Pathway of vesicular stomatitis virus entry leading to infection

J

Mol. Rd.

(1982)

156, 609-631

Pathway of Vesicular Stomatitis Virus Entry Leading to Infection KARL S. IMATLIS, HUBERT REGGIO, ARI HELENIUS ASI) KAI QIMONS Division of Cell Biology Molecular Biology Laboratory Postfach 10.2209 6900 Heidelberg Federal Republic of Oermcrny

Europeun

(Received

I1 August

1981, and in revised form 3 LVovember 1981)

The entry of vesicular stomatitis virus into Madin-Darby canine kidney (MDCK) cells was examined both biochemically and morphologically. At low multiplicity and 0°C viruses bound to the cell surface but were not internalized. Binding was very dependent on pH. More than ten times more virus bound at pH 65 than at higher pH values. At the optima1 pH, binding failed to reach equilibrium after more than two hours. The proportion of virus bound was irreproducible and low, relative to the binding of other enveloped viruses. Over 90% of the bound viruses were removed by proteases. When cells with pre-bound virus were warmed to 37% a proportion of the bound virus became protease-resistant with a half-time of about 30 minutes, After a brief lag period, degraded viral material was released into the medium. The protease-resistant virus was capable of infecting the cells and probably did so by an intracellular route, since ammonium chloride blocked the infection and slightly reduced the degradation of viral protein. When the entry process was observed by electron microscopy, viruses were seen bound to the cell surface at 0°C and, after warming at 37”C, within coated pits, coated vesicles and larger, smooth-surfaced vesicles. No fusion of the virus wit,h t,he plasma membrane was observed at pH 7.4. When pre-bound virus was incubated at a pH below 6 for 30 seconds at 37”(‘, about 40 to SO?, of the pre-bound virus became protease-resistant. On the basis of this result’ and previously published experiments (White et al.. 1981). it was c.oncluded that vesicular stomatitis virus fuses to the MDCK cell plasma membrane at low pH. These experiments suggest that vesicular stomatitis virus enters MDCK cells by rndocytosis in coated pits and coated vesicles, and is transported to the lysosome where the low pH triggers a fusion reaction ultimately leading to the transfer of the penome into the cytoplasm. The entry pathway of vesicular stomatitis virus thus resembles that described earlier for both Semliki Forest virus and fowl plague virus.

1. Introduction To infect membrane cytoplasm.

a cell, an enveloped virus must transfer its genome across a douhlrbarrier: that is, from within the virus particle into the cellular where it can be replicated. Two locations are recognized where 609

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penetration of the virus genome can occur (Dales, 1973) : the cell plasma membrane and an intracellular vacuole. Transfer at an intracellular location is distinct from that at the plasma membrane because it must be preceded by endocytosis of the virus particle, and because t,he intracellular environment. may be quite different from that at the cell surface. Penetration through the plasma membrane is exemplified by Sendai virus, which is known to fuse its envelope with the plasma membrane (Poste & Pasternak, 1978). In this way the genome is transferred into the cytoplasm, where replication can commence. The fusion is dependent upon cleavage of the spike protein F into two sulfhydryl-linked glycopolypeptides (F, and F,), which are joined by disulfide bonds (Homma & Ohuchi, 1973; Scheid & Choppin, 1974). The best-characterized example of penetration at an intracellular location is the alphavirus Semliki Forest virus (Helenius et al., 1980 ; Marsh & Helenius, 1980). Our results have shown that, after binding to microvilli, it is taken up by coated vesicles and routed through endosomes to the secondary lysosomes. The acidic environment in the lysosome induces fusion of the virus membrane and lysosomal membrane, releasing the genome into the cytoplasm (Helenius et a2.. 1980,1981 ; White et al., 1980). Fowl plague virus, an influenza virus, was recently shown by us to follow a very similar pathway into the cell (Matlin et at.: 1981). For vesicular stomatitis virus, a rhabdovirus, evidence exists for both fusion at the plasma membrane (Heine & Schnaitman, 1969,1971) and endocytosis (Simpson et al., 1969; Dahlberg, 1974; Fan & Sefton. 1978). The observation that lysosomotropic agents block infection (Shimizu et al., 1972 ; Miller & Lenard. 1980) has been interpreted as evidence for lysosomal involvement in productive entry (Miller & Lenard, 1980). Recent studies have shown that at low pH vesicular stomatitis virus, like Semliki Forest virus and influenza virus, can fuse cells together and fuse to the baby hamster kidney cell plasma membrane (White et al., 1981). The relationship of these findings to cell infection has not been investigated. In this paper, we describe a combined morphological and biochemical study of the entry pathway of vesicular stomatitis virus into Madin-Darby canine kidney cells. Our results suggest that infection begins with endocytosis and that the mechanism for penetration is probably identical t,o that found for both Semliki Forest virus (Helenius et al., 1980: Marsh B Helenius. 1980) and the influenza virus fowl plague (Matlin et al., 1981).

2. Materials and Methods (a) Cells Madin-Darby canine kidney cells were grown as described (Matlin et al., 1981) in Eagle’s MEM with Earle’s salts (Earle’s MEM) supplemented with 10% fetal calf serum, 10 mMHEPESt (pH 7.3), penicillin (100 units/ml), streptomycin (100 &ml) and Fungizone (@025 pg/ml). t Abbreviations used: HEPES, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; MDCK, Madin-Darby canine kidney cells; p.f.u., plaque-forming units: BSA, bovine serum albumin; PBS, [bis-(2-hydroxyethyl)imino]-Tris-[(hydroxymethyl)methaIle] ; phosphate-buffered saline ; bis-Tris, PIPES, piperaziue-N, N’-bis(%-ethane suifonic acid); MOPS, 3-(N-morpholino) propane sulfonic acid ; BHK, baby hamster kidney-21 cells.

