Entry of murine retrovirus into mouse fibroblasts

VIROLOGY

125, 85-98

(1983)

Entry of Murine Retrovirus into Mouse Fibroblasts KLAUS

B. ANDERSEN’

The Fibiger-Laboratwyry,2

Nordre Received

July

AND

Frihawsgade

BJBRN A. NEX0 70, DK-2100

8, 1982; accepted

October

Copenhagen

S; Denmark

22, 1982

We have studied the entry of murine retrovirus into mouse fibroblasts by following the fate of both radioactively (protein) labeled virus particles and infectious virus particles. Physical and infectious particles bound to the cell surface with a half time of 1.52 hr. Both types of particles were internalized with a half time of approximately 3 hr as measured by the resistance to externally added proteases. The binding proceeded both at 37 and O”, whereas the internalization was blocked at 0’. The internalized physical particles followed two routes: they either were degraded or remained stable in the cell. Degradation was blocked by lysosomotropic bases and is therefore believed to occur in the lysosomes. Infection could also be inhibited by lysosomotropic bases when present in the first hours after the internalization, indicating that the infectious route also is leading through the lysosomes or another acidic compartment of the cell.

by receptor-mediated endocytosis (Goldstein et d, 1979; Willingham and Pastan, 1980; Pastan and Willingham, 1981). The endosome (receptosome) content is collected in the lysosomes where some of the virus particles are degraded. Other virus particles are believed to enter the cytoplasm by a low-pH dependent membrane fusion between the virus membrane and the lysosomal membrane, which thus offers the uncoating. Possibly the fusion already occurs in the receptosome, since these vacuoles acidify before their fusion with the lysosomes (Tycko and Maxfield, 1982). The process by which C-type retroviruses enter their host cells is less clear. It is known that the viruses bind to the cell surface by attachment of the viral glycoprotein gp70 to receptors on the cell surface (DeLarco and Todaro, 19’76; Fowler et a& 1977), but from electron microscopic studies both membrane penetration (Miyamoto and Gilden, 1971) and viropexis (Dales and Hanafusa, 1972) have been suggested as mechanisms of internalization. Both studies, however, observed the presence of intact and disintegrated virus particles inside vesicles. Also the retrovirusassociated enzyme, reverse transcriptase has been followed during the infection

INTRODUCTION

Viral infection of cells involves attachment of the virus particle to the cell surface and entry of parts of the virus particle into an intracellular site where replication can occur. The physical translocation is essential to successful infection, and is thereby of decisive importance to host range and pathogenicity. Two alternative routes of entry of enveloped animal viruses have been recognized (see review by Lenard and Miller, 1982): plasma membrane penetration and endocytosis (viropexis). The membrane penetration occurs by a fusion between the plasma membrane and the virus membrane, whereby the virus core is uncoated and enters the cytoplasm. This route is followed by Semliki Forest virus entering its host cells at low pH (White et al, 1980). Semliki Forest virus at neutral pH (Helenius et aL, 1980; White et aL, 1980), influenza virus (Matlin et ak, 1981), and vesicular stomatitis virus (Matlin et al, 1982) follow the route of endocytosis: The viruses are bound to receptors on the cell surface and are endocytosed, presumably 1 To whom reprint ‘Under the Danish

requests Cancer

should Society.

be addressed.

85

0042-6822/83/030085-14$03.00/O Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved

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(Aboud et aL, 1979). The reverse transcriptase was shown to be present inside recently infected cells, in large cellular structures as well as particles sedimenting with the density of intact virus particles. The study did not clearly show in which cellular compartments the reverse transcriptase existed. As is the case with most animal virus (Lonberg-Holm and Philipson, 1974), only a low fraction of the physical retrovirus particles give successful infection. It is therefore not a priori certain that the majority of the physical particles follow the route of the infectious particles. In this study we have examined the infection process of retrovirus using prelabeled radioactive virus particles and quantitative biochemical methods. We could therefore study the kinetics of entry of the physical particles, and compare it directly to the kinetics of entry of the infectious particles. Using radioactive particles we were furthermore able to study the route of infection at virus particle to cell ratios below 1, approaching realistic conditions of infection. METHODS

AND

MATERIALS

Cells. Mouse fibroblasts of the lines BALB 3T3 (Aaronson and Todaro, 1968) and SC-1 (Hartley and Rowe, 1975) were used. The cells were grown in monolayer cultures in minimal essential medium (Gibco, N. Y.) supplemented with 10 and 5% fetal calf serum (Gibco), respectively, 0.5 mg/ml glutamin, 0.03 mg/ml streptomycin, and 250 units/ml penicillin. All cultures were grown at 37”, 5% COa, and 90% relative humidity unless otherwise noted. Virus. The B tropic virus C57MC pool 1636 (Schuh et al, 1976) was used. The virus was propagated in SC-l cells. Labeled virus was obtained from infected SC-l cultures grown in T 75 flasks (NUNC, ROSkilde, Denmark). The cultures were first labeled with 200 &i rH]leucine (130 Ci/ mol) (The Radiochemical Center, Amersham, England) in 7 ml leucine-free medium, supplemented with 5% dialyzed fetal calf serum for 1 hr. This medium was discarded in order to remove unlabeled vi-

