J. Mol. Biol. (1980) 142, 439454

Adsorptive Endocytosis of Semliki Forest Virus

MARK MARSH AND ARI HELENIUS

The European Molecular Biology Laboratory Postfach 10.2209

6900 Heidelberg, G’erman,y

(Received 18 April 1980)

Endocytosis of Semliki Forest virus into BHK-21 cells has been studied at multiplicities varying between one virus and 4 x lo5 viruses per cell. Uptake requires attachment to the cell surface, it is very rapid (half-life 10 to 35 min), it occurs in coated vesicles, it is inhibited at temperatures below 15°C and it does not elevate the fluid-phase uptake of the cells. Inhibitor studies using colcemid, cytochalasin B, sodium azide, dinitrophenol, 2-deoxy-n-glucose and lysosomotropic weak bases show that the uptake is largely independent of cytoskeletal and lysosomal function, but partially dependent on oxidative phosphorylation. Together the biochemical and morphological results indicate that SFVt uptake occurs by adsorptive endocytosis in a manner very similar to the receptor-mediated endocytosis of serum low-density lipoprotein and other physiologically important extracellular proteins (Goldstein et al., 1979). SFV thus appears to exploit a facultative cellular process in gaining entry into cells.

The uptake of SFV has been used to study quantitative aspects of the adsorptive endocytosis process. At high multiplicities of viruses to cells, 1500 to 3000 virus particles can be internalized per minute per cell. Electron microscopy indicated an average of 1.3 viruses per virus-containing coated vesicle.

1. Introduction We have recently shown that Semliki Forest virus, a simple membrane-containing alphavirus, infects BHK-21 cells by an endocytotic pathway (Helenius et al., 1980a,b). Our evidence suggests that the final penetration takes place in the lysosomes by fusion between the viral membrane and the lysosomal membrane, resulting in release of the viral nucleocapsid into the cytosol (Helenius et al., 198Ou; White & Helenius, 1980). From electron microscopy it is well known that other membrane viruses are also internalized by endocytosis (for a review, see Dales, 1973) but, as detailed biochemical data are lacking, the mechanism and the significance of the phenomenon have remained unclear (see Lonberg-Holm & Philipson, 1974).

In this paper we have extended the studies on SFVt endocytosis. We have characterized the kinetics under various conditions, the temperature dependence, the cellular capacity, the fate of the viral proteins, the effects of inhibitors, and the effects of virus on the cellular fluid-phase endocytosis. [35S]methionine-labelled

t Abbreviations used: SFV, Semliki Forest virus: SDS, sodium dodecyl sulphate; PBS, phosphatr- bllffered saline.

439

0022-2836/80/270439-16 $02.00/O 0 1980 Academic Press Inc. (London) Ltd.

440 ESDOCYTOSIS OF SEMLIKI FOHEST \‘lKUS

virus was used, which made it possible to work with concentrations ranging from one virus per cell to 4 x lo5 viruses per cell. The results show that the mechanism by which SFV enters the cell is very similar to the receptor-mediated uptake of physiological macromolecules (Goldstein et al., 1979). In addition, data obtained at high multiplicities of viruses to cells demonstrate that the cellular capacity for adsorptive endocytosis is extremely high.

2. Materials and Methods (a) Ce12s and virus

BHK-21 cells were grown as previously described (Helenius rt a/.. 1980~) in MEM-Glasgow medium using 72 cm’ plastic cell-culture bottles (Falcon), 24.well Linbro plates (Flow Laboratories), glass coverslips and 35 cm diameter plastic Petri dishes (Falcon). A prototype strain of SFV was grown and purified as described (Kairiiinen et al., 1969). For intrinsic radioactive labelling of SFV, [3sS]methionine, [3H]uridine or 32P (all from Amersham . . Buchler) were used (Kaariamen & Siiderlund, 1971). The carrier-free [35S]methionine-labelled virus preparations had specific activities between 5 x 1 O3 and 16 x 1 O3 virus particles per ct/ min. Viral protein, in 0.1% SDS was determined bv the Lowry procedure (Lowry et al.; 1951) using bovine serum albumin as a standard. A Viral protein content of 57% and a viral molecular weight of 64 x lo6 are assumed for the calculations (Laine et al., 1973). Virus was stored at -80°C in 065 M-Tris.HCl (pH 7.4) containing 0.1 M-N&I and (for radioactive virus only) 42% (w/w) sucrose. From two preparations of SFV a particle to plaque-forming unit (p.f.u.) ratio was determined. Plaque assays on BHK-21 cell monolayers (Porterfield, 1960) and protein determination gave values of 2.4 and 2.8 particles per infective unit.