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(b) Stock virus Vesicular stomatitis virus, Indiana serotype, was provided by Dr Larry Altstiel of thr Rockefeller University, and was plaque-purified 3 times on MDCK cells. For virus stock production, MDCK cells were infected in a 5% CO, atmosphere (901 p.f.u./cell) for 20 h at, 37°C in Earle’s MEM containing 02% (w/v) bovine serum albumin. The medium was cleared of cell debris by centrifugation at SOOOgmax for 15 min at 5°C and the virus concentrated by cemrifugation at 16,000g max for 2 h at 5°C in the Sorvall HB4 rotor (Dupont Instruments. Newtown, Corm.) Virus pellets were resuspended overnight at 0°C in phosphate-buffered saline (Dulbecco formulation, PBS) containing 92% BSA, and stored in equal samples at. -X0?‘. (c) Radioactive virus Vesicular stomat’itis virus labeled with [35S]methionine was prepared by infecting slightly subconfluent flasks of MDCK cells with 20 p.f.u./cell in Earle’s MEM, without bicarbonatcx. buffered to pH 6.3 (10 mw-bis-Tris, 10 mM-PIPES, 10 m&r-MOPS, 10 mM-NaHzPO,), and supplemented with 62% BSA at 37°C. After 1 h, the inoculum was aspirated and replaced with 5 ml Earle’s MEM per flask containing ~PM-methionine (01 the normal MEM concentration) and 15 mM-HEPES (pH 7.3). After another 1.5 h, 1 mCi of [35S]methionine was added to each flask, and incubation was continued for 7-5 h in an incubator in an atmosphere of 506 C02. After a spin to remove debris, the virus was concentrated by centrifugation at 16,000g max for 2 h in a Sorvall HB4 rotor and resuspended overnight at 0°C in 50 miw-Tris. HCl (pH 7.4) containing 50 mM-NaCl. Finally, the virus was purified on sucrose gradients prepared by overlaying a SO-ml 25”i, to 55”i, (w/v) isopycnic gradient with an SO-ml loo/; to 200/, (w/v) velocity gradient, both in Tris/NaCl. The gradients were centrifuged for 3 h at 284,000 g max in a Beckman SW40 rotor. The peak of radioactivity in t,he lower part of the gradient was pooled and stored in portions at - 80°C. This material was identified as virus and judged free of contaminating labeled material by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography. The titer of preparations was approx. 2 x 10’ p.f.u./ml, which corresponded to 1 to 2 p.f.u./ct per min. (d) Unlabeled virus preparations Large quantities of vesicular stomatitis virus were grown in BHK cells and purified either as described earlier (White et al., 1981) or by an alternative procedure that reduces aggregation (Dr Frank Landsberger, The Rockefeller University, personal communication). In this method the medium from BHK cells infected for 20 h at 601 p.f.u./cell was centrifuged at 8090 g for 15 min at 5°C in a Sorvall HS4 rotor to remove cell debris, and the virus concentrated hy centrifugation at 16,000g max in a Sorvall GSA rotor. The pellets were resuspended overnight in Tris/NaCl buffer, dispersed with 5 strokes of a Dounce homogenizer, and purified by banding to isopycnic density on 5% to 50% (w/v in TrisNaCl) potassium tartrate gradients at 81,500 g for 90 min at 5°C. The salt was removed by dialysis in the cold overnight against 300 vol. of Tris/NaCl, and the virus was frozen in equal samples at a concentration of 1.5 mg protein/ml at -80°C. The preparation was pure virus as judged by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels and contained about 1.7 x 10’ p.f.u./pg protein. The particle-to-p.f.u. ratio was about 100. assuming a particle molrvular weight of 5% x 10-i’ (Miller & Lenard, 1980). (e) Plaque titrations Plaque titrations were performed in duplicate on MDCK cell monolayers (Gaush & Smith. 1968) grown in 60 mm plastic Petri dishes (Falcon Plastics, Oxnard, Calif.). Experimental samples were diluted serially in PBS containing 02% BSA and 625 ml samples were adsorbed to cell monolayers for 1 h at 37°C in 5% CO,. The inoculum was then removed and the cells overlaid with 4 ml of Earle’s MEM containing 61% BSA, O.lo/, fet,al calf serum. penicillin. streptomycin and 0.9% agarose (Litex, Denmark). After 2 days incubation at 37Y’ in 5”,, CO,, plaques were detected by staining with 902% neutral red in PBS.

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(f) Binding and uptake assays To measure binding, MDCK cells were grown to confluency ( 106 to 2 x lo6 cells/dish) in 35 mm diameter plastic Petri dishes (Falcon), washed twice with 1 ml binding medium per dish, and cooled for 10 to 15 min on an ice-bath. Approx. 20,000 to 40,000 cts/min of radioactive vesicular stomatitis virus in 200 ~1 of the appropriate ice-cold binding medium were added to each dish, and the dishes were gently shaken for various times at 0°C. After this incubation, the free virus was removed and the cell monolayers washed twice with 1 ml cold binding medium. Cells were t,hen scraped from the dish, the dish was washed twice with 1 ml cold binding medium and the cells were pelleted in a conical glass centrifuge tube at 2000 revs/mm for 5 min at 4°C. After removal of the supernatant, pellets were solubilized directly in Rotiszint 22 (Carl Roth, Karlsruhe, G.F.R.), a detergent-based scintillation fluid. and counted in a Searle mark III liquid scintillation counter (Searle Analytic Des Plaines. Ill.). Variable pH binding media were prepared by supplementing Earle’s MEM containing 62% BSA with the series of buffers suggested by Eagle (1971). Media with a pH less than 6.0 were buffered with 20 mM-succinate. Bicarbonate was omitted from all media used at 0°C or at pH values other than 7.4. Adjustment of pH was done at room temperature. All measurements of virus uptake were done after warming sample dishes for various periods in media, buffered either to pH 7.4 or 6.3. After incubation of labeled virus with cells, the medium was collected, and the medium and cells were analyzed separately. The cells were washed, scraped from the dish, pelleted, and counted directly, or washed and incubated with proteinase K to remove surface-associated virus. In most cases. proteinase K was added to monolayer-s at a concentration of 65 mg/ml in PBS. and the dishes shaken at 0°C for 45 min. Afterwards, the proteinase K solution was removed and the monolayers were scraped and pelleted in PBS containing 020/6 BSA and 1 mM-phenyl methane sulfonyl fluoride. Finally, the cell pellet was washed twice with 5 ml PBS/Ort% BSA, and the pellet was counted. Medium removed after the warming step was precipitated with an equal volume of 10% trichloroacetic acid on ice for 1 h, centrifuged for 5 min in a microfuge (Eppendorf. Hamburg. G.F.R.). and the supernatant was counted for radioactivity. (g) Infection by internalized virus To determine if cell-associated, proteinase K-resistant virus was capable of infecting the host cell, MDCK cells were grown to confluency in 2 24.well plastic trays (5 x lo5 to 6 x lo5 cells/well) (Falcon). The cells were washed with binding medium (pH 6.3) and cooled to 0°C. Stock virus was suspended in 0.2 ml ice-cold binding medium with or without 20 rn,vammonium chloride, and allowed to bind to the cells as described above (see Table 2). After 1 h, the inocula were removed from 1 of the 2 trays, and 15 ml pre-warmed medium at pH 7.4 (with bicarbonate, and with or without ammonium chloride) was added to each well. The tray was incubated for 10 min at 37°C in an atmosphere of 5% CO,. and then cooled to 0°C by placing it on ice and exchanging the warm medium for cold medium. The second tray was not taken through this warming step. Both trays were then processed together throughout the rest of the experiment. To remove surface-associated virus, proteinase K (05 mg/ml in Earle’s MEM with or without 20 mM-ammonium chloride) was added to each well and the trays were shaken at 0°C for 30 min. Bfter digestion, the protease solution was removed and the monolayer washed 3 times in cold medium (pH 7.4) with or without ammonium chloride. Next, pre-warmed complete MDCK cell growth medium with or without ammonium chloride was added to each well for 1 h to help the cells recover, and finally, medium (pH 7.4) with bicarbonate was added and the trays incubated for 45 h at 37°C in 5% C02. The amount of virus produced was measured by plaque titration. (h) Antibodies Antibodies against vesicular stomatitis virus G protein were prepared by immunizing rabbits with G protein complexes. These were prepared by modifying a procedure used for