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rus (Forchhammer et aZ., 1976). New medium (7 ml) containing 1 mCi [3H]leucine was then added for 3-5 hr. In this period, the majority of the radioactivity was incorporated into the cells and virus. The medium was centrifuged at 500 g for 5 min in order to remove cells, and was then centrifuged at 38,000 rpm for 1 hr in a Beckman Ti 50 rotor in order to collect the virus. The virus pellet was carefully rinsed with phosphate-buffered saline (PBS), resuspended in PBS, and stored at -80”. The purity of the preparations was examined by electrophoresis on SDS-polyacrylamide gels (22%) (Laemmli, 1970). The gels were treated with 2-5-diphenyloxazol (PPO) as a scintillator and autoradiographed.

Infection experiments with radioactive and nonradioactive virus. Twenty-square centimeter Petri dishes or 2 or 0.4 cm2 multiwell dishes were seeded with respectively 600,000 cells in 3 ml medium, 60,000 cells in 0.5 ml medium, or 12,000 cells in 100 ~1 medium. The medium was supplemented with 10 m&f HEPES, pH 7.3 (N-2 -hydroxy-ethyl-piperazine-N’-2-ethanesulfonic acid). The following day, the media were removed and new medium supplemented with 10 mlMHEPES, pH 7.3, and 5 pg/ml polybrene was added. After at least 1 hr, radioactive or nonradioactive virus was added.

Assays of the cellular processing of radioactive virus. Total radioactivity in the media was measured by counting aliquots in scintillation fluid. To quantitate trichloric acid (TCA) soluble material in the media, aliquots were precipitated with 8% TCA for 1 hr on ice. The samples were centrifuged and the supernatants were then filtered through nitrocellulose filters (Schleicher and Schiill, Dassel, West Germany; BA 85) and counted. For the analysis of the radioactivity in the cells and of the viral proteins in the cells, the media were removed and the cultures were washed with PBS. The cells were then lysed in gel sample buffer (10% v/v glycerol, 5% v/v 2-mercaptoethanol, 80 mg/ml sodium dodecyl sulfate (SDS), and 125 mMTris-(hydroxymethyl)-aminomethane, pH 6.8). The TCA-soluble radio-

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activity in the cells was measured as above after lo-fold dilution of the lysate in 5% calf serum. Individual viral proteins in the cells were examined by gel electrophoresis. The lysates were sonicated and 500 mg/ ml urea was added to lower the viscosity, and they were then electrophoresed on 22% SDS-polyacrylamide gels. Trypsinization was used for the analysis of the distribution of virus on the surface and inside the cells. The media were removed from the cultures and they were washed with PBS, whereafter 2.5 mg/ml trypsin (Difco, Detroit, Mich.) and 1 mM EDTA in PBS were added for 25 min at 0” unless otherwise noted. This treatment also loosened the cells from the cultures. The cells were collected by centrifugation (150 g) for 5 min in a refrigerated centrifuge and were washed once with PBS. Trypsinsensitive cellular radioactivity was measured as the radioactivity in the combined supernatants, and trypsin-resistant cellular radioactivity was measured as the radioactivity remaining in the cells. Assays of iv&ectim. The infection of the cells was analyzed 34 to 48 hr after addition of virus, by either a plaque assay detecting p30 (Nexe, 197’7) or by the production of virus. The production of virus was measured as the reverse transcriptase activity which could be pelleted from the culture media (Pedersen et al, 1981). Inhibitors. The effects on virus degradation and infection of the following substances were tested: chloroquine (Sigma, Saint Louis, MO.), amantadine (Sigma), tributylamine (Merck, Darmstadt, West Germany), methylamine (Merck), and ammonium chloride (Merck). These substances were added in solutions with pH adjusted to 7.

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preparations had specific biological activities of 0.05 to 0.1 PFU/cpm. Figure 1 shows a separation of a typical virus preparation. The individual proteins were identified by their size and isoelectric point in two-dimensional gel electrophoresis (O’Farrell, 1975). From electron microscopy of the virus preparations it appeared that the majority of the material was intact virus particles. No significant loss of biological activity was observed during the period of collection of radioactive virus: In one test, virus collected over 20 min had a specific activity of 0.054 PFU/cpm, whereas virus collected over a period of 4 hr had a specific activity of 0.048 PFU/cpm. The specific radioactivity of the virus particles was calculated to be approximately 0.1 cpm/virus particle, using as molecular weight of the particle 10’ and 8% of this as the leucine content of the virus (Tooze, 1973; Oroszlan and Gilden,

xwwa

~12, u12EK p15, 412

RESULTS

Virus Preparations A purification procedure using only one step, centrifugation, was chosen in order to obtain preparations of virus which were both reasonably pure and retained their biological activity. Forty to 100% of the plaque-forming units (PFU) was recovered after the centrifugation, and the

of ‘H-labeled C57MC. Ten FIG. 1. Autoradiogram microliters of a C57MC preparation (25,996 cpm/rl) labeled with FHjleucine in SC-1 cells was electrophoresed on a SDS-polyacrylamide gel and autoradiographed. As molecular weight markers phosphorylase B (92,566), albumin (69,666), ovalbumin (46,OW.Q carbonic anhydrase (3O,ooO), and cytochrome c (12,309) were used.