(b) Analysis of cell-associated S’F k’ with BHK-21 cells

For binding and uptake studies, confluent monolayers of cells on 3.5-cm Petri dishes were used on the second day after seeding (seeding density 7 x lo4 cells/cm2). The cells, washed twice with R-medium (RPM1 1640 : containing 20 miw-4-(2.hydroxyethyl)1 -piperazine ethane sulphonic acid, HEPES (S erva, Germany) pH 6% and 0.2% bovine serum albumin), were incubated (with slow rocking) with carrier-free radioactive virus, or with radioactive virus plus a known amount of unlabelled virus, in 05 ml R-medium at either 4°C for 1 h or 37°C. The supernatant was removed and the cells were washed twice with 1 ml R-medium. Cells, incubated at 4°C to bind virus, were covered with 1 ml R-medium and incubated at 37°C to internalize the bound virus (Helenius et al., 1980a).

After incubations with virus the cell supernatants were centrifuged at 1500 revs/min for 5 min at 4°C to remove cell debris. The total radioactivity and acid-soluble radioactivity of the remaining supernatant were determined in Triton Xl00 toluol scintillation fluid (Rotiszint 22, Carl Roth KG, Karlsruhe. West Germany, was used throughout) in a ?iuclear Chicago mark III scintillation counter. Cell-associated radioactivity was assayed by scraping the cells (using a Teflon scraper) into 3 ml of R-medium at O”C, and rinsing the plates with an additional 4 ml R-medium. The suspended cells were pelleted by centrifugation at 1500 revs/min for 5 min at 4”C, washed once in 6 ml of R-medium at 0°C and repelleted. The cell pellet was solubilized directly in scintillation fluid.

The acid-soluble fraction ofthe cell-associated radioactivity was assayed bysolubilizing the cells in phosphate buffered saline containing 1% SDS, and making the samples lO%with cold trichloroacetic acid. After 1 h at 4°C the samples were centrifuged at 3000 revs/min for 10 min and a sample of the supernatant was taken up into scintillation fluid.

The intracellular virus was determined using Proteinase K (Helenius et al., 198Oa). Cell monolayers, after treatment with viruses, were incubated with 1 ml of 0.5 mg Proteinase K/ml (Boehringer, Germany) in PBS at 0°C. After 45 min shaking at 4”C, 1 ml of PBS at 0°C. containing 1 mnr-phenyl methylsulphonyl fluoride (PMSF, Sigma) and 30 mg bovine serum albumin, was added. The cells were removed by pipette, made up to 5 ml with PBS at 072, and

M. MARSH AND A. HELENIUS 441

centrifuged. The pellet was washed once in 6 ml PBS at 0°C containing 02% bovine serum albumin and dissolved in scintillation fluid. Proteinaae K removed 97 to 98% of the bound viruses from the cell surface. The remaining 2 to 3% w&s not internalized as the value remained the same regardless of the time the cells were at 4°C. No cell-associated viruses were seen in thin sections of treated cells. The enzyme disrupted the monolayers but most (98%) of the cells remained viable as judged by trypan blue exclusion and their ability to grow normally when replated. Additionally, after cells with bound virus were treated with Proteinase K no cell-associated viral antigens could be seen by indirect immunofluorescence using anti-SFV antibodies.

(c) Bulk JEW&phase endocytosis

[3H]sucrose (Amersham Buchler) was used to assay cellular fluid-phase endocytosis (Wagner et al., 1971). BHK cell monolayers were incubated with 1 ml R-medium containing 4.0 nr+[‘H]sucrose(spec. act. 2.1 Ci mmol- ‘) and 100 nM unlabelled sucrose. To determine the amount of the fluid phase endocytosed at different time-points, cells were washed 5 times with cold PBS containing 10 mnr-sucrose, scraped, washed 3 times with cold PBS-sucrose and the final pellet dissolved in scintillation fluid, aa described above. The effect of virus uptake on fluid-phase endocytosis was assayed by adding 10 ~1 [‘2P]SFV to confluent monolayers of cells to which [‘HIsucrose had been added 30 min earlier. After subsequent incubation periods the cells were assayed for internalized 3H and 32P radioactivity using the Proteinase K assay.

(d) Lysosomotropic agents and other inhibitors

Stock solutions of lysosomotropic agents and inhibitors were made up in R-medium as follows : chloroquine-HCl (Sigma), 10 mM ; tributylamine (Merck), 10 mM ; amantadine-HCI (Sigma), 10 mM; methylamine (Merck), 100 mM; NH,Cl (Merck), 100 mre; sodium azide (Merck), 100 mM; 2-deoxy-n-glucose (Sigma), 1 M; dinitrophenol (Sigma), 10 IIIM; cyclohex- imide (Sigma), 2 mg ml- ’ ; cytochalmin B (Sigma) in dimethyl sulphoxide (Merck), 40 pg ml-‘; colcemid (Sigma), 1 mM. Where necessary the solutions were adjusted to pH 68 with NaOH or HCl and appropriate dilutions made into R-medium.

Cells on 3.5 cm diameter petri dishes or in Linbro plates were washed once with R-medium, or serum-free MEM-Glasgow medium (Gibco Bio-Cult), and incubated with 0.5 ml or 0.1 ml, respectively, of medium containing SFV and the lysosomotropic agent or the inhibitor at 4°C. After washing away unbound virus, uptake was initiated by incubating the cells at 37°C in the presence of the inhibitor.