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the preparation of spike protein complexes from other enveloped viruses (Simons at al.. 1978). Vesicular stomatitis virus (1.5 mg) was dissolved at room temperature in 2% Triton X-109 containing 50 mu-Tris.HCl (pH 8) and 200 m&f-NaCl, mixed for 15 min, and centrifuged over a 20% to 50% sucrose gradient containing a zone of 15% (w/v) sucrose, 1‘?. (v/v) Triton X-100 in the same buffer for 24 h at 284,OOOg max in an SW40 rotor at 20°C. The spike protein complexes were located by gel electropboresis, dialyzed against 500 vol. 50 mw-ammonium chloride at 5”C, and lyophilized. To purify the complexes further, they were redissolved in 50 mw-Tris . HCl (pH 8) containing 200 mM-NaCl and centrifuged over a second gradient identical to the first, except for the absence of the Triton zone. G protein complexes isolated in this manner were judged free of M protein and other viral components by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Rabbits were injected directly in the popliteal lymph nodes with 10 to 20 rg protein in Freund’s complete adjuvant (Goudie et al., 1966), boosted subcutaneously 3 weeks later in Freund’s incomplete adjuvant, and bled after 10 days. Anti-G protein immunoglobulin G (IgG) was prepared by standard procedures of ion-exchange chromatography (Fahey, 1967). (i) Immuno$uorescence lmmunofluorescence was performed by the indirect technique. MDCK cells were grown to a slightly subconfluent density on glass coverslips in 35 mm plastic dishes. The dishes (1 coverslip per dish) were washed in binding medium (pH 63) and cooled on ice. Vesicular stomatitis virus (1 pg/dish) in ice-cold binding medium at pH 6.3 was added and allowed t*o bind in the cold for 1 h. After removing the free virus, 2 coverslips were washed 3 times with cold PBS at pH 6.3 (Eisen formulation), covered with cold formaldehyde (3% (w/v) formaldehyde, 61 mM-NaCl, 61 miw-M&l, in PBS), incubated at room temperature for 20 min, and quenched for 10 min in 50 mM-NH,Cl in PBS. The other dishes were warmed at 37°C for various times in medium at pH 7.4, and the coverslips washed 3 times with PBS at pH 7.4 (Eisen formulation), incubated for 20 min in fixative, and quenched for 10 min. Viral antigens on the cell surface were visualized by inverting the coverslips over 25 ~1 of anti-(;protein IgG (300 pg/ml), washing and then staining with goat anti-rabbit IgG conjugated to rhodamine. To see both surface and intracellular antigens, fixed monolayers were freezrthawed once on solid CO1 and stained with antibodies (Ash et al., 1976,1977). Coverslips were mounted in 900,;, (v/v) glycerol in PBS and viewed through a Zeiss photomicroscope III equipped with a Planapo 63 oil-immersion objective (Zeiss, Oberkochen, G.F.R.). The rhodamine-conjugated antibody, a gift from Dr Daniel Louvard, was affinity-purified and selected by ionexchange chromatography to have 2 to 3 rhodamine molecules per Ig(: molecule (Brandzaeg, 1973; Blakeslee & Baines, 1976). (j) Electron microsempy To visualize virus hinding and internalization, cells in 35 mm Petri dishes were washed and 60 pg virus bound at 0°C in 200 ~1 binding medium (pH 6.3) for 1 h. The samples were then either fixed directly with cold 2.5% glutaraldehyde in 50 mw-sodium cacodylate (pH 7.2), 50 mM-KCl, 2.5 mM-MgCl, for about 30 min, or warmed at 37°C and then fixed at room temperature. Post-fixation with osmium tetroxide, dehydration and embedding were as described earlier (Helenius et al., 1977), except that the cell monolayer was scraped aft,er osmium treatment with a Teflon blade, dehydrated, and embedded in Epon 812 aft,er centrifuging in Beem capsules. (k) Low-pH-dependent association To measure the low,-pH-induced, protease-resistant association of vesicular stomatitis virus with MDCK cells, radioactive virus was bound at 0°C for 1 h and the monolayer washed free of unattached virus. The dishes were then transferred from an ice-bath to the surface of a water bath or to a hot-plate at 37°C and flooded with warm medium at various pH values for different times. The medium was then aspirated and the monolayers rapidly cooled on ice and treated with L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-

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trypsin (1 mg/ml in PBS). After 45 min the trypsin was inhibited by addition of an equal weight of soybean trypsin inhibitor and the cells were scraped and counted as described earlier. (1) Miscellaneous techniques Solutions of ammonium chloride were freshly prepared prior to each experiment by dissolving the salt directly in the appropriate medium, and, when necessary, correcting the pH to the original value. Protein determinations were performed as described by Lowry et al. (1951). To check for virus aggregation, small quantities of radioactive vesicular stomatitis virus were suspended in 200 ~1 of medium, at either pH 6.0, 63 or 7.4 at 0°C and loaded on 15% to 30% (w/v) linear sucrose gradients in Tris/NaCl with a 55% sucrose cushion. The gradients were centrifuged for 30 min at 102,OOOg max in an SW40 rotor as described by Keller et al. (19’78). Viral preparations were analyzed on sodium dodecyl sulfate/polyacrylamide gels utilizing the Laemmli (1970) buffer system. The gels were prepared with a 10% to 15% (w/v) acrylamide gradient (acrylamide to bisacrylamide, 75 (w/w)) and contained 8 M-urea for improved resolution (Matlin, 1979).

(m) Sources of reagents Cell culture media and reagents were purchased from Gibco-Biocult (Glasgow, Scotland). Fetal calf serum was obtained either from Gibco or Boehringer Mannheim (Mannheim, G.F.R.). Proteinase K was from either Boehringer or Merck (Darmstadt, G.F.R.). All buffers were from Sigma Chemicals (Muenchen, G.F.R.). Soybean trypsin inhibitor and TPCKtrypsin were purchased from Worthington (Freehold, N.J.). Reagents for electron microscopy were purchased either from Ladd Research (Burlington, Vt) or Serva (Heidelberg, G.F.R.). All other chemicals were reagent grade. [%]methionine (> 600 Ci/mmol) was obtained from Amersham Radiochemicals (Amex-sham, U.K.).

3. Results (a) Binding

at 0°C

To study the entry of vesicular stomatitis virus into MDCK cells, we first examined the interaction of virus and cells at 0°C. Previous measurements of vesicular stomatitis virus binding to cells at 0°C by Miller & Lenard (1980) indicated that less than 10% of the added virus became cell-associated in one hour and that binding did not easily reach an equilibrium value. This result contrasts with the relatively efficient binding we observed for both Semliki Forest virus and the influenza A fowl plague virus (Fries & Helenius, 1979 ; Helenius et al., 1980 ; Marsh & Helenius, 1980; Matlin et al., 1981). In an effort to optimize vesicular stomatitis virus binding to MDCK cells, virus association with cells after one hour at different pH values and 0°C was examined. Surprisingly, binding of trace quantities of virus was sharply dependent on pH (Fig. 1). At pH values between about 5.5 and 6.5, binding was maximal. At pH 6.8 and above, binding was over ten times less. Below pH 5.5, intermediate values were obtained. The proportion of added virus bound at the optimal pH varied considerably between 2% and 20% from experiment to experiment. Our measurements of the kinetics of binding confirmed the earlier observation by Miller & Lenard (1980). Binding at pH 6.3 and 0°C increased throughout the experiment without reaching a plateau (Fig. 2). After incubation for 150 minutes,

only about 7% of the added radioactive

virus was cell-associated in this experiment

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6.0 PH

FIG. 1. Effect of pH on virus binding. Vesicular stomatitis virus ( - 20,000 cts/min) was suspended al 0°C in 200 ~1 of medium buffered to the indicated pH values and allowed to bind to MDCK cells for 1 h at 0°C with gentle shaking. Afterwards, the cells were washed twice with cold medium at the same pH scraped and counted. Maximal binding in this experiment (pH 65) was 19%.

0

30

90 Time (min)

150

FIG. 2. Rate of virus binding. Vesicular stomatitis virus (- 20,009 cts/min) was suspended at 0°C in 200 ~1 of medium buffered to pH 6.3 containing (0) or not containing (0) 20 mM-ammonium chloride. and allowed to bind to MDCK cells for various times. After the binding step, the cells were washed twice with cold binding medium with or without ammonium chloride, scraped and counted.