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1980). According to this specific activity less than 3 virus particles were added per cell in the experiments of which less than 0.7 bound per cell. From the specific biological activity (0.05 to 0.1 PFU/cpm) and the specific radioactivity it appears that 0.5 to 1% of the virus particles gave successful infection. Binding

Figure 2A shows the binding of tritiated retrovirus particles to SC-l and BALB 3T3 cells at 37“. As will be seen later, some of the bound virus was excreted into the media as TCA-soluble material, whereas little or no TCA-precipitable radioactivity (including loose cells) was released from the cell layer. The amount of TCA-soluble radioactivity plus the amount of bound radioactivity is therefore a reasonable measure of the material which is or has been bound to the cells. As seen from Fig. 2A, a final level of 50 to 55% was bound to both the SC-l and the BALB 3T3 cells. The remaining material consisted presumably of nonviral material and disrupted virus particles. The final level of binding varied with the different virus preparations from 30 to 70% of the added radioactivity. The absolute amount of virus bound was proportional to the amount added, and the course of binding followed reasonably well the line of an exponential function with a half time of 1.8 hr (shown in Fig. 2B). These observations indicate that the binding is a firstorder process and is far from saturation. The rates of binding as well as the final levels attained were similar for the two cells lines. In other experiments performed under similar conditions the half time of binding varied from 1.5 to 2 hr. Binding also occurred at 0”, but the rate was lower. The binding of infectious particles was also examined. We analyzed both the infection resulting from the virus particles which were bound at various times and the infectivity of the residual particles. It should be noted that a loss of infectivity in the course of the experiment could in-

TIME,

HOURS

FIG. 2. Binding of C57MC to BALB 3T3 and SC-1 cells. (A) %-labeled C57MC [lO,OOO cpm (0, l ), 3000 cpm (0, n ), or 1000 cpm (A, A)] was added to 2 cm2 cultures of BALB 3T3 cells (filled symbols) or SC-l cells (open symbols). The cultures were harvested at the indicated times, and the radioactivity in the cells and in TCA-soluble material in the media was measured. The results are shown as percentage of the added radioactivity. The dashed lines show the TCAsoluble radioactivity in the media and the solid lines show the sum of the cellular radioactivity and the TCA-soluble radioactivity in the media. The background of TCA-soluble radioactivity in the virus preparation (9.0%) was subtracted from the results. (B) Nonradioactive C57MC (approximately 2000 PFU) was added to 2 cm* cultures of SC-1 cells. At the indicated times the media were transferred to new cultures and new media was added to the first cultures. Forty-eight hours after infection the amount of virus produced in both sets of cultures was measured as the pelletable reverse transcriptase. The results are presented as the percentage of reverse transcriptase in the first cultures in comparison to the sum of reverse transcriptase in the first plus the second cultures. The experiment was performed twice. In the first experiment (0) the sum of reverse transcriptase activity was 26,000 f 7500 cpm and in the other (0) 160,000 + 26,000 cpm. The curve is the function 100% . (1 - ect’n2/1.8hr)).

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fluence the results of this experiment but we have not observed any significant loss of infectivity of the virus preparations at 37”. The infectious particles also bound to the cells with a half time of 1.8 hr (Fig. 2B), and binding also occurred at 0” (see Fig. 3B). The similarity of binding of the radioactive and the infectious particles suggests that the involved mechanisms are similar. Internalization Trypsin treatment of the cells at 0” was used to examine the distribution of virus between the cell surface and interior. The protease treatment removes proteins and virus bound to the cell surface (Schlessinger et ah, 1978; Helenius et al, 1980), whereas no cellular degradation of virus occurs at low temperature (results not shown). In one experiment (Fig. 3A), radioactive virus was bound to cells at 0”, and unbound virus was washed away. Initially after the washing procedure, 20% of the bound material was free in the media. This material was TCA precipitable, and was presumably virus material, which was loosened and disrupted in the washing procedure. More than 97% of the virus material, which was bound to the cells at 0”, could be removed by trypsin treatment even after prolonged periods at 0”, and was hence surface bound. The lack of internalization at low temperatures is in agreement with the findings for other animal viruses and receptor bound hormones (Kohn, 1975; Helenius et al, 1980; Schlessinger et al, 1978). After the binding of virus at 0”, the cultures were rewarmed to 37”. The amount of surface bound virus then decreased with a half time of approximately 3 hr. Not all surface bound material disappeared, thus after 20 hr approximately 15% of the bound virus remained on the surface. The amount of internal virus material increased to approximately 40% in the first 2 hr, whereafter the amount remained rather constant. Degraded material appeared in the media after a lag time of approximately 1.5 hr,

