunofluorescence. In-terestingly, accumulation ofrabies virus antigens of experi-mentally infected animals occurred in the muscular stretch propioceptors displaying unmyelinated threads of sensory nerve endings (35).

FIG. 8. Axoplasmic transport ofCVS nucleocapsids. Immuno-fluorescence using FITC-labeled rabbit anti-rabies virus nucleo-capsid serum for

demonstr-ation

of CVS nucleocapsids in neurites

fromaneuron24 h p.i.

Magnification.

x3,200.

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FIG. 9. The neurons and neurites which constitute the neuronal network in the inner chamber of the cylinder were infected and subsequently incubated (in the presence ofanti-rabies virus antibodies). The cultures were stained with anti-rabies virus nucleoprotein fluorescent conjugate 72 h after virus inoculation. Magnification, x3,200.

At present it is difficult to evaluate to what extent this binding involves virus-specific receptors. The rabies virus

receptorhasnotbeen identifiedasyet.Arole forglycolipids

in rabies virus binding (42), which would be mediated by

Q 100

o

A.,

CL

4,,.

0

X~~~~~~:

A:

_

0 2 4 6 8 24

HOURS FIG. 10. Rateofaxoplasmictransportof rabiesvirus in neurites. NeuritesintheouterculturecompartmentwereexposedtoCVS.At various intervals ranging from 1 min to 24 h p.i., all neuritic extensions outside the diffusion barrier of the culture were

elimi-natedby treating theouterculture compartmentwith H2S04. The

culturesweresubsequently incubatedat37°C foratotal period of72

h, after which time the number of fluorescent foci in the inner

culture compartment was recorded. The results are the mean of

three experiments. The number offluorescent foci isexpressedas

the percentageof the resultsobtained withanuntreatedbutinfected

control.

their sialic acid residues

(40),

appears

likely.

Consequently,

cellular

gangliosides might

be an

integral

part of the rabies virus receptor

(41).

It is worth mentioningthat thenatureof

gangliosides

varies

according

tonerve celltypes (2).

Although

neurons were

predominantly infected,

infection also affected other cell types (23, 34, 39). In a

cells,

some of which were identified as Schwann cells (47). It appears to us that rat Schwann cells can also be

infected,

although

their relative susceptibility to rabies virus is dif-ficult to evaluate

(unpublished data).

The possible involve-ment ofSchwann cells in rabies pathogenesis is still tobe established.

Itis welldocumented that theaxonaltransportmachinery

is also capable ofdirectingmovement offoreign

organelles

and particles such as injected synaptic vesicles and inert beads with charged surfaces (1, 6, 37). The rate of axonal transport of rabies virus in neuritic extensionswasestimated tobecloseto12mm/day,as2mm wastheminimaldistance TABLE 1. Neuritic transfer of rabiesvirus after treatment of

neurites with cytochalasinB andvinblastine"

Treatment" Time p.i. No.sc f LogPFU/ml

(h) fluorescent foci LgPUm

None 0 205(100) 2.54

Cyt 0 9(4) 0

Vin 0 12(6) 0

Cyt + Vin 4 22(16) 0

Cyt + Vin 24 190 (93) 2.17

"Rabies virus was inoculated into the outer culture compartment. The

presenceofviral antigens (fluorescent foci)orinfectious virus in the inner

culturecompartment at72 hp.i.wasconsideredasanindication ofneuritic

transfer of virus across thebarrierbetween thecompartments.

'Finalconcentrations ofcytochalasin B(Cyt) and vinblastine (Vin)were

0.1and 0.01mM, respectively.

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FIG. 11.

Neurites in

colchicine (( after(-)ino

innercultur4

centfoci der Theresults;

percentages

tobe runf

barrier of t hwasthet

isconsider simplex vii (11). The r

acetylcholi mm/dayin direction, c axonaltrar

axonal trar

since the ti events, i.e

port. In the tion by ne

fusion ofe arapidrea

is likelyto transport.

not yet kr

microscope

shortly aft enveloped

may be me

E. Lyke, a pits were infection o

cervical ga

involve co, some time

rateofvirn

Recently ro-9-3-(2-hl intracellula

various wa

axons

were

found

to block the transport of the lectin wheat A germ

agglutinin

and of herpes

simplex virus

in neurites of DRGcells(30). Thiseffect wasascribed to the action of the drugs on microtubular transport. Consistent with the prob-able importance of plasma membrane and cytoskeleton

A \ activities for neuritic uptake and transport of viruses, we

found that both cytochalasin B and vinblastine efficiently blocked the neuritic transferofrabies virusinthe DRGcell cultures. Cytochalasin B inhibits microfilament formation by

v preventing actin from assembling and consequently blocks

endocytosis (9, 33). Cytochalasin B could also block axonal transport by abolishing microtubule formation (22).

Micro-A tubule inactivation resulting in transport dysfunction can

A also be caused by vinblastine, which is supposed to induce

A microtubule aggregation (7). In addition,

colchicine,

which is

... most commonly used for

blocking

axonal transport,

exer-A cises a

depolymerizing

effect on microtubules. We verified

A that colchicine inhibited neuritic transfer of rabies virus

l I when it was administered either

before,

or at the same time

-4 -2 0 +2 +4 as, orup to 1 hafter virusinoculation. At 10FM

concentra-HOURS tion. maximal inhibition of microtubule

assembly

in

HOURS neuropils required 1h(36). Weconclude from these data that Effect ofcolchicineonneuritictransport of rabiesvirus. both tubulin- andactin-containing structures appeared to be the outer culture compartment were treated with involved in the neuronal transport of rabies virus.