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(Fig. 2). These results differ sharply from the observed binding kinetics of both Semliki Forest virus and fowl plague virus, where an equilibrium level of 40 to 600/, binding to BHK and MDCK cells. respectively. was reached in one to two hours at, 0°C (Helenius et al., 1980; Matlin et al., 1981). A possible explanation for the increased binding at low pH would be low-pHinduced virus aggregation. If such aggregates bound to cells, viru+cell association would appear higher without any actual increase in the interaction between individual viruses and the cell surface. This possibility was ruled out by velocity centrifugation of radioactive virus suspended in media at pH 6.0, 6.3 and 7.4 in gradients of 15% to 30:/, sucrose in Tris/NaCl, with a cushion of 55y; sucrose. Virus aggregates were expected to sediment to the level of the cushion (Keller et al., 1978). Under all pH conditions tested, however, the radioactivity in the virus was located in the top third of the gradient and not at the level of t,he cushion (data not shown). It was also possible that the increase in binding was due to virus fusion to the plasma membrane rather than a reversible interaction between the virus and the cell surface. Low-pH-induced membrane fusion activities have recently been described for vesicular stomatitis virus (White et al., 1981), Semliki Forest virus (White & Helenius, 1980; White et al., 1980,198l) and influenza virus (Maeda 8: Ohnishi, 1980; Huang et al., 1981 ; White et al., 1981 ; Matlin et a,Z., 1981). To examine whether fusion was occurring, [ 35S]methionine-labeled virus was allowed to bind to cells at 0°C and pH 6.3 for one hour, and the cells were washed and treated in the cold with proteinase K. Since the virus preparation was labeled with [35S]methionine, most of the radioactive label was concentrated inside the viral envelope in M protein and capsid-associated proteins as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography. If fusion with the plasma membrane had occurred during the incubation at O”C, most of t,he radioactivity would be inside the plasma membrane and protected from proteinase K. Conversely. virus bound to the plasma membrane was expected to be removed by the protease treatment, because the spike glycoprotein G is sensitive to proteinase K (Mudd, 1974). In fact, we observed that over go”/,, of the cellassociated virus was released by treatment with various concentrations of proteinase K for 20 minutes at 0°C (Table 1). This experiment indicated that, at 0°C and pH 6.3, vesicular stomatitis virus was not fused to the plasma membrane of MDCK cells but was bound to the cell surface. Furthermore, the experiment showed that no viruses were endocytosed under these conditions. because endocytosed virus would be resistant to the effects of proteinase K. In later experiments treatment with proteinase K in the cold was used to distinguish between bound and internalized viruses.

(b) Internalization

at 37°C

The interaction of virus and cells at 37°C was examined next. To assess the effect of pH on internalization, trace quantities of radioactive virus suspended in warm medium at either pH 6.3 or 7.4 were added to MDCK cells at 37°C. At various times,

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1

Rdea,sP of vesicular stomatitis virus from MDCK

‘Prratment

617

VIRUS

Incubation time at 0°C (min)

crlln by proteinasP K

Cts/min remaining

o/o (‘ontrol remaining

None (binding control)t PBS MEM

40 40

2628 906 881

100 34 34

Proteinase K (mg/ml) OC25 0.5 0.75 1.0

40 40 40 40

189 133 110 89

7.2 5.0 4.2 3.4

t Trace quantities of radioactive virus were allowed to bind to MDCK cells in pH C3 medium for 1 h at OY’. Binding in this experiment was about 6%. After removing unattached virus. the cells were treated as indicated. washed, scraped and counted.

one dish of cells was washed and the cells scraped and counted, and a parallel dish treated with proteinase K to remove surface-bound virus, was washed, scraped and counted. This procedure permitted both surface-associated and internalized radioactivity to be measured. As illustrated in Figure 3, both the surface and the proteinase K-resistant radioactivity increased with time at both pH values. The amount of surface-associated virus at pH 6.3 exceeded that at pH 7.4 at all times examined (Fig. 3(a)). The differences, however, between the amounts of virus resistant to proteinase K at pH 6.3 and at pH 7.4 were minute (Fig. 3(b)). The proportion of virus internalized at either pH was small. After 30 minut,es incubation, only about 1% of the added radioactivity was proteinase K-resistant. In contrast, about 50% of the added Semliki Forest virus entered BHK cells in 30 minutes under similar conditions (Helenius et al., 1980 ; Marsh & Helenius, 1981). This experiment indicated that the increased binding observed at pH 6.3 does not lead to a proportionately higher amount of virus becoming proteinase Kresistant. Uptake of vesicular stomatitis virus in MDCK cells, therefore, may not be limited by the rate of binding but by the rate of internalization. Another possibility is that the rate of internalization at pH 6.3 is reduced in comparison with that at pH 7.4. In the previous experiment, the internalization of virus into cells depended on both the rate of binding and the rate of uptake. To study uptake independently. the internalization of pre-bound virus was examined. Trace amounts of radioactively labeled vesicular stomatitis virus were added to cells at pH 63 and 0°C and incubated on ice for one hour. After washing, fresh medium at pH 63 and 37°C was added, and the uptake of virus measured at various times by the proteinase K assay (Fig. 4). Under these conditions, vesicular stomatitis virus entered the cells with an initial rate measured within the first 15 minutes

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Y 0

30 60 Time (min)

90

0

I I 30 60 Time (mln 1

(al

I

90

(b)

FIG. 3. Cell association of vesicular stomatitis virus at 37°C. Vesicular stomatitis virus (w 26,666 cts/min) was suspended in 65 ml of medium at 37°C buffered to either pH 63 (0) or pH 7.4 (0) and added to MDCK cells. At various times, the medium was removed from 2 duplicate dishes and the dishes cooled on ice. The cells in one dish were washed twice with cold medium at the same pH, scraped and counted to determine total cell-associated radioactivity. The cells in the other dish were treated with proteinase K (65 mg/ml) on ice for 45 mm, washed, scraped and counted to determine proteinase Kresistant counts. (a) Cell surface-associated virus (proteinase-K-sensitive, cell-associated radioactivity). (b) Internalized virus (proteinase K-resistant, cell-associated radioactivity). (0) pH 63, (0) pH 7.4.

corresponding to a half-time of about 30 minutes. Uptake during the experiment, however, never exceeded about 50% of the bound virus radioactivity. Similar bursts of endocytosis at 37°C by pre-cooled cells have been observed (Anderson et al., 1977; Marsh & Helenius, 1980). Acid-soluble radioactivity began to appear in the medium within 15 minutes of warming. If virus taken into the cells is calculated as the sum of proteinase K-resistant radioactivity and acid-soluble radioactivity in the medium, the maximal value attained is 42% internalized after 60 minutes (Fig. 4(a)). In contrast, Semliki Forest virus under similar conditions has a halftime on the surface of about 10 minutes and 60% is internalized after 15 minutes (Marsh & Helenius, 1980). Uptake of vesicular stomatitis virus is, therefore, relatively slow and inefficient when binding and internalization are dissociated by pre-binding the virus at 0°C. (c) Morphology

of virus uptake

To obtain an overall idea of the entry pathway followed by vesicular stomatitis virus into MDCK cells, virus uptake was examined by immunofluorescence. Cells were grown on coverslips, incubated for one hour at 0°C and pH 6.3 with large quantities of virus (1 pg/dish) and warmed at 37°C for different times. After fixation, vesicular stomatitis virus G protein on the cell surface was visualized by