‘/L, 0

4 TIME,

8 HOURS

FIG. 3. Internalization of C57MC in SC-l cultures. (A) ‘H-labeled 57MC (65,006 cpm) was bound to 20 cm2 SC-l cultures on ice for 2 hr. Unbound virus was removed and new ice-cold media was added, 26,000 f 4400 cpm attached to the cells. The cultures were incubated further on ice (dashed lines, closed symbols) or at 37” (solid lines, open symbols) and were harvested at the indicated times. The total and the TCA-soluble radioactivity in the media and the trypsin sensitive and trypsin-resistant cellular radioactivity were measured. The results for each culture are given in percentage of the radioactivity attached after the binding period at 0”. TCA-soluble radioactivity in the media (A, A). TCA-soluble radioactivity in the media plus trypsin-resistant cellular radioactivity (0, 0). TCA soluble radioactivity plus total cellular radioactivity (0, n ). (B) 70,000 PFU of nonradioactive C57MC was added to 20 cm2 SC-l cultures on ice for 2 hr. After 2 hr unbound virus was washed away and new media was added. The cultures were incubated on ice (0) or at 37” (0, 0). At the indicated times the cells were trypsinized off the culture dish, washed, and reseeded in new cultures (0, 0). As a control the trypsin treatment was omitted (0). After 2 days the infection was analyzed as the amount of pelletable reverse transcriptase activity in the media.

and after 20 hr approximately 40% of the initially bound virus was degraded. A similar experiment was performed with BALB

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3T3 cells. Approximately the same half time of internalization and lag time of degradation was observed, whereas 75% of the bound material was degraded. Trypsination was also used to analyze the internalization of infectious virus particles (see Fig. 3B). Virus was bound to the cells at 0”; unbound virus was washed away, whereafter the cultures were incubated at 37 or 0”. After various times of incubation, the cultures were trypsinized and the subsequent infection was measured. When the trypsination was performed immediately after the binding or after further incubation at 0”, no infection was observed. After warming of the cultures the fraction of virus which was trypsin resistant increased with a half time of approximately 3 hr. After 20 hr at 37” trypsin treatment no longer reduced the infection. The analogy of the conditions and kinetics of internalization of the radioactive and infectious particles suggest that their mechanisms of internalization are similar. Degradation Degraded radioactive material appeared extracellularly after a lag time of l-2 hr (Figs. 2A and 3A) in amounts proportional to the amount of virus added (Fig. 2A), indicating that several steps are involved in the route leading to degradation and that each step has first order kinetics. Degradation of virus was blocked at 0” and no degradation occurred in fresh medium or medium in which the cells had grown (results not shown). We therefore conclude that the degradation is cell-associated. The rate and degree of degradation of bound virus were examined: Radioactive virus was added to cultures, and after 1 hr (in which period no TCA-soluble material appeared) unbound virus was washed away. The radioactivity in the cells and in the media was then followed (see Fig. 4). Immediately after the wash, lo-15% of the material was TCA precipitable in the media. This material is believed to be viral material, which is loosened from the cells

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FIG. 4. Viral material in the cells. %-labeled C57MC was added to 20 cm’ cultures of BALB 3T3 (140,000 cpm) (filled symbols) and SC-l (340,000 cpm) (open symbols). After 1 hr of incubation, the unbound virus was washed away and new media was added. After this incubation, 62,100 f 3000 cpm and 34,600 + 5360 cpm were bound to the BALB 3T3 and SC-l cultures, respectively. After further incubation, the cultures were harvested at the indicated times, and the radioactivity as total and TCA-soluble material in the media and in the cells was measured. The results for each culture are presented in percentage of the total radioactivity, which attached to the cells after the initial incubation period: TCA-soluble radioactivity in the cells (0, 0), total radioactivity in the cells (D, q ), and total radioactivity in the cells plus TCA-soluble radioactivity in the media (A, A).

during the washing procedure. The amount of the TCA-precipitable material in the media did not increase significantly after further incubation which means that the cells did not excrete TCA-precipitable material into the media. The degradation in this experiment showed first-order kinetics with a half time of approximately 4 hr for both the SC-1 and BALB 3T3 cells. In identical experiments, the half time for the degradation varied from 3 to 5 hr. Less than 3% of the cellular radioactivity was TCA soluble, which means that the viral material, which was degraded in the cells, was rapidly released. The viral material in the cells did not degrade totally, but when the degradation had ceased, a fraction of the initially bound material remained in the cells. This fraction differed between the two cell lines. At the end of the experiment, the ratio between the TCA-soluble material in the me-

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dia and the viral material in the cells was 1.5 for the BALB 3T3 and 0.5 for the SC1 cells. The absolute rate of degradation was hence higher in the BALB 3T3 cells than in the SC-l cells. The different degree of degradation in the two cell lines indicates that the degradation was not a feature of the virus preparations (e.g., a fraction of partly disrupted virus particles), but that it was inherent to the cell lines. Degradation of internalized virus was examined separately: Radioactive virus was added to cell cultures at 37’; after 0.5 hr, unbound virus was washed away, and the cells were trypsinized at 0” in order to remove surface bound virus. The procedure loosened the cells from the culture dishes, but they rapidly reattached on further incubation. The production of TCAsoluble material in the media, the amount of viral material remaining in the cells, and viral material in loose cells were followed. The results are shown in Fig. 5. A part of the internalized material was, as seen, degraded. The degradation proceeded as a first-order process and had half times of approximately 0.75 and 2 hr for BALB 3T3 and SC-1 cells, respectively, and at the end of the experiment, the ratios between the TCA-soluble material in the media and the viral material in the cells were 2.8 for the BALB 3T3 and 0.75 for the SC-l culture. These latter values were slightly larger than those observed in Fig. 4. The difference can be explained by the finding that some surface bound virus material remained on the cells in Fig. 4 (see Fig. 3A). Degradation, when surface bound virus was removed (Fig. 5) was faster than when the surface bound virus was present (Fig. 4). This suggests that internalization normally is limiting the rate of degradation, which is often observed for lysosomal degradation (see below) of external material (Lloyd, 1980). The observed half time of degradation when surface bound virus was present (3-5 hr) thus belongs to the internalization, in agreement with the results of Fig. 3. It is possible that the trypsin treatment in the experiment (Fig. 5) by changing the