However,

D.1 mM)eitherbefore (+).oratthesame timeas

(0),

or the

relative

participation

of each structure is not known at culation of CVS.The amountof virus transportedto the

ecompartment was recorded as the number of fluores- present.

nonstrable after atotalincubationperiod of 72 h at37C. The rat DRG sensory neuron can be described as being arethe mean ofthreeexperiments and areexpressed as permissive but only moderately susceptible torabies virus, of the results obtained with untreatedcontrol. which is suggested by the large multiplicity of infection necessary for establishing a productive infection. This con-trastswith thehigh susceptibility usually found with primary ortransferring infection across the diffusion-tight cultures ofneurons derived from the mouse and rat CNS (47; he compartmentalized culture system and 3.5to 4 unpublished data). Furthermore, the rat sensory neuron ime necessary forthistransferto occur.Thisrate appears capable ofproducing only relatively small amounts ably less thanthatpreviously reported forherpes of virus. We estimated that there was about 1 PFU of rus (50 mm/day), using the same culture system infectious virus produced per DRG neuron over a 48-h rapid phase of the axonal transport of molecular period, provided that no additional DRG cells in the culture ine esterase hasbeen estimated to be close to 150 became productively infected. The observation of a rela-theretrograde and410mm/day in the anterograde tively low virus production by the neuronal cells correlates and the corresponding slow phase of anterograde well with the scarcity of virus when infected neurons were

isport is estimated as 2.9 to 5.1 mm/day (13). The examined by electron microscopy (M. Derer, unpublished nsport rates are difficult to evaluate for viruses, data). Again, it is interesting to compare these results with me measured isusually theresult of asequence of the production of high yields of infectious virions by mouse ., attachment, internalization, and axonal trans- and rat CNSneurons (10- to 100-fold) (47; unpublished data)

ecase ofherpes simplex virus, whoseinternaliza- and to note the incapacity of superior cervical ganglia urites and nonneuronal cells seems to occur by neurons to replicate infectious rabies virions (45). Taken all nvelope and plasma membrane (18, 32), which is together. these results are in favor of the existence of various ction, the experimentally estimated transport rate levels in neurotropism. To what extent the low production of reflect more accurately the actual rate of axonal virus isrelated to the stability of neurons ininfected cultures The neuritic uptakemechanism for rabies virus is and to the prolonged production of virus remains to be iown. However, from our preliminary electron specified. The finding that rabies virus-infected DRG

neu-e observations, the virus found in the nerve cells rons can be maintained in culture for over 4 weeks demon-ter inoculation of the cultures appeared to be strates the capacity of this neuronal cell type to harbor the and internalized by coated pits, and thus uptake virus for long periods of time. Consequently, DRG neurons diated by an endocytosislike process (M. Derer, aregood candidates as sites for the protracted harboring of nd H. Tsiang, unpublished data). In fact, coated virus in naturally occurring cases of rabies exhibiting pro-observed in the early phases after rabies virus longed incubation times.

ofchicken embryo-related cells (40) and superior The observation that isolated neurons or isolated groups Lnglia neurons (45) If virus internalization were to of neurons can be infected through the neuritic network ated pits, virus uptake by neurites could require despite the presence of

neutralizing

antibodies deserves to and thus be responsible for the seemingly slow be discussed. This finding is tobe related tothe differential is axonal transport. cell-to-cell virus transmission of

pathogenic

and

apathogenic

, threesubstances [taxol, nocodazole, and eryth- rabies virus strains (17). In our

study,

we observed the ydroxynonyl)adenine] which are known to inhibit infection of neurons

presumably

connected to the neurites

tr transport of endosomes and to interfere in extending in the external compartment, followed

by

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ment. Since this infection occurred in the presence of

neutralizing antibody even when the neurons were not contiguous,wepostulate that viral spreadoccursby neurite-to-cellcontact. This clearly shows that once rabies virus is

hidden inaneuronalcircuitry, itescapestothe host defenses andcanbe transported fromone neurontothe next. In this respect, theculture device described in thispapermimicsa brain model of rabies virus infection in which there is an

entrypathway and a neuronal network.

The relatively low susceptibility of DRGneurons,coupled

with alow capacity for producing viral particles,can bean advantage for this celltype, enablingitto sustainlong-term infection. DRGneurons have already been shown to repre-sent an excellent environment for persistent infection with other viruses, such as herpesvirus (29). It is tempting to

assume that rabies virus preferentially infects central

neu-rons(47) andmotorneurons(44), resultingin theproduction ofhigh yields of rabies virus, whereas DRG neurons should rather be considered as sites for virus entry in a persistent infection cycle. We have already shown that myotubes, which allow an abortive rabies virus infection to develop, could be a site where rabies virus canpersist extraneurally (44) andthat thesuperiorcervicalganglianeurons,whichare relatively resistant to rabies virus infection, could also harbor the virus forlong periodsof time withoutexcretingit (45). Thusavariety of celltypeswhicharenaturally

suscep-tible to rabies virus infection could harbor the virus and protect it from the host's natural defenses until an event triggers viral replication.

There is increasing interest in the investigation of virus circulation in the nervous system. As for rabies virus, herpesvirus transport has been reported in the brain (5, 12, 29)as wellasin culturedneurons (32, 49). The involvement of the neuralpathwaysinthetransportofmanyother viruses has been invoked, including the scrapie agent(28),

poliovi-rus (14), reovirus (48), and Semliki forest virus (27). The compartmentalized cell culture system described here for rabies virus is a useful tool for the investigation of neurotropic virustransport incultured neurons.

ACKNOWLEDGMENTS

We gratefully acknowledge the technical assistance of Janine Cadinuand Simone Guillemer, and we thank Bernard Bizzini for reviewingthemanuscript.

Thestudywassupported bygrants from the Institut National de LaSantdetdelaRecherche Medicale (CRE 846019)and from the

Swedish Medical Research Council(4514).

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