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-0-o P

0-O

/d 0

15

30

/ I

I

I

I

45

60

75

90

Time (mm)

FIG. 4. Cell association of pre-bound vesicular stomatitis virus. Vesicular stomatitis virus ( - 27.000 ctsjmin) was suspended at 0°C in 200 ~1 of medium buffered to pH 63 without (a) or with (b) 20 mM-ammonium chloride and allowed to bind to MDCK cell monolayers for 1 h at 0°C with gentle shaking. Binding in this experiment was about 3% in the absence of ammonium chloride (810 cts/min) and 2% (540 cta/min) in the presence of ammonium chloride. The cells were then washed twice with cold binding medium with or without ammonium chloride, and warmed at 37°C with medium at pH 6.3 containing or not containing ammonium chloride. At various times, the medium was removed and acidsoluble radioactivity determined. The cells were cooled, and treated with proteinase K to measure proteinase K-resistant radioactivity. (a) Minus ammonium chloride. (b) Plus ammonium chloride. (0) Cell-associated. proteinase K-resistant radioactivity. (0) Acid-soluble radioactivity.

indirect staining. To see both surface and intracellular G protein, cells were freezethawed once prior to staining. As shown in Figure 5, the surface of cells kept at 0°C’ was covered with bright dots of different sizes (Fig. 5(a)). This pattern was not altered by freeze-thawing (not shown). After warming for one hour, most of the surface fluorescence had gone (Fig. 5(b)). Internally, however, a large number of vesicles distributed throughout the cytoplasm were stained (Fig. 5(c)). Warming times shorter than one hour gave intermediate results. This result clearly indicated that the majority of the viral antigens were internalized. It did not, however, distinguish bet,ween endocytosis of whole virus particles and internalization of viral G protein after fusion to the plasma membrane. Electron microscopy of vesicular stomatitis virus entry was performed to provide more detailed morphological data. As in the immunofluorescence experiment, cellassociated virus was examined after binding at 0°C and at different times aRer warming. Various stages characteristic of the endocytotic uptake of other enveloped

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FIN:. 5. Binding and uptake of vesicular stomatitis virus by MDCK cells. Vesicular stomatitis virus (1 pg) was suspended in 200 ~1 of ice-cold medium at pH 6.3 and allowed to bind for 1 h to MIEK cells grown on glass coverslips at 0°C. After washing away free virus with cold binding medium, one set of dishes was fixed directly while the others were warmed at 37°C for various times in medium at pH 7.4 and then fixed. G protein on the cell surface was detected by staining with anti-G IgG followed by a rhodamine-conjugated second antibody. G protein both on the surface and inside the cell was detected by staining after freeze-thawing the fixed cells. (a) Surface staining after binding but no warming. &cause the cells are flat. nuclei are visible in some cells as an out-of-focus bulge. (b) Surface staining after warming for 1 h at 37°C. (c) Surface plus internal staining after warming for 1 h. The bar represents 5 pm ; magnification, 2200 x

viruses (Baechi, 1970: Dourmashkin & Tyrell, 1974; Patterson et al., 1979; Dales, 1978; Helenius et al., 1980; Matlin et al., 1981) were observed (Fig. 6). Due to low binding of virus to the cell surface and because of the slow rate of internalization, the number of observations was, however, relatively limited. Virus particles bound to the plasma membrane (Fig. 6(a) to (c)) did not appear to be attached preferentially to any one area of the membrane. such as the microvilli. In addition, contact with the membrane was not made by any special portion of the virus virus particles entered coated pits particle (Fig. 6(a) to (c)). Upon warming, (Fig. 6(d) and (e)) and coated vesicles (Fig. 6(d). (f) and (g)). The coated vesicles were, in some cases, partially smooth (Fig. 6(f) and (g)). These vesicles may be in the process of losing their coats. Viruses were also rarely observed in irregularly shaped smooth-surfaced vacuoles (Fig. 6(h), ( i ) and (j)). At no time was fusion between vesicular stomatitis virus and the plasma membrane seen. To observe entry of the virus, it was necessary to work at very high multiplicities (about 5000 particles added per cell). For this reason, these morphological results are not directly comparable to the biochemical data described above. They do. however, demonstrate that endocytosis is an important viral entry mechanism in

FIG. 6. Endocytosis of vesicular stomatitis virus in MDCK cells. Vesicular stomatitis virus (60 fig) was suspended in 200 ~1 of ice-cold binding medium (pH 6.3) and allowed to bind to MDCK cell monolayer-s for 1 h at 0°C. Cells were then fixed directly in cold glutaraldehyde or warmed at 37°C. Various stages of endocytosis including ((a) and (b)) coated pits ((c) to (e)), coated vesicles ((d), (f) and (g)) and smooth vesicles ((h) to (j)) were observed. Note the partially coated vesicles in (f) and (g). Magnification: (a) and (0 50.000 x : (b), (c) and (g) to (j) 75,ooO x ; (d) 62,500 x ; (e) 100,000 x ; the bar represents 62 pm.

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ET AL.

these cells and that, under these conditions, fusion between the virus envelope and plasma membrane does not occur to a great extent (see Fan & Sefton, 1978). (d) Inhibition

of infection by Eysosomotropic agents

Infections by vesicular stomatitis virus (Miller & Lenard, 1980) and a number of other enveloped viruses are inhibited by lipophilic weak bases (for a review, see Helenius et al., 1981) such as ammonium chloride, chloroquine and amantadine. These compounds are so-called lysosomotropic agents, which accumulate in the lysosome where they raise the lysosomal pH (Ohkuma & Poole, 1978). They are also known to inhibit lysosomal hydrolases and alter other cellular functions (Liu & Schofield, 1973; Wibo & Poole, 1974; Helenius et al., 1981). Figure 7 shows that infection of MDCK cells was almost completely blocked by 20 mM-ammonium chloride, a concentration similar to that found to be inhibitory in other virus-cell systems (Helenius et al., 1981; Matlin et al., 1981). When 10 mMammonium chloride was added one hour after the virus, instead of at the same time, the degree of inhibition was reduced. As reported earlier (Miller & Lenard, 1980), chloroquine also inhibited vesicular stomatitis virus infection of MDCK cells. Neither binding nor internalization of vesicular stomatitis virus was significantly affected by inhibitory concentrations of ammonium chloride (Figs 2 and 4). Degradation of virus into trichloracetic acid-soluble material in the medium was.

+b-TkY

0

[NH,CII (mM)

FIG. 7. Effect of ammonium chloride of vesicular stometitis virus production. MDCK cells were infected with 20 p.f.u./cell of vesicular stomatitis virus in the presence of various concentrations of ammonium chloride. After 6 h, the medium w&s removed and virus production measured by plaque titration.