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FIG. 5. Viral material and degradation of viral material in trypsinized cells. %labeled CX’MC (190,000 cpm) was added to one 20 cm2 BALB 3T3 culture (filled symbols) and to one 20 cm* SC-l culture (open symbols). After 0.5 hr of incubation, unbound virus was washed away, the cells were trypsinized for 25 min on ice, and the cells were collected and washed by centrifugation. Of the added radioactivity, 40,350 and 49,150 cpm bound to the BALB 3T3 and SC-l cultures, respectively, and of the bound radioactivity 7500 and 11,200 cpm, respectively, were trypsin resistant. The trypsin-treated cells were seeded each in one 20 cm2 culture dish with 3 ml prewarmed media. At various times, aliquots of the media were taken and the aliquots were immediately centrifuged in order to collect loose cells. The radioactivity in the loose cells, in the centrifuged media, and as TCA-soluble material in the media was measured. At the end of the experiment, the radioactivity remaining in the cells was measured. From these data, the radioactivity in the cells at the various times of incubation was calculated. The radioactivity in loose cells (0, 0), radioactivity in all cells (m, fl), and the radioactivity in the cells plus totally produced TCA-soluble radioactivity in the media (A, A) are presented in percentage of the reseeded material.

state of the cells also disturbed viral processing. This is however unlikely to be a serious problem because (i) the degree of degradation was comparable to that observed when trypsin treatment was not performed (see above). (ii) Only little viral degradation was observed when the cells were trypsinized 24 hr after virus addition (results not shown). (iii) The same results as seen in Fig. 5 were obtained when the trypsin treatment was performed for 5 min at 21’ (not shown). The kinetics of binding, internalization and degradation was examined for varying

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virus particle to cell ratios. Within a range from l/30 to 30/l bound virus particles per cell, no significant differences were observed (results not shown) indicating that no saturation of the involved steps occurred. The Individual fection

Viral Proteins

during

In-

Radioactive virus was added to cell cultures for 1 hr, unbound virus was washed away, and after various additional times of incubation, the cultures were lysed and the lysates were separated on SDS gels (see Fig. 6). It can be seen from the autoradiogram that (i) all viral proteins were present in the cell lysates 1 hr after the virus was added. This indicates that whole virus particles bound to the cells and not fractions thereof. In contrast, only a small amount of nonviral material was bound to the cells. (ii) All the viral proteins were degraded to some extent. No striking differences in the kinetics and in the degree of degradation occurred. The synchronous degradation of all virus proteins indicates that whole virus particles are degraded, TIME,

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P30 m

PI~,PI’=~ p15t

FIG. 6. Viral proteins in BALB 3T3 cells after infection. The cellular lysates of the BALB 3T3 cultures presented in Fig. 4 were electrophoresed on an SDSpolyacrylamide gel and autoradiographed for 21 days. The times indicate the time of harvest of each culture after the initial incubation period of 1 hr.

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and not only parts of the virus particles. (iii) No intermediate peptides (with molecular weights above 8000) occurred in the cells. Nor were similar peptides observed in the media (results not shown). These observations show that the degradation of the protein molecules, once started, is rapid. (iv) Even after 2 days of incubation, viral proteins occurred in the cells. This shows that a fraction of the viral proteins remained stably associated with the cells. (v) The background of the gels did not increase significantly with incubation time, indicating that the degraded material was not to any large extent used for resynthesis of cellular material. The radioactivity in the cells is, therefore, a reasonable measure of the amount of viral proteins in the cells. In a similar experiment the gp70 and p30 bands were cut out of the gel, dissolved, and counted. The results can, however, only be considered as semiquantitative, because it was difficult to determine the background radiation in the area of gp70. The ratio of gp70 to p30 in the virus preparation was 0.08. After 4 hr of binding the ratio in the cells was 0.09, and after an additional 18 hr with unbound virus removed the ratio decreased to 0.04. After 4 hr of binding 10% of the gp70 and 35% of the p30 resisted trypsination of the cells, whereas essentially all gp70 and p30 resisted trypsination of the cells 18 hr after removal of unbound virus. The Degradation Is Lysosnml Lysosomotropic bases are believed to inhibit lysosomal protein degradation fully and selectively (Seglen et al, 1979). These substances are known to be collected in the lysosomes, where they raise the pH (DeDuve et aa, 1974; Ohkuma and Poole, 1978), thereby inhibiting the lysosomal hydrolases, which have acid optima. The lysosomotropic bases tested were observed to cause a vacuolation of the cells as previously rapported (DeDuve et al., 1974). At concentrations higher than those shown in Table 1, the inhibitors caused the cells to shrink, round up, and fall off the culture dish.