(i) Gel filtration (e) Other methods

Samples lyophilized and redissoved in 70% formic acid were analyzed on a 50 cm x 0.7 cm Biogel P-2,200 to 400 m-h (Bio-Rad laboratories) column in 70% formic acid. The columns were calibrated using c$ochrome c 1.9 mg), two peptides, Met-Trp (2.0 mg) and Leu-Trp-Met (2.4 mg) (Serva), and [ ‘Slmethionine. The eluted fractions were assayed by spectrophoto- metry at 280 nm or 400 nm and for their radioactive content.

(ii) SDS/polyacrylamide gel electrophoresia

To analyze [“S]SFV-infected cell samples, cell pellets from a Linbro well were dissolved in 50 ~1 of 50 mM-Tris (pH 8.0) containing 2% SDS and 2 m&r-PMSF at O”C, and quickly heated to 95°C for 5 min. Samples (10 ~1 were taken up in sample buffer (100 mm-Tris- HCl, pH 8.8,4% SDS, 1 mM-EDTA, 50 mu-methionine, 15% (w/v) sucrose and bromophenol blue) and analyzed without reduction or alkylation (to improve the separation of the viral spike proteins El and E2) on modified Laemmli 10 to 15% gradient gels (Blobel & Dobberstein, 1975). For fluorography the gels were fixed in 10% trichloroacetic acid and treated with EN3HANCE (New England Nuclear) for 1 h. The proteins were visualized by autoradiography on Kodak X-Omat film.

442 ENDOCYTOSIS OF SEMLIKI FOREST VIRUS

(iii) Electron microscopy Electron microscopy was carried out, as described, on BHK cells grown to confluence on

glass coverslips (Helenius et al., 198Oa). The cells were fixed with ice-cold 25% glutaral- dehyde in 0.05 M-sodium cacodylate buffer (pH 7.2) containing 50 mM-KC1 and 2.5 mM- MgCl,. Dehydration, embedding, sectioning and uranyl acetate staining were performed as described (Helenius et al., 1977) and silver-grey sections were examined in a Philips 400 electron microscope.

3. Results (a) Saturation of binding and uptake of Semliki Forest virus

To determine whether the process of SFV internalization into BHK-21 cells is saturatable, [35S]methionine-labelled SFV was bound to cell monolayers in the cold. The number of viruses added to individual dishes ranged between one virus and 4 x lo5 viruses per cell, the lower limit being set by the specific activity of the radioactive virus and the upper limit by the amount of available unlabelled virus. After one hour at 4°C the cells were washed and, from one set ofdishes, the number of cell-bound viruses determined. Parallel dishes were placed in an incubator at 37°C for 30 minutes to allow endocytosis to occur, whereafter the number of viruses internalized was determined using the Proteinase K assay.

The results in Figure 1 (a) show that the fraction of viruses bound to the cells in the cold decreases when more than 4 x lo4 viruses per cell are added (this is consistent

m m % 60- % 60- .- .- , 0 0 : :

i :;iE&t

ln ln a-” 0 a-” 0 I I 1 1 I I I I 1 1 I I I I 1 1 I I I

IO0 IO’ IO0 IO’ IO’ I03 IO4 IO5 IO’ 103 IO4 IO5 IO’ IO’ 102 IO3 IO4 IO5 102 IO3 IO4 IO5

SFV added per cell SFV added per cell SFV bound per cell SFV bound per cell

(a) (b)

FIG. 1. Binding and endocytosis of SFV at different multiplicities. Binding was performed for 1 h a.t 4’C and internalization for 30 min at 37°C. (a) The percentage of SFV bound at 4°C (-m--m-), and the percentage of SFV internalized at 37°C (-•-•-). (b) The percentage of cell-bound SFV internalized at 37°C (--&-A--).

with our previous binding data; Fries & Helenius, 1979). A concomitant decrease is also observed in the fraction of viruses internalized during the 30-minute warming period. However, this decrease results from saturation of the cell surface rather than saturation of the endocytosis process, since the fraction of bound viruses in- ternalized remains constant up to the highest multiplicities (Fig. l(b)). The highest calculated uptake is 5 x lo4 virus particles per cell, which amounts to an average rate of 1500 viruses endocytosed per minute. The results suggest that although high, this amount of SFV is not sufficient to saturate the endocytosis process.