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(Fig. 4. compare

623

(a) and

@I). This experiment shows that vesicular stomatitis virus infection of MDCK cells can be blocked by lysosomotropic agents and that the stage affected follows internalization. It suggests, therefore, that the infect,ious pathway traverses an intracellular route. (e) Infection

by internalized

IBM

Our previous experiments with trace quantities of pre-bound radioactive virus showed that the virus quickly became resistant to proteinase K (internalized) upon warming to 37°C (Fig. 4), and that at high multiplicities (20 p.f.u./cell) infection could be inhibited by a lysosomotropic agent (Fig. 7). To demonstrate directly that virus. defined biochemically as internalized by prot’einase K resistance, was actually capable of infecting MDCK cells by the route sensitive to ammonium chloride, the following experiment was performed. Vesicular st*omatitis virus (30 p.f.u./cell) was incubated with MDCK cells for one hour at 0°C’ and pH 6.3. One half of the cells was then warmed for 10 minutes at 37°C and rapidly cooled. The second half of the cells was kept cold. To remove surface-bound virus. both sets of cells were digested with proteinase K. washed extensively, and t’hen incubated at 37°C for 5.5 hours to permit any viral infection to develop. In both cases, ammonium chloride (20 mM), which was previously shown not to effect binding and internalization. was present until the final incubation period. Two controls were included. In one, a positive control, ammonium chloride was absent throughout the experiment. In the second, negative control, ammonium chloride was present at all times. If the virus that had entered the cells during the ten-minute warming period was caapal)le of infecting the cell after removal of ammonium chloride, we expected to det,ect significant amounts of virus production at the end of the incubation period. Altjernativelg. if this virus was following a non-infectious route, the level of virus production wit’h or without warming was expected to be about the same. As the results in Table 2 demonstrate, only cells warmed with pre-bound virus for ten minutes were infected at all. Furthermore, virus production initiated by the warming step was sensitive to ammonium chloride. This experiment shows that proteinase K-resistant (internalized) virus is infectious, and that the pathway used by this virus traverses a compartment sensitive to ammonium chloride. A similar result’ was previously obtained with both Semliki Forest virus and fowl plague virus (Helenius et ~1.. 1981 ; Matlin et al., 1981).

(f) ;1snociation

of vesicular

stomatitis

virus with the plasma

membran,e at low pH

The data presented in the previous experiments suggest that vesicular stomatitis virus transfers its genome to the cytoplasm at an intracellular 1ocat)ion after endocytosis. Other enveloped viruses known to penetrate cells at an intracellular site do so when the low pH of the lysosome triggers fusion between the viral and Iysosomal membranes (Helenius et al., 1980,1981 ; Matlin et al., 1981). These viruses 22

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TABLE 2

Infection by internalized

Well Bind, OC, 60 min, pH 6.3 Warm, 37”C, 10 min, pH 7.4 Proteinase K, o”C, 30 min Wash, 0°C 1 2 3 MDCK medium, 37”C, 60 min pH 7.4 medium, 37°C. 4.5 h

vesicular atomatitis viru,s

Tray A (warmed) y---7 2 1 3 -t -

+ + -I+ + + + + A

Multiplicity

(p.f.u./cell) 1 30

100 (815)

2 O( -)

+ + + + + -

Tray B (not warmed) -7 1 2 3 -

+ (no warming + + + + + +

Virus production as percentage of controlf (log p.f.u.) 3 1 70~4(860)

O(3.60)

+ step) + + + -

B

2 O(-)

3 0(400)

t + and - . used to denote presence and absence of 20 mwammonium chloride in the medium. 1 Virus production is given as percentage of the positive control, Al, and as log p.f.u. in the medium at the end of the experiment (in parentheses).

are also able to fuse to the plasma membrane of cells when the pH of the medium is artificially lowered (White et al., 1980,198l; Maeda & Ohnishi. 1980: Huang et al.. 1981 ; Matlin et al., 1981). We have previously shown that vesicular stomatitis virus can fuse to the plasma membrane of BHK cells (White et al., 1981). To determine if vesicular stomatitis virus could also fuse to the MDCK plasma membrane at low pH, we performed the following experiments. In the first experiment, trace quantities of vesicular stomatitis virus were allowed to bind to MDCK cells at 0°C and pH 6.3. The cells were then washed to remove free virus, and flooded with media of different pH values at 37°C for 30 seconds. After cooling the cells to O”C, they were treated with trypsin to differentiate between fused or endocytosed viruses, and viruses simply bound to the plasma membrane. At pH values between 5.2 and 5.8, about 70% of the bound virus was trypsin-resistant, indicating an association between the virus and cells that was different from binding (Fig. 8). At pH values higher than 6.0, less than 10% was resistant. In the second experiment, the kinetics of low-pH-induced cell association was compared with uptake at higher pH values. As illustrated in Figure 9, after one minute at pH 5.4, about 45% of the cell-bound virus was protease-resistant. At both pH 6.3 and pH 7.4 less than half that’ amount was resistant. The results of these experiments resembled those obtained in similar experiments by fusion of Semliki Forest virus or fowl plague virus to the plasma membrane of cells at low pH (White et al., 1980; Matlin et al., 1981). They suggest that the rapid,

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IO -

3 PH

FIG. 8. Association of virus with cells at low pH. Vesicular stomatitis virus (- 40,000 cts/min) was suspended at 0°C in 200 ~1 of medium buffered to pH 63 and allowed to bind to MDCK cells for 1 h at O”C, with gentle shaking. Binding in this experiment was 10%. Afterwards, the cells were washed twice with cold binding medium to remove unattached virus. Prewarmed medium (37°C) at different pH values was then added to the cells for 3Os, and the cells cooled and treated with TPCK-trypsin (I.0 mg/ml) for 45 min at O”C, washed, scraped, and counted to determine protease-resistant cell association

20

40

60

80

100

120

Time I s )

FIG. 9. Rate of virus association to cells at low pH. Vesicular stomatitis virus (- 47,000 cts/min) was suspended in cold medium buffered to pH 63 and allowed to bind to MDCK cells for 1 h at 0°C. Binding in this experiment was about 6%. Afterwards, the cells were washed twice with cold binding medium, and prewarmed (37°C) media at pH 5.4 (a), 63 (0) or 7.4) (A) were added to the cells for the indicated times. The cells were then quickly cooled and treated with TPCK-trypsin to determine proteaseresistant radioactivity.

protease-resistant association at low pH is also due to fusion between the vesicular stomatitis virus envelope and the MDCK plasma membrane. The low level of cell association observed at pH 6.3 and 7.4 is probably a result of endocytosis during the 30-second warming period. Low-pH-induced fusion of Semliki Forest virus to the

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BHK cell plasma membrane leads to infection that is resistant to lysosomotropic agents (Helenius et al., 1980; White et al., 1980). Several attempts to infect MDCK cells with vesicular stomatitis virus by brief incubation at low pH in the presence of ammonium chloride were, however, unsuccessful.

