Dissociation of Adsorbed Virus Particles with Dithiothreitol ... Sendai virus particles bind to human erythrocytes at 4°C and fuse with them at 37°C. The present ...
Vol. 49, No. 3
JOURNAL OF VIROLOGY, Mar. 1984, p. 1009-1013 0022-538X/84/031009-05$02.00/0
Attachment of Sendai Virus Particles to Cell Membranes: Dissociation of Adsorbed Virus Particles with Dithiothreitol NOR CHEJANOVSKY, MICHEL BEIGEL, AND ABRAHAM LOYTER* Department of Biological Chemistry, Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel Received 20 May 1983/Accepted 23 November 1983
Sendai virus particles bind to human erythrocytes at 4°C and fuse with them at 37°C. The present work describes a new method by which adsorbed virus particles can be removed from human erythrocytes, allowing quantitative determination of the number of virus particles which can bind and fuse with human erythrocyte membranes. Through the use of 125I-labeled Sepdai virus particles, it is shown that incubation with 50 mM dithiothreitol removed about 90 to 95% of adsorbed virus particles. Fused virus particles were resistant to treatment with dithiothreitol. Negligible amounts of 125I-labeled Sendai virus particles were removed by treatment with dithiothreitol after incubation of virus-cell complexes at 37°C. Trypsinized virus particles were able to attach to, but not fuse with, human erythrocytes even after prolonged incubation at 37°C. Treatment with dithiothreitol removed as much as 80 to 85% of trypsinized virus particles incubated with human erythrocytes at 37°C. A quantitative determination revealed that about 1,000 to 1,200 and 600 to 800 Sendai virus particles can bind to or fuse with human erythrocytes, respectively.
from the allantoic fluid of fertilized eggs and then radioiodinated with chloramine-T and Na125I as described previously (16). The 125I-SV particles (specific activity, 4 x 105 to 1 x 106 cpm/mg) were washed once in solution A (160 mM NaCl buffered with 20 mM Tricine, pH 7.4) and then loaded on a Sephadex G-25 (fine) column (10 mg of SV particles per 5 ml of packed Sephadex G-25 beads). Before each binding experiment, the virus preparations were briefly sonicated in a bath sonicator for 15 to 30 s at room temperature. These two steps were essential to avoid aggregation of the 1251-SV particles and to obtain a homogeneous preparation. Human blood, type 0, recently outdated, was washed three times and then suspended in solution A as previously described (10). 125I-SV particles (50 pLg of protein) were added to 0.5 ml of HE (2.5%, vol/vol) suspended in solution A. After incubation for 10 min at 4°C, unbound virus particles were removed by centrifugation in the cold at 800 x g for 5 min. For reduction with DTT, the pellet containing agglutinated erythrocytes was suspended in 0.5 ml of solution A containing various concentrations of DTT and 2 mM EDTA. The suspension obtained was incubated first for 15 min at 4°C and then for 30 min at room temperature. Unbound virus was removed by centrifugation, and the erythrocytes were then hemolyzed with water. The amount of radioactive material in the different fractions (unbound virus in the supernatant and bound virus in the erythrocyte pellet) was determined by a gamma counter. The amount of virus that remained attached to the HE after the first 10 min of incubation in the cold and after the washing step was considered 100%. This amount (100%) reached about 60 to 75% of the total added virus. Protein was determined by the method of Lowry et al. (6), with bovine serum albumin used as a standard. When virus-cell complexes were treated with 10 mM DTT, only 30% of the bound virus particles were released from the cells (Table 1). As much as 50 mM DTT was required to remove 85 to 90% of the bound virus particles from the erythrocyte membranes. It appears that the higher concentration of DTT required to remove erythrocyte-bound SV particles (50 mM) than to inactivate the viral binding protein in a suspension of free virus particles (5 to 10 mM) (9; data
Enveloped viruses penetrate and infect animal cells essentially via two routes: endocytosis of the whole virus particles (2) or fusion between the virus envelope and the plasma membrane of the recipient cell (11). It is conceivable that by endocytosis, an almost unlimited number of virus particles can penetrate each recipient cell. On the other hand, the number of enveloped viruses which can fuse with each recipient cell is as yet unresolved (4, 11). Elucidation of the quantitative aspects of virus-cell interaction is a prerequisite for a better understanding of the molecular mechanism of virus-cell fusion and the entire process of infection of animal cells by enveloped viruses. Furthermore, knowing the number of virus particles which can be fused with each targeted cell may pave the way for the construction of fusogenic vesicles that may serve as efficient biological carriers (7, 15). When Sendai virus (SV) particles are incubated with human erythrocytes (HE), they cause agglutination at 4°C (virus-cell binding) and induce hemolysis (virus-cell fusion) and cell-cell fusion at 37°C (10, 11). The interaction between 125I-labeled SV particles and HE has been used as a model for studying the quantitative aspects of virus-cell adsorption (16). It was shown that about 1,500 virus particles can adsorb to each erythrocyte at 4°C, giving a Kin for interaction of 0.277 min-1 (16). Because of the multivalent binding, it appeared that the SV particles were adsorbed to the erythrocyte membrane in an irreversible manner, since neither soluble sialic acid-containing polypeptides, which potentially serve as virus receptors, nor cold virus particles were able to release the virions from the membrane (16). This evidently makes impossible a clear differentiation between the number of virus particles that are adsorbed or fused with the erythrocyte membranes. In the present work we incubated virus-cell complexes with the reducing agent dithiothreitol (DTT). DTT has previously been shown (9) to affect the binding properties of free SV particles. Addition of DTT caused an almost quantitative release of erythrocyte-bound 1251-SV particles and of bound, radiolabeled reconstituted envelopes. DTT-induced dissociation. SV particles were obtained *