We have previously studied the binding and endocytosis of SFV by electron

M. MARSH AND A. HELENIUS 443

microscopy and shown that uptake occurs mainly, if not exclusively, through coated pits and coated vesicles (Helenius et al., 198Oa). However, the multiplicities used were considerably lower than the highest multiplicities in the above experiments. It was therefore of interest to look at cells treated with such high multiplicities (4 x 10’ viruses per cell) by thin-section electron microscopy. Vertical sections of cells kept in the cold show that the viruses bind predominantly to the top surface of the cells, access to the lower surfaces being restricted by close contacts between the cells. As previously documented, most of the bound viruses are associated with the microvilli (Fig. 2(a)) and frequently viruses are seen in coated pits (Fig. 2(b) and (c)). When cells, briefly warmed to 37°C are fixed and sectioned, the viruses are seen in coated vesicles (Fig. 2(d) to (g)). We examined 248 coated vesicles located at the cell periphery, all of which were larger than 80 pm. A total of 108 vesicles contained viruses; 90 had 1 virus, 12 had 2,5 had 3 and 1 had 4 viruses, which gives an average of 1.3 viruses per virus-containing coated vesicle. The remainder of the vesicles were apparently virus-free. The existence of virus-free coated vesicles at the cell periphery is consistent with the result that uptake from the cell surface is not saturated at concentrations of 4 x 10’ viruses per cell. The average internal diameter of a coated vesicle is 90 nm, the diameter of a virus is 70 nm (Siiderlund et al., 1979) and the thickness of the section is about 60 nm, making the probability of missing a virus in a coated vesicle small, when equatorial sections are viewed.

(b) Internalization and degradation of prebound Semliki Forest virus

In BHK cells SFV infection occurs rapidly: viral RNA synthesis is detectable after 1.5 hours and the progeny viruses are released after 2.5 hours (Kglliriliinen & Soderlund, 1978). We followed the uptake and degradation of surface-bound viruses for four hours using high and low multiplicities. 3sS-labelled SFV was bound to cell monolayers for one hour in the cold, the unbound viruses were removed by washing and the cells warmed to 37°C by placing the dishes in an incubator. At different time- points dishes were removed and the cells and the media analyzed for acid-soluble and acid-precipitable radioactivity. Proteinase K resistance was used to assay the amount of intracellular virus. In some cases theproteinase K-resistant radioactivity was analyzed by SDSJpolyacrylamide gel electrophoresis to determine the intact- ness of the intracellular viral proteins. The medium was subjected to gel filtration to determine the form in which the degraded proteins are released from the cells.

At low multiplicity (1 virus bound/cell) the number of cell surface viruses declines to zero within four hours; half of the viruses become internalized in 35 minutes (Fig. 3(a)). Concomitant with the decrease in cell surface radioactivity, an increase in intracellular radioactivity is observed rectching its peak between one and two hours. The appearance of acid-soluble radioactivity shows a lag of 30 to 45 minutes and virtually all of it can be found in the medium, increasing linearly with a rate of about 15% of the initial bound radioactivity per hour. Of the radioactivity released into the medium, over 90% elutes at the position of methionine on a Biogel P-2 gel filtration column equilibrated with 70% formic acid. The rest is acid-precipitable and elutes in the void volume of the gel ; SDS/polyacrylamide gel electrophoresis shows that this fraction consists of intact viral proteins and probably represents virus released from the cell surface.

-- 1

Fl(:. 2. Thin-section electron microscopy of BHK cells incubated with sFV at a multiplicity of4 x 16)“ viruses/cell. (a) Viruses on the cell surface after binding for 1 h at 4’(‘: most viruses were bound to th,> microvilli, but some associated with coated pit regions (b) and (c). se arrow in (a). .4f%er warming to 37°C for 30 s viruses were internalized in coated vesicles ((d) to (g)). Occasionally 2. 3 or 4 viruses were observed in a single coated vesicle. Viruses rxternal to the ~11 after 4 h at 37°C could be localized in regions of close approach between ~11s (h).

M. MARSH AND A. HELENIUS 445

(b)

Time (hl

PIG 3. llptake and degradation of pre-bound ~35S]methionine-labelled SFV at low ((a) and (b)) and high ((r) and (d)) multiplicities (1 virus/cell and 1.5 x lo4 viruses/cell, respectively). The viruses were allowed to bind for 1 h in the cold. unbound viruses were washed away and the cells warmed to 37°C. (‘el1 surface 35S radioactivity (-e-O--); internal activity (-O-O-): internal acid-soluble activit\r (-A-A-); acid-precipitable activity in the medium (-m--O--); acid-soluble activity in the medium (--m-m-): total recovered radioactirit,y (-n-n-).

Although the degradation of viral protein occurs intracellularly, the amount of acid-soluble radioactivity inside the cells remains low. SDS/polyacrylamide gel electrophoresis performed at different time-points after warming up indicates that the intracellular radioactivity corresponds to intact viral proteins, the amount of which decreases with prolonged incubation (Fig. 4). Partial degradation products are not observed. Of the viral proteins, E2 appears to be more sensitive to degradation than the capsid protein or El ; E2 virtually disappears after two hours incubation at 37°C.