4. Discussion The interaction of vesicular stomatitis virus with MDCK cells differs in many respects from that of Semliki Forest virus with BHK cells (Helenius et al., 1980; Marsh & Helenius, 1980), and that of fowl plague virus with MDCK cells (Matlin et al., 1981). The main dissimilarities are in binding and endocytosis, but the process of fusion may also be distinctive. The weight of our evidence, however, suggests that vesicular stomatitis virus enters MDCK cells by a sequence of events similar to that followed by both Semliki Forest virus and fowl plague virus. The salient steps are : (1) binding to the plasma membrane ; (2) endocytosis in coated pits and coated vesicles; (3) transport to the lysosome; and (4) low-pH-induced fusion of the viral and lysosomal membrane, followed by release of the genome into the cytoplasm and replication. Each of these stages is discussed below. (a) Binding At pH 6.3 and 0°C vesicular stomatitis virus binds to the surface of MDCK cells. Binding is highly pH-dependent ; the amount of virus bound at pH 6.5 is about ten times that at pH 7.4. Even at the optimal pH, binding remains relatively low and irreproducible. Thus, after one hour at pH 6.3 and O”C, a maximum of only 2 to 20% of the added virus was bound in separate experiments. The proportion of virus bound continued to increase, however, with incubation beyond one hour and did not reach a constant value after even two hours incubation. Our results are in good agreement with those of Miller & Lenard (1980), who reported that the binding of vesicular stomatitis virus to BHK cells is low, and that a plateau is not quickly reached. The extreme dependence of vesicular stomatitis binding to MDCK cells on pH may be a property of the apical microvillar surface of the cell. The MDCK cell line has characteristics in common with cells from differentiated epithelial tissues and has retained many properties of kidney tubule cells (Cerejido et al.. 1978; Louvard, 1980). Only the apical surface is exposed to the medium in monolayer culture. Increased binding of various macromolecular ligands to the apex of epithelial cells at slightly acidic pH has been observed. Rodewald (1976) found that IgG bound to the microvillar membrane of neonatal rat enterocytes at pH 6.0 but not at pH 7.4. Roth & Linden (1978) have also reported low pH-dependent association of IgG and phosvitin (T. Roth, University of Maryland, personal communication) to the yolk sac of the developing chicken oocyte. The dependence of ligand binding on pH is believed to be important in binding and release during transepithelial transport of proteins from the luminal to vascular surfaces (Rodewald, 1976). Presumably, a ligand such as IgG binds to a receptor on the microvillar membrane where the pH is low in many tissues (Rodewald, 1976;

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Guyton. 1976), is endocytosed in coated vesicles (Rodewald. 1973), and is released on the basolateral side, where the pH is near neutrality. The existence of such a pathway in kidney tubule cells is controversial (Carone, 1978). It is known that’ the normal pH of the kidney tubule is between 6.0 and 6.9 (Guyton. 1976). It is possible. therefore. that receptors exist on MDCK cells with increased affinity for t’hr vesicular stomatitis virus G protein at low pH. Another possibility is that the pH dependence of binding is a propert,y of the (: protein. Because vesicular stomatitis virus possesses a low-pH-induced fusion activity. the G protein might also have a higher affinity for its receptor(s) at acidic2 pH. Viruses incubated with cells at 0°C and pH 6.3 are not. however, fused t,o the plasma membrane since they can be removed by proteinase K. ln contrast to the observations reported here, both Semliki Forest virus and fowl plague virus bind efficiently to BHK cells and MDCK cells, respectively. reaching a czonst,ant value of about 40 to 6OO/oof the applied virus in one to two hours at WY’ (Helenius ef nl.. 1980; Matlin et al., 1981). Although both these viruses show increased association at slightly acidic pH values. the effect of pH is less dramatic than that seen for vesicular stomatitis virus (Fries & Hetenius. 1979: Mat#lin rt r/l.. 1981). &At0°C’. no vesicular stomatitis virus enters MDCK cells. Over 90(& of t,he bound virus can be removed by incubation in the cold with proteinase K, a treatment, that neither kills the cells nor removes them from the plastic dish. Trypsin is also effective in removing bound virus (Miller & Lenard, 1980). It. is not clear if the virus rrcept,or on the cell surface, the G protein spike. or both are attacked by the proteases, although G protein is known to be sensitive to both proteinase K antI trypsin (Mudd, 1974). (b) Endocytosis Our morphological observations of vesicular stomatitis virus uptake into MD(‘K (aells are consistent with endocytosis rather than fusion at, the plasma membrane. At 37°C’ virus particles were observed in four distinct cellular locations: (1) at’tached to the cell surface; (2) associated with coated pits: (3) contained within coated vesicles: and (4) included in smooth-surfaced vacuoles. The sequence in which the virus enters these various compartments cannot be definitely determined from the present studies. From comparison with other work on the endocytosis of vesicular stomatitis virus (Dahlberg, 1974) and other enveloped viruses (Helenius of NI.. 1980: Marsh & Helenius, 1980; Matlin et al., 1981), it seems likely that the primary endocytotic vesicles are coated and that the secondary ones are smooth. So concentration of virus particles in any intracellular structure was observed. In fact; it was difficult to find the virus in any vacuole other than coated vesicles (SW Dahlberg. 1974). This problem may be partly due to the low binding and slow rate of endocytosis in MDCK cells, which results in small particle-to-cell ratios. A further complication was that t,he concentrated virus preparations used for electron microscopy (15 to 10 mg/ml) had a tendency to aggregate, t’hus reducing the effective particle concentration. Efforts to prevent aggregation by using low-speed c*t~ntrifupation t,o concentrate the virus were only partially surcessfut.

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Our morphological results are in agreement with the studies of Simpson et al. (1969) and Dahlberg (1974). Both of these reports concluded that the primary mode of entry of vesicular stomatitis virus into L-cells was by endocytosis. They also failed to notice accumulation of virus particles inside the cell. In contrast, both Semliki Forest virus and fowl plague virus, which enter cells by endocytosis more efficiently t’han vesicular stomatitis virus, were seen concentrated inside large smooth-surfaced vacuoles, some of which contained over 50 particles (Helenius et nl., 1980; Matlin et al.. 1981). Rabies virus, which is related to vesicular stomatitis virus. has also been shown by electron microscopy to ent,er cells by endocytosis (Iwasaki et al., 1973). The biochemical data also support an endocytotic entry pathway similar to that utilized by Semliki Forest virus and fowl plague virus, although a number of differences bet,ween the viruses are apparent. When cells with pre-bound vesicular stomatitis virus are warmed (Fig. 4), the initial half-time of entry is about 30 minutes. Internalization levels off within 15 to 45 minutes, however, so that no more t,han half the bound virus is taken up in 90 minutes. When added to cells at 37°C’. virus uptake is linear and at a rate such that only about 1% of the added virus is internalized in 30 minut’es. Similar results have been obtained by Miller & Lenard (1980). After examining the infe&ion of BHK cells by both wild-type vesicular stomatitis virus and a temperature-sensitive mutant’. they concluded that) about one-third of the virus pre-bound at 0°C‘ became trypsin-resistant in 15 minutes, and that internalization at 37°C’ was dependent both on the rate of binding and the rate of uptake. Ent,ry of vesicular stomatitis virus into MDCK cells is, therefore, slower and less rxt’enxivc than the endocytosis of Semliki Forest virus and fowl plague virus in thcair respective cell systems. Surface-bound Semliki Forest virus and fowl plague virus enter cells with a half-time of less than 15 minut’es (Marsh & Helenius, 1980: Matlin rt crl., 1981 ). The txxtent of internalization of these viruses is much greater t)han that of vesicular stomatitis virus (Helenius et al.. 1980; Marsh & Helenius. 1980; Matlin et al.. 1981). At 37°C Semliki Forest virus and fowl plague virus uptake is linear, like that of vesicular stomatitis virus. but the rate is faster. The precise reasons for the slow endocytosis of vesicular stomatitis virus in MDCK cells are not known. The virus may bind t’o several classes of receptors on the cell surface. Perhaps t,hese recept,ors are either very slowly endocytosed or endocytosed at different rat,es. Bret,scher rt al. (1980) have recently shown that certain plasma membrane proteins are not in coated pits. Virus particles bound to such receptors may be endocytosed at a low rate or not endocyt’osed at all. Miller & Lenard (1980) concluded that’ some vesicular stomatitis virus bound to the BHK cell surface but was not internalized. An additional restriction on t)he endocytosis of vesicular stomatitis virus may be t’he size of coat,ed vesicle required for the virus particle. Both Semliki Forest virus (diam. 650 a) and fowl plague virus (diam. 1000 A) can fit into a 1000 to 1500 .A coated vesicle. III fact, at high multiplicities, more than one Semliki Forest virus particle (ban be seen in a single coated vesicle (Marsh & Helenius. 1980). In contrast, vesicular stomat,itis virus has a shape and size that coated vesicle (see Fig. 6). Formation of these require a large and elongated st,ructures b.v the cell may be difficult, and therefore both slow and limited.