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TABLE EFFECT

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1

OF LYSOSOMOTROPIC BASES ON BINDING, INTERNALIZATION, OF C57MC AND THE RESUCTING INFECTION”

TCA-soluble radioactivity in the media in percentage of added radioactivity

1 hr

4 hr

14.8 + 1.2

34.9 * 1.3

12.7 k 1.4

3.3 + 0.3

15.1 f 0.2

456 + 24

38.4 35.9 37.2 30.2 33.0

16.8 14.9 12.7 9.3 6.0

2.6 1.5 0.9 0.0 0.0

15.7 11.6 11.6 2.0 1.4

464 504 356 192 32

Concentration

None, mean of four samples f SD

AND DEGRADATION

Trypsininsensitive cellular radioactivity in percentage of added radioactivity 4 hr

Total cellular radioactivity in percentage of added radioactivity

bM)

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4 hr

10 hr

Infection, virus titer 10 + 24 hr

Chloroquine

0.001 0.003 0.010 0.032 0.10

15.4 15.2 13.8 13.5 8.1

Amantadine

0.01 0.03 0.10 0.32 1.0

16.2 14.0 14.2 15.0 12.8

34.0 35.4 36.8 40.7 36.7

10.7 10.3 11.0 12.7 10.3

3.0 2.3 1.1 1.3 0.4

14.9 14.7 11.3 8.1 3.9

512 408 540 540 280

0.25 0.79 2.5 7.9 25

15.1 13.7 14.9 13.0 9.4

36.8 35.7 37.0 34.5 36.1

10.9 12.7 12.3 10.5 7.7

3.0 2.1 1.7 0.8 0.2

16.9 13.4 11.9 9.0 3.4

400 332 224 176 108

0.5 1.6 5.0 16 50

13.3 14.5 10.7 12.1 13.2

34.0 36.0 36.9 33.4 30.9

10.8 10.5 8.3 8.9 8.7

0.7 0.0 0.0 0.0 0.0

9.0 1.9 1.2 0.1 0.0

352 464 340 152 120

1 hr

6 hr

6 hr

6 hr

10 hr

10 + 24 hr

19.4 + 1.3

36.7 + 2.3

17.9 f 2.2

8.8 f 0.7

21.2 f 1.4

200 + 10

19.2 17.2 21.7 23.2 16.1

39.2 41.9 43.7 44.1 38.5

17.5 17.1 20.9 18.6 10.9

5.9 2.4 0.9 0.1 0.2

15.7 6.6 3.7 0.8 0.4

178 169 181 113 21

Methylamine

None, mean

of four

samples

+ SD

Ammonia

0.56 1.8 5.6 18 56

“The inhibitors were added in the indicated concentrations to 2 cm* BALB 3T3 cultures. After 2 hr of incubation %labeled C57MC (7740 epm) was added to each culture. The cultures were then harvested at 1.4 (or 6), and 10 hr after virus addition. The radioactivity in the TCA-soluble material in the media, in the trypsin-sensitive cellular material, and in the trypsin-insensitive cellular material was measured. The background of TCA-soluble material of the virus preparation was subtracted from the respective values. The values are presented as percentage of the added radioactivity. After the harvest of the media from the lo-hr incubations, new media without inhibitors were added and the cultures were incubated for an additional 24 hr, whereafter the infection was measured as infected cell plaques.

The effect on virus binding, internalization, and degradation of the following lysosomotropic bases was tested: chloro-

quine, amantadine, tributylamine, methylamine, and ammonia. As seen in Table 1 all these substances inhibited degrada-

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tion of the virus. The concentrations giving 50% inhibition (observed 10 hr after virus addition) were 0.02, 0.4, 10, 0.6, and 1 mlM, respectively. They did not inhibit binding or internalization significantly. It can therefore be concluded that the degradation occurs in the lysosomes. The Route of In$ection To investigate if lysosomes (or other acidic vesicles of the cell) also were a part of the infectious route of entry, we quantitated the effects of several lysosomotropic bases in infection assays. The lysosomotropic bases amantadine, methylamantadine, and chloroquine have previously been shown to inhibit the replication of retrovirus (Wallbank et al, 1966; Wallbank, 1969; Pazmino et aZ., 19’74), but it was not shown at which step(s) the inhibition occurred. As seen in Table 1 chloroquine, amantadine, tributylamine, methylamine, and ammonia all inhibited infection when present during the first 10 hr following addition of virus. The concentrations giving 50% inhibition were 0.02, 1, 2.5, 8, and 20 rn$ respectively. These results agree with a lysosomal role in the infectious process. However, the inhibitors may also have nonlysosomal effects. Specifically, primary amines and ammonia are known to block some types of surface clustering and internalization of receptor bound compounds, presumably by inhibiting transglutaminase activity (Maxfield et ah, 1979; Pastan and Willingham, 1981), which could explain the effects of amantadine, methylamine, and ammonia, but not chloroquine and tributylamine. We therefore further investigated the effect of two compounds, chloroquine and ammonia (Table 2). The compounds had little or no effect on general cellular DNA and protein synthesis. They did not destroy infectivity when incubated with virus preparations, nor did they exert any effect on infection when present in cells only before virus addition. When the compounds were present during binding of virus at 0” little or no effect was observed. In contrast, when the inhibitors were present from 0 to 6 hr af-