At high multiplicity (1.5 x lo4 viruses bound/cell) the uptake and degradation are remarkably similar to that at low multiplicity (Fig. 3(c) and (d)). The main difference is that about 10% of the viruses are not internalized during the four hours incubation. Thin-section electron microscopy at two hours (later time-points are precluded by the appearance of progeny virus) shows that most of the cell surface is

446 ENDOCYTOSIS OF SEMLIKI FOREST VIRUS

A 6 30 60. 120. 240.

C dr FIG. 4. SDS/polyacrylamide gel electrophoresis of [3sS]methionine-labelled SFV internalized by

BHK cells. Viruses (5 x 10’ cts/min, carrier-free) were bound to cells in Linhro wells for 1 h at 4°C. After warming to 37°C for varying time periods (30, 60, 120, 240 min) 0°C PBS containing 0.5 mg ml-‘. Proteinase K was added to stop internalization and to remove extracellular SFV. The cells were prepared for electrophoresis as described. A, Cells kept at 0 to 4”C, no Proteinase K treatment; B, cells kept at 0 to 4”C!, with Proteinase K treatment. (El, E2, viral spike proteins; C, viral capsid protein).

free of viruses but some are trapped in regions of close contact between neighbouring cells (Fig. 2(h)). If the 10% non-internalized radioactivity is deducted from the total internalized radioactivity, the rate of internalization at high multiplicity comes very close to the values observed at low multiplicity, and again illustrates the fact that the entry of the viruses into the cells does not appear to be dependent on the multiplicity.

The kinetics of uptake and degradation were also observed using [3H]uridine- labelled viruses. As expected the rate of uptake is the same as for [35S]methionine- labelled viruses but less than lO%of the total radioactivity is found in the medium after four hours; however, about 30% of the intracellular r3H]uridine activity is acid-soluble.

(c) Uptake of prebound Semliki Forest virus after rapid warming

In the experiments described above the uptake of prebound viruses was initiated by placing the cooled dishes into an incubator at 37”C, a procedure that gives relatively slow warming. To study the initial rate of virus uptake after instan- taneous warming, plates were immersed into medium at 37°C. The results shown in Figure 5 show that in this way a half-life of five to ten minutes for uptake at low multiplicity (1 virus bound/cell) and 20 minutes for uptake at high multiplicity (6 x lo4 viruses bound per cell) can be measured. The reason for the difference between high and low multiplicity is not clear. It may be partly explained by the previously described trapping of viruses between the cells. It is also possible that the virus load during the first minutes after warming exceeds the capacity of endocytosis. The initial rate of uptake at high multiplicity amounts to 3000 viruses per cell per minute.

(d) Semlik% Forest virus uptake without prebinding

By prebinding viruses it is possible to characterize the uptake of viruses independently of the binding. If viruses are added directly to the medium at 37°C

M. MARSH AND A. HELENIUS 447

Time (min)

FIG. 5. Internalization of pre-bound [35S]methionine-labelled SFV by rapid warming at low (1 virus/cell, -m-e--) and high (6 x lo4 viruses/cell, -m-m--) multiplicities. Viruses were bound to the cells for 1 h at 4°C and unbound viruses removed by washing. The cells were warmed rapidly by immersing the plates in R-medium at 37°C.

Time (h)

(a) (b)

FIG. 6. Uptake and degradation of [%]methionine-labelled SFV added to the cell medium at 37°C’ (1 virus/cell. (a) Acid-precipitable 35S radioactivity in the medium (-@-a-): cell surface activity (-O-O-); total radioactivity recovered (-A-A-). (b) Intracellular activity (--n-O-); acid-soluble activity in the medium (-W-m-).

binding and endocytosis occur in parallel. Figure 6(a) and (b) shows that the viral radioactivity can, under such conditions, be followed, with time, from the acid- precipitable pool in the medium to a surface-bound fraction, further to an internalized fraction, and finally back into the medium as acid-soluble activity. Half of the viruses are removed from the medium in 30 minutes, the cell-surface-bound viruses reach a maximum level after one hour, and the amount of intracellular viruses increases steadily up to 1.5 hours. The acid-soluble radioactivity increases in the medium linearly after a one-hour lag phase and amounts to 30% of the total radioactivity by 25 hours. Over 92% of the initial radioactivity is internalized during the first 2.5 hours, of which about half is degraded and released into the medium. The rate of internalization of the viruses into the cells at any time-point is

448 ENDOCYTOSIS OF SEMLIKI FOREST \‘tltl:S

directly proportional to the number of viruses bound bo the cell surface, and viruses attached to the cell surface are rapidly internalized. Experiments of this type were performed at low (Fig. 6) and intermediate multiplicity (8 x 1 O3 viruses per cell) with very similar results.

(e) Temperature dependence of Semliki Forest viru.s upta,ke

The temperature dependence of SFV uptake was determined. Viruses were bound to the cells at 4°C and, after washing, the cells were overlaid wit,h R-medium at temperatures between 0°C and 40°C. The dishes were incubated at the appropriate temperature for 30 minutes and subsequently assayed for cell-associated and internalized radioactivity. Proteinase K-resistant radioactivity remained at a 39b background level between 0°C and 10°C but increased thereafter with increasing temperature (Fig. 7).