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of lysosomes

Two lines of evidence implicate lysosomes in the infective process. Firstly. ammonium chloride and chloroquine, which are known to raise lysosomal pH (DeDuve et al., 1974; Ohkuma & Poole, 1978), prevent productive infection of MDCK cells by vesicular stomatitis virus (Fig. 7), but have no significant effect cm virus binding (Fig. 2) or uptake (Fig. 4). Miller & Lenard (1980) have shown that chloroquine inhibits viral RNA synthesis in BHK cells, but has no direct effect on t’he viral particle. These results suggest that endocytosed vesicular stomatitis virus not only passes through the lysosome, but the lysosome plays an active role in the infective process. Secondly, when pre-bound virus is taken into MDCK cells at 37”C, acid-soluble radioactivity is released into the medium within 15 minutes (Fig. 4(a)). In the presence of ammonium chloride, the amount of acid-soluble material released is slightly diminished (Fig. 4(b)). The observed degradation could occur after endocytosed virions enter lysosomes. Ammonium chloride might reduce the degree of degradation by raising the lysosomal pH (Ohkuma & Poole, 1978) and inhibiting or altering lysosomal hydrolases (Liu & Shofield. 1973; Wibo & Poole. 1974). Semliki Forest virus degradation is also partially inhibited by the lysosomal drug chloroquine (Helenius et al., 1981), and release of acid-soluble material after fowl plague virus endocytosis is totally prevent,ed by ammonium chloride (Matlin ef al., 1981). Lysosomotropic agents have been shown to block the degradation of several natural ligands that are taken into lysosomes, including low-den&J lipoprotein (Goldstein et al., 1975), insulin (Marshall R: Olefsky. 1979) and epidrrmal growth factor (King et al., 1980). (d) The role of fusion Vesicular stomatitis virus rapidly associates with the MDCK cell in a probeaseresistant form when the pH of the medium is lowered. Under similar conditions. we have observed virus-initiated cell-cell fusion, and fusion between the vesicular stomatitis virus membrane and the plasma membrane of BHK cells (White et trl., 1981). We conclude, therefore, that the low-pa-induced interaction between thrx MD(:K cell and vesicular stomatitis virus is also a membrane fusion. Fusion with the plasma membrane may be inefficient when artificially induced by lowering th(A medium pH. Indeed, the extent of low-pH-induced association after 30 seconds is less than that of fowl plague virus or Semliki Forest virus under significant,ly similar conditions (White et al., 1990; Matlin et al., 1981). We have obtained no evidence for fusion at neutral pH. Heine & Schnaitman (1969.1971) have seen fusion profiles of vesicular stomatitis virus with the plasma membrane of L-cells at neutral pH. These authors. however, adsorbed virus to cells by centrifugation. which, as demonstrated by Dahlberg (1974). leads t’o an increased amount of fusion. The existence of a lowpH-induced fusion activity in vesicular stomatitis virus oxplains bot,h how the viral genome is released into the cytoplasm and whJ lysosomotropic agents block the infection. Aft,er endocytosis the virus enters the Iysosomr, where the acidic environment causes the viral and lysosomal membranes t,o fuse. After the fusion event the inside of the virus membrane is continuous wit,11 thta cytoplasmic face of the vacuolar membrane, thereby exposing the rapsid to

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cytoplasmic enzymes. When the lysosomal pH is raised by. for example, ammonium chloride, no fusion and hence no penetration is possible. Since we have not directly demonstrated virus particles in lysosomes, we cannot exclude that a pre-lysosomal vacuole with a low internal pH is responsible for inducing virus penetration by fusion. No organelle except the lysosome has, however, been defined that has both low pH and the proteolytic enzymes necessary to degrade the virus (DeDuve et al., 1974). In addition to low pH, other factors may be necessary for vesicular stomatitis to fuse efficiently with membranes. Lysosomal hydrolases may, for example, cleave the spike glycoprotein G, making it an even more effective fusogen. The influenza haemagglutinin must be cleaved to HA1 and HA2 (White et al., 1981), and the Sendai F glycoprotein to F, and Fz (Scheid & Choppin, 1974), before they can mediate membrane fusion. Cleavage of PE2 to E2 may also be required before Semliki Forest virus can fuse (White & Helenius, 1980; White et al., 1980), although this has not been demonstrated. Infection of cells by endocytosis is probably a strategy employed by many viruses. Both Semliki Forest virus and the influenza A fowl plague virus have been shown to infect by this route. Reovirus (Silverstein & Dales, 1968) also enters cells by endocytosis and accumulates in lysosomes. Recent reports indicate that other invasive agents mav also exploit endocytosis to enter cells. The action of diphtheria toxin is prevented by lysosomotropic agents, and the block can be bypassed by lowpH treatment (Sandvig & Olsnes, 1980; Draper & Simon, 1980). Further work should clarify the details and control mechanisms of this cellular pathway. Hilkka Virta, Barbara Skenr and Shirley Robinson provided excellent technical assistance, Eva Bolzau and Annamette Ohlsen performed the electron microscopy, and Annie Biais and Wendy Moses typed the manuscript. The authors are also grateful to Mark Marsh, Barbara Skene and ?Judy White for helpful discussions, to Mark Marsh and Judy White for reading the manuscript, to Daniel Louvard for helping with immunological techniques, to Larry Altstiel for giving us the original virus isolate, and to Mary Holmes and Susan Mottram for cheerfully and rapidly digging up many references. REFERENCES Anderson, R. G. W., Brown, M. 8. & Goldstein, J. L. (1977). Cell, 10, 351-364. Ash, J. F., Vogt, P. K. & Singer, S. J. (1976). Proc. Nat. Acad. Sci.. U.S.A. 73, 3603-3607. Ash, J. F., Louvard, D. & Singer, S. J. (1977). Proc. ,Vat. Acad. Sci., U.S.A. 74, 5584-5588. Baechi. T. (1970). Path. Microbial. 36, 81-107. Blakeslee, D. & Baines, M. G. (1976). J. Zmmunol. Meth. 13, 30.5320. Brandtzaeg, P. (1973). &and. J. Immunol. 2, 273-290. Bretscher, M. S., Thomson, J. N. & Pearse, B. M. F. (1980). Proc. Nat. Amd. Sci., rJ.S.A .77, 41564159. Carone, F. A. (1978). Ann. Clin Lab. Sri. 8, 287-293. Cereijido, M., Robbins, E. S., Dolan, W. J.. Rotismo, <‘. A. & Sabatini, D. D. (1978). J. Cell Biol. 77, 853-880. Dahlberg, J. E. (1974). Virology, 58, 250-262. Dales. S. (1973). Bactrriol. Rro. 37, 103-135. Dales, 8. (1978). Lije Sciences Rexarch Report 11, Dahlem Conference, pp. 47-67, Abakon Verlagsgesellschaft, Berlin. DeDuve, C., DeBarsy, T., Poole, B., Trouet, A., Tulkens, P. & Van Hoof, F. (1974). Biochem. Pharmacol. 23, 2495-2531. Dourmashkin, R. R. 8r Tyrell, D. A. .I. (1974). J. Oen. c’irol. 24, 129-141.

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by W’. Frnnke