AND NEX0

ter rewarming of cultures with bound virus a high degree of inhibition was observed. Less inhibition was again observed when the compounds were present from 6 to 12 and 12 to 18 hr after rewarming of cultures with bound virus. To further characterize the sensitive period, virus particles were bound at 0”, the cultures were incubated for 1 hr at 37”, trypsinized, and incubated further at 37”. Chloroquine did not inhibit infection when present in the 1-hr period between binding and trypsination, whereas ammonia reduced infectivity moderately. In contrast, both compounds strongly inhibited infection when present after the trypsin treatment. Thus the two lysosomotropic bases tested interfere with a process occurring shortly after internalization. We tested the effects of the lysosomotropic compounds on reverse transcription in vitro. Little or no inhibition was observed with chloroquine and ammonia (Table 2). Amantadine, tributylamine, and methylamine had no effect when added in the highest concentrations shown in Table 1. It is interesting that the time of sensitivity toward the inhibitors follows the time of degradation (which is inhibited by the same compounds): No inhibition of infection by chloroquine, nor degradation was observed in the first hour after binding (Table 2, Fig. 3A), whereas both full degradation and full inhibition of infection with chloroquine and ammonia were observed in the first 6 hr after trypsination (Table 2, Fig. 5). The moderate inhibition of infection observed with ammonia in the l-hr period is not parallel to the degradation. This inhibition can possibly be explained from the finding of Maxfield et al. (1979) that ammonia besides its lysosomotropic effect can inhibit internalization. The simultaneity of the passage through the inhibitor-sensitive step and the degradation suggests that the infectious particles and the physical particles which are degraded follow the same steps before they pass through the inhibitor-sensitive step and/or are degraded. It appears that these latter steps are not identical, but rather parallel, because their sensitivity toward

ENTRY

OF

RETROVIRUS

INTO

TABLE

EFFECT OF AMMONIA

AND CHLOROQUINE

MOUSE

95

CELLS

2

ON CELL GROWTH,

INFECTION,

AND REVERSE Ammonia

No addition mean + SD Parameter

measured,

conditions

Protein synthesis: Incorporation into TCA-precipitable cellular 5 hr

Infection, culture addition

when inhibitors were from 6.5-0.5 hr before

Infection, together at 0”

when with

inhibitors were virus in a 2-hr

Infection, when inhibitors 0 to 6 hr after a 2-hr Infection, when inhibitors 6 to 12 hr after a 2-hr

present virus

were binding

present period

from at 0”

were binding

present period

from at 0”

were present from binding period at 0”

assay

15,000

f 500

67

108

90

100

66,500

f 2,000

62

82

84

100

49,000

f 6,000

67

75

74

108

34,000

+ 6,500

56

101

91

117

19,100

f 5,800

62

87

129

127

15,500

+ 5,000

2

14

17

40

27,500

+ 7,500

36

86

52

92

14,400

f 4,000

41

:, 95

90

125

27,400

f 7,000

20

47

86

134

27,400

f 7,000

1

21

2

26

105

117

57

82

with at 37’,

Infection, when cells were incubated with virus 2 hr at 0”, 1 hr at 37”, trypsinized, and incubated 6 hr with inhibitors transcriptase

40 pM

in the

period

Infection, when cells were incubated virus 2 hr at 0”, 1 hr with inhibitors and trypsinized

Reverse

80 /LM of control)

with

present binding

Infection, when inhibitors 12 to 18 hr after a 2-hr

40 mM (percentage

of rH]leucine material over

DNA synthesis: Incorporation of [*H]thymidine into TCA-precipitable cellular material over 5 hr Infection with virus preincubated inhibitors for 3 hr

Chloroquine

80 mM

(cpm)

TRANSCRIPTASE”

247,000

-+ 52,000

a Protein and DNA synthesis were measured by adding respectively 600,000 cpm of [3H]leucine or 430,000 cpm of [3H]thymidine to 0.4 cm* SC-l cultures for 5 hr. The cell layers were thereafter washed twice with phosphate-buffered saline and once with 8% TCA for 0.5 hr on ice. The cell layers were then dissolved in 0.1% SDS and counted in scintillation fluid. Virus infection was analyzed the following way: Approximately 2000 PFU of C57MC were added to 2 cm2 SC-l cultures. The cultures were incubated at 37’ unless otherwise noted and inhibitors were present in the indicated time periods. The resulting infection was measured 48 hr after virus addition as the pelletable reverse transcriptase activity in the media. Each value represent the average of at least four determinations. The effect of the inhibitors on the reverse transcriptase was tested when added directly to the reverse transcriptase assay. Media from infected cultures were used as a source of reverse transcriptase. the

bases differ up to 20-

lysosomotropic

fold (see Table 1). DISCUSSION

The large discrepancy between the number of infectious units and the actual par-

titles present in most stocks of animal viruses has limited the studies of the infectious process, a major criticism being that microscopic and biochemical observations might not be relevant to the minority of particles successful in infection. The prob-