80 -

Temperature PC)

FIG. 7. The temperature dependence of [35S]methionine-labelled SFV uptake. The virus (1 virus/c~vll) was bound to the cells for 1 h at 4”C, after which medium wa,s added to the cells at the indicat,ed temperatures. Internalization after 30 min was determined using Proteinase K.

( f) Effects of eeWar inhibitors

Inhibitors of oxidative phosphorylation (sodium azide and dinitrophenol) are capable of partially inhibiting the uptake of prebound viruses at the concentrations indicated in Table 1; however, neither inhibitor affects the cell surface binding of viruses in the cold. 2Deoxy-n-glucose, an inhibitor of glycolysis, has a minimal effect on binding but no effect on uptake, and does not enhance the effect of sodium azide when used simultaneously. These results demonstrate a dependence on oxidative metabolism for viral endocytosis in BHK-21 cells.

Cycloheximide, an inhibitor of protein synthesis, has no effect on uptake or binding of viruses. Colcemid, which disrupts the microtubular systems, has only a

minimal effect on uptake (Fig. 8(a)) and no effect on binding in the lo-’ M to 10e4 M

concentration range. Indirect immunofhrorescence with anti-tubuiin antibodies, after fixation and treatment of the cells with Triton X100, shows that 10W5 M-

colcemid totally disrupts the microtubular system within 30 minutes. Cytochalasin B, which inhibits actin gelation and microfilament function in the

M. MARSH AND A. HELENIUS

TABLE 1

Effect of antimetabolites on Semliki Forest virus-BHK cell interactions

449

Inhibitor Concentration

(n-f)

Binding Endocytosis (4”C, 60 min) (37”C, 60 min)

(%) (%)

None 100 100 Sodium aside 10 100 37

50 76 40 Dinitrophenol 610 100 45

Dinitrophenol + sodium aside [ 0.10 1 10.0 100 31

%Deoxy-D-glucose + sodium aside [ 50 1 50 83 40

d-Deoxy-o-glucose 50 100 106

concentration range from 0.1 to 30 pg ml - ’ (Spudich, 1972; Weihing, 1976) does not affect binding of SFV to BHK-21 cells in the concentration range from 5 to 40 pg ml-’ (Fig 8(b)). Th ere is, however, partial inhibition of uptake, which increases linearly with increasing cytochalasin B concentration. At 10 pg cytochalasin B ml- 1

(where endocytosis is inhibited by 20% both at high and low multiplicities of virus) immunofluorescent experiments using anti a-actinin indicate that the organization of cw-actinin-containing cytoskeletal structures is totally disrupted. The production of infective viruses (assayed by plaque titration) in the presence of 10 pg cyto- chalasin B ml -’ is reduced by SO%, but this is not due to an effect on the early stages of infection, since addition of cytochalasin B 20 minutes after the inoculum

. $ 80-

% - z 60- 4 -

:\

g 40-

E z 20-

lO-6 IO-' 10-q IO 20 30 40

Colcemld (M ) Cytocholasin B (pg ml-‘)

(0) (b)

FIG. 8. The effect of colcemid and cytochalasin B on binding and uptake of [35S]methionine-labelkxl SFV (1 virus/cell). The percentage binding compared to uninhibited cells during 1 h at 4°C is shown in (a) for colcemid (-m-m-) and in (b) for cytochelasin B (-@-a-). The percentage of uptake, determined using Proteinese K, during 30 min at 37°C is shown in (a) for colcemid (-n-o-) and in (b) for cytochalasin B (-O-O-).

450 ENDOCYTOSIS OF SEMLIKI FOREST VIRL’S

virus gives the same inhibition as cytochalasin B present throughout the course of infection.

We have previously shown that lysosomotropic weak bases inhibit some early stage of SFV infection. The effect of chloroquine on binding and internalization was tested but no significant inhibition was observed (Helenius et al., 1980a). There is evidence, however, that other weak bases can inhibit receptor-mediated endoc,ytosis (Maxfield et al., 1979; Sando et al., 1979). We therefore tested five lysosomotropic amines known to inhibit SFV infection (chloroquine. amantadine, methylamine, tributylamine and ammonium chloride) and found no significant effect on binding of SFV to cells at low temperature nor in the subsequent uptake upon warming (Table

2).

TABLE 2

Effect of lysosomotropic weak bases on Semliki Forest virus-BHK cell interactions

Inhibitor Concentration

(mM)

None Tributylemine Amantadine Methylamine Chloroquine NH,CI

1 05

10 0.1

10

Binding Endocytosis (60 min, 4°C) (30 min: 37°C‘)

(%I (“6)

100 96 93 90 97 9%

100 107 104 103 106 87

(g) Effect of Semliki Forest virus err jluid-phase endocytosis

To determine whether the virus stimulates its own internalization, fluid-phase en- docytosis in the presence or absence of viruses was measured using [3H]sucrose as a fluid-phase marker. Cells were incubated at 37°C with 1 ml R-medium containing a trace amount of [3H]sucrose. After 30 minutes incubation 10 ~1 32P-labelled SFV was added to half of the plates and the internalization of [3H]sucrose and [32P]SFV was determined at different time-points. In the dishes without viruses the sucrose uptake was approximately linear over a six-hour incubation period. From the slope of the line an uptake of 0.37 ~1 h-i per 10’ cells can be calculated (Fig. 9). The addition of viruses (3.2 x lo5 particles/cell) results in a 15 to 20:/, reduction in the sucrose uptake during the period of virus endocytosis.

The average internal diameter of equatorially sectioned coated vesicles containing SFV is 90 nm giving a volume of 3.8 x lo-i3 ~1 for an endocytotic coated vesicle. The uptake of 80% of the added viruses (cf. Fig. 7) during the two hours following virus addition corresponds to 2100 viruses internalized per minute per cell. If 1.3 viruses are present per virus-containing coated vesicle, we calculate a value of 1600 vesicles per minute per cell. The volume of the virus is O-9 x lo-i3 ~1, which means that 13 viruses per coated vesicle occupy one third of the internal volume. The uptake of viruses must therefore be accompanied by the uptake of bulk fluid. With 2.5 x lo5 viruses per cell this would correspond to 0.7 ~1 of bulk fluid in addition to the basal

M. MARSH AND A. HELENIUS

-5

0 123456 lime(h)

451

FIG. 9. Fluid-phase endocytosis of [“HIsucrose in the absence (-@-a-) or presence (-n-A-) of [32P]SFV. [3H]sucrose was added at time zero and the cells incubated at 37”C, after 30 min (arrowed) [32P]SFV was added to half the plates (3.2 x 10’ viruses/cell). Plates were sampled at 30-min intervals for cell associated ‘H radioactivity and internal 32P radioactivity (-O-O-).

endocytosis. Thus a clearly detectable increase in sucrose uptake would be expected after the addition of virus. The fact that such an increase does not occur indicates that the viruses are internalized as a part of the basal endocytotic activity. The slight decrease observed in the sucrose uptake may correspond to the displacement of fluid from endocytotic vacuoles by the viruses.

4. Discussion From electron microscopic data three main mechanisms for endocytosis of

membrane-containing viruses have been proposed : (1) a particle-induced en- docytosis process where the cellular microfilaments play a central role, i.e. phagocytosis (Dales, 1973); (2) a particle-induced mechanism independent of microfilaments (the virus may, for instance, induce its own uptake by wrapping the plasma membrane around itselfto form a tight-fitting endocytotic vacuole stabilized by multiple virus glycoprotein-receptor interactions; Patterson et al., 1979); (3) endocytosis in coated vesicles (Dahlberg, 1974; Pathak et al., 1976; Dales, 1978), a process that is generally considered particle and microfilament-independent (Goldstein et al., 1979; Silverstein et al., 1978).

Our present results on the endocytosis of SFV into BHK-21 cells make it possible to define the type of process responsible for the uptake. The salient features are as follows :

(i) Efficient endocytosis requires virus-binding to the cell surface. We have identified the main receptors on mouse and human cells as the major histocompat- ibility antigens (Helenius et al., 1978) but the receptors on BHK cells are unknown.

(ii) The uptake is saturatable at high virus concentrations. However, the saturation observed is due to saturation of binding rather than of endocytosis.

(iii) The uptake is rapid and efficient. The high affinity of viruses for the cell surface (apparent K,= 10” to 10” M- 1 ; Fries & Helenius, 1979) the relatively high binding capacity (about lo5 viruses per cell), and the fast rate of clearing from the

452 Eh-DOCYTOSIS OF SEMLIKl FOREST \‘IR1’8

cell surface (half-life 10 to 35 min) ensure rapid and virtually complete removal of virus from the medium over a wide range of virus multiplicities.

(iv) The uptake is not sensitive to colcemid and only marginally sensitive to cytochalasin B at concentrations (10 pg ml- ‘) that totally inhibit phagocytosis (Davis et al., 1971). This suggests that microtubules and microfilaments do not play an important role.

(v) Uptake is completely blocked at low temperature. (vi) Uptake occurs via coated pits and coated vesicles. (vii) From the coated vesicles the endoeytosed viruses are rapidly routed into

endosomes (larger uncoated vacuoles devoid of lysosomal enzymes; Helenius et al.. 1980a; Marsh, Louvard & Helenius, unpublished results) and finally into lysosomes. The bulk of the endocytosed viruses enter the lysosomal compartment after a lag of about 30 minutes or more, whereafter viral proteins degraded to single amino acids start to appear in the medium.

(viii) Virus uptake decreases fluid-phase uptake, which is consistent with virus uptake occurring by a continuous virus-independent process.