96

ANDERSEN

lem has often been accentuated by the high particle inputs needed for detection. We have tried to control these sources of error by performing quantitative studies of infectious and radioactive particles in parallel, at virus particle to cell ratios below one. We find similar kinetics for both binding and internalization of physical and infectious particles and simultaneity of passage of infectious particles through a step sensitive to lysosomotropic bases and degradation (which is inhibited by the same substances). Thus at least in our system the early processing (binding, internalization, and the steps leading to the inhibitorsensitive step) seems to proceed by the same mechanisms. The individual steps of viral processing followed first-order kinetics. The half times were 1.5-2 hr for binding to both cell lines, approximately 3 hr for internalization in both cell lines, and 0.75, respectively 2 hr for degradation of internalized virus particles in BALB 3T3 and SC-1 cells. The observed kinetics in our study imply a slower process than suggested by Aboud et al. (1979). Our data place the protein degradation and the passage through the inhibitor-sensitive step roughly simultaneous with reverse transcription of the viral genome in analogous avian systems (Varmus et al, 1974). It is, however, unlikely that the reverse transcription is the sensitive step, because it is not inhibited in vitro. A relative large portion of the proteins from the internalized particles survived within the cells for days. We do not at present know where these virus components are located and how they relate to the infectious particles. It was observed that BALB 3T3 cells had a higher rate as well as degree of degradation of internalized virus proteins than the SC-1 cells. This could mean that the stabilization of the undegraded proteins takes place in competition with the degradation at an internal site. If one makes this assumption it is possible to calculate the specific rate constants of degradation (kd) and “stabilization” (k,) of internalized virus proteins

AND NEX0

in the two cell lines. Forty-three and 74% of the internalized proteins were degraded in SC-l and BALB 3T3 cells with a half time of 2 and 0.75 hr, respectively (Fig. 5). kd is thus 0.43. In 2/2 hr = 0.15 hr-’ for SC-l and 0.74. In 2/0.75 hr = 0.68 hr-’ for BALB 3T3, and k, is 0.57. In 2/2 hr = 0.20-l for SC-l and 0.26. In 2/0.75 hr = 0.24 hr-’ for BALB 3T3. If the assumption is correct it would seem that both cell lines have equal intrinsic abilities to “stabilize” the proteins but that BALB 3T3 has a four- to fivefold higher ability for degradation. We propose that the internalization of retrovirus in fibroblasts occurs by endocytosis and that the viruses enter and infect the cells through lysosomes or other acidic vesicles. Conceivable uncoating and transfer from the acidic vesicles then occurs by a low pH-dependent membrane fusion, as has been suggested for Semliki Forest virus (White et al, 1980). This model is supported by two sets of observations: First, physical particles were rapidly degraded in the lysosomes. Though the outcome of degradation possibly is abortive infection (because all proteins in the particles apparently were degraded, which will expose the viral RNA genome to lysosomal RNases) it shows that virus particles can enter the lysosomes. Second, infection was inhibited by lysosomotropic substances at a step shortly after internalization which does not appear to be the reverse transcription. Since these compounds accumulate in and interfere with the functions of acidic vesicles (DeDuve et d, 1974) the data imply an acidic organelle in the infectious route. An endocytic mechanism of entry is compatible with most previous data presented (Dales and Hanafusa, 1972; Miyamoto and Gilden, 1971; Wallbank et aL, 1966; Wallbank, 1969; Pazmino et al, 1974). A similar mechanism of entry has been proposed for several other animal viruses (Helenius et al, 1980; White et cd, 1980; Matlin et al, 1981, 1982). The model of entry can explain the stabilization of virus proteins as the entry of virus particles (core particles) from the acidic vesicles to the cytoplasm before the proteolysis reaches an intolerable level.

ENTRY

OF

RETROVIRUS

According to the model whole virus particles are internalized and the virus core enters the cytoplasm whereas the envelope remains in the acidic vesicles/lysosomes, where it presumably is susceptible to degradation. This is in accord with the preliminary finding that both the envelope protein gp70 and the core protein p30 were internalized, and that more gp70 than p30 was degraded. However, precise measurements of the degradation and localization of the individual virus components are needed to determine how the different virus parts are processed. The degradation of virus particles can in part explain why the efficiency of infection is poor. For each event of successful infection 50 to 100 physical particles bound to the cells, and l/4 to l/2 of the virus material avoided degradation, corresponding to at least 12 virus particles. We do not know whether this higher, but still low efficiency of infection at this step is due to mechanisms of the cell (see Steeves and Lilly, 1977) or to a low infectivity of the virus preparations. ACKNOWLEDGMENTS

The authors are indebted to Christina Aalvik and Helle Jensen for excellent technical assistance, and to Annie Methling and Birgit Elk for typing the manuscript. The work was supported by the Danish Cancer Society, Grant 40/‘78,81/78, and 74/81; by the Danish Medical Research Council, Grant 12-3112; and by the Vera and Carl Johan Michaelsen Foundation.

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