On the basis of these results we can exclude phagoqytosis. which is a particle- dependent process and highly sensitive to qytochalasin B (Silverstein et al., 1978), as a mechanism for SFV uptake. A virus-induced uptake process independent of microfilaments also seems unlikely. What the results strongly suggest is a mechanism of uptake similar to that previously described for the uptake of extracellular proteins such as yolk proteins and serum lipoproteins (Roth & Porter, 1964: Goldstein & Brown, 1977), polypeptide hormones (Gordon et al., 1978), lysosomal enzymes (Neufeld et al., 1977), %2-macroglobulin (Willingham et a.2.. 1979) and asialoglycoproteins (Tolleshaug et al., 1977). This form of uptake, generally called receptor-mediated endocytosis (see review by Goldstein et al., 1979) is a constitutive property of most cell types and plays an important part in cellular regulation, nutrition and disease. Receptor-mediated uptake of physiological ligands is, like SFV uptake, saturable, receptor-dependent, fast and highly efficient. It is relatively insensitive to agents that disrupt cytoskeletal elements and to other inhibitors, it leads in most cases to the delivery of the ligand into lysosomes and, in cases where ultrastructural information exists, it, occurs in coated vesicles (Silverstein et al., 1978 ; Goldstein et aE., 1979). Endosomes as an intermediate station on the way to lysosomes have also been described (Ostlund et al., 1979: Tolleshaug et al., 1979). The main distinction between SFV uptake and the uptake of ligands with physiological function is the specificity of the receptors. Since we do not know enough about the molecular details of SFV binding to the cell surface we prefer to term the SFV endocytosis adsorptive rather than receptor-mediated. It is clear. however, that the virus can exploit existing cellular mechanisms in its entry into the cell. Previous studies (Fan & Sefton, 1978; Helenius et al., 1980a) have shown that endocytosis is needed for productive infection of cells by SFV and related viruses.

Biochemical and kinetic data on endocytosis exist also for reovirus, a naked RR’A virus (Silverstein & Dales, 1968). This virus binds to L-cells and is internalized and degraded in lysosomes in a very similar way to RFV. Morphological studies with other membrane-containing viruses are also consistent with an adsorptive en docytosis mechanism. Coated pits and vesicles with viruses inside have been

M. MARSH AND A. HELENIUS 453

observed with myxo-, rhabdo- and retroviruses as well as alpha viruses (Dales & Hanafusa, 1972; Dahlberg, 1974; Pathak et al., 1976; Dales, 1978; Patterson et al.. 1979).

In addition to helping us understand the stratagem by which viruses infect cells, the SFV system can be used to characterize the mechanisms of adsorptive endocytosis, the molecular details of which are not known. It is possible that SFV binds to cell-surface molecules that normally function as receptors in the internaliz- ation of physiological macromolecules. However, two considerations speak against this possibility: (1) the major histocompatibility antigens have been identified as the main receptors for SFV on murine and human cells, but are not known to have a physiological receptor function involving endocytosis; and (2) other non- physiological multivalent ligands such as antibodies directed against cell-surface antigens (Louvard, 1980; Schneider et al., 1979), lectins (Willingham et al., 1979) toxins (Dorland et al., 1979) and, as discussed above, a variety of other viruses appear to be internalized by adsorptive endocytosis. In view of the apparent non- specificity in adsorptive uptake, we favour the notion that aggregation of surface receptors into a cluster by a virus or other multivalent ligand will suffice to trap the ligand-receptor complex into coated pits and thereby facilitate endocytosis.

At high multiplicity our results demonstrate that the capacity for adsorptive endocytosis in BHK cells is very high. We measured rates of uptake as high as 3000 virus particles per minute per cell. If all the viruses are internalized by coated vesicles and if an average of 1.3 viruses enter per virus-containing coated vesicle, the uptake corresponds to the internalization of 1 to 2% of the total surface area of the cell per minute (the area of the cell being determined approximately as 5000 pm2 from scanning electron micrographs). The actual value for membrane uptake in coated vesicles may be even higher as an equal number of large virus-free coated vesicles are seen in the cell periphery. The membrane uptake in coated vesicles alone is of the same order as the total membrane uptake estimated by Steinman et al.

(1976) from fluid-phase uptake in macrophages and fibroblasts. The high rates of coated vesicle endocytosis that we observe may not, however, represent the steady state in the cell at 37°C. In agreement with Anderson et al. (1977) we have observed that warming of the cells from 4°C to 37°C leads to a burst of coated vesicle formation. To determine the degree of involvement of coated vesicles in the basal cellular fluid-phase uptake, the uptake of virus and fluid-phase markers is presently being studied at constant temperatures.

We thank Eva Bolzau and Jennifer Schijnherr for excellent technical assistance, Wendy

Moses for typing the manuscript, Drs Kai Simons, Karl Matlin, Graham Warren and Judy White for helpful discussion and r&ice, and Dr Daniel Louvard for assistance with the immunofluorescence microscopy.

(One author M. M.) acknowledges the Royal Society of London for the provision of a European Science Exchange Programme Fellowship.

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