Corresponding author. 1009
1010
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TABLE 1. DTT-induced dissociation of HE-associated SV DTT concn (mM)
particles % 1251-SV bound
0 5 10 15 25 50
100 87.2 69.8 36.0 28.1 15.7
Agglutination
+ +
+ -
-
not shown) is due to the consumption of most of the DTT by the erythrocytes (Fig. 1). Effect of free erythrocyte concentration. HE (3.75 x 107 cells) were incubated for 10 min at 4°C with 30 pLg of 1251-SV
'00
A
N~~ N,
N,
N,
N
50
0
O
10
20
30
40
particles, in a final volume of 0.5 ml of solution A. At the end of the incubation period, unattached virus was removed and increasing amounts of washed HE (3.75 x 107, 10.5 x 107, and 31.5 x 107 cells) were added to the virus-HE complexes. After addition of DTT and EDTA (final concentration, 2 mM), the various systems were suspended in a final volume of 0.5 ml of solution A. In a second experiment, increasing amounts of HE were incubated with 125lISV at 40C for 10 min, keeping the virus-cell ratio constant (297 p.g of 1251-SV added per 28 x 107 cells). The unbound virus was washed, and the samples were resuspended in solution A, and then different concentrations of DTT and EDTA at a final concentration of 2 mM were added. The samples were incubated for 30 min at room temperature, after which the numbers of free and bound radioactive virus were estimated. The addition of untreated, washed erythrocytes to a suspension of virus-erythrocyte complexes caused a gradual increase in the concentration of DTT required for efficient release of bound virus particles (Fig. 1). About 25 mM DTT was sufficient to release up to 85% of the virus particles from the virus-cell complexes formed between 30 p.g of virus and 3.75 x 107 HE (Fig. 1A). Very few of the bound virus particles were released by addition of 25 mM DTT when 31.5 X 107 washed HE were added to the system. Under these conditions, even incubation with as much as 50 mM DTT resulted in removal of only 50% of the bound virus particles.
50
cr) _ '-4
In
CYJ
100
B 0
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0
am
'I
501 t
B
100
N~-
'a~~~~~~~~~ 'a~~~~~~~~-"
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50s 10
20
30
40
50
DTT [mM] FIG. 1. Effect of increasing concentrations of free erythrocytes on the amount of virus particles released by DTT from virusagglutinated erythrocytes. (A) HE (3.75 X 107 cells) (0) were incubated with 1251-SV particles. Unattached virus was then removed, and increasing amounts of washed HE (3.75 x 107 [0], 10.5 X 107 [A], and 31.5 x 107 [A] cells) were added to the virus-HE
complexes. After the addition of DTT and EDTA, the various systems were suspended in solution A. (B) Increasing amounts of HE were incubated with 1251-SV, keeping the virus-cell ratio constant. The unbound virus was washed and the samples were resuspended in solution A, and then various concentrations of DTT and EDTA (2 mM) were added. Number of erythrocytes: 0, 3.75 x 107; *, 7 x 107; A, 14 x 107; K, 28 x 107; A, 42 x 107.
4
.
20
30
40
TEMPERATURE (°C) FIG. 2. Effect of temperature on DTT-induced dissociation of HE-associated SV particles. (A) Samples incubated for 30 min at different temperatures. At the end of the incubation period, DTT and EDTA were added to final concentrations of 50 mM and 2 mM, respectively. (B) DTT and EDTA at final concentrations of 50 mM and 2 mM, respectively, were added, and the samples were first incubated for 10 min at 4°C and then for 30 min at different temperatures. System incubated with (0) or without (0) DTT.
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VOL. 49, 1984
The results clearly show that the percentage of virus particles released from the erythrocyte membrane by DTT was highly dependent on the total number of HE present. The results of the experiment in Fig. 1B confirmed this observation. The quantitative ratio between HE and virus was kept constant, whereas the total amount of HE and, consequently, SV particles was increased. An increase in the number of HE necessitated a parallel increase in DTT concentration for efficient removal (85 to 95%) of the bound virus particles. Effect of temperature and trypsinization of virions. Attachment of virus particles to cell surface receptors is a temperature-independent process, whereas virus-cell fusion is temperature dependent, being optimal at 37°C (4, 11). Therefore, the ability of DTT to release bound SV particles after incubation for 30 min at different temperatures was studied. Several systems containing 23 ,ug of 1251-SV particles were incubated with HE (0.5 ml) at 4°C for 10 min. After unbound virus was removed, the pellets were resuspended in solution A and incubated for 30 min at different temperatures (Fig. 2A). At the end of the incubation period, DTT and EDTA were added to a final concentration of 50 mM and 2 mM, respectively. Unbound virus was removed from the erythrocytes (in the systems incubated at temperatures above 30°C, hemolyzed and fused erythrocytes were obtained) by centrifugation through 0.3 M sucrose (1 ml in solution A) in Eppendorf tubes, as described previously (14). After centrifugation, the amount of radioactive material in the pellet and in the supernatant was determined by a gamma counter. In another experiment (Fig. 2B), DTT and EDTA at final concentrations of 50 mM and 2 mM, respectively, were added and the samples were first incubated for 10 min at 4°C and then for 30 min at the different temperatures. The numbers of free and bound virus were then determined. Up to 85% of the bound virus particles were released by 50 mM DTT from virus-cell complexes incubated for 30 min between 4 and 27°C (Fig. 2A). However, DTT was much less effective when it was added to virus-cell complexes incubated at temperatures higher than 30°C, indicating irreversible binding of the virus particles. This result may be due to fusion between the viral envelopes and the cell plasma membranes (10). The results show that 93% and 62% of the to
cell-associated SV particles remained attached to the HE membranes when 50 mM DTT was added to the virus-cell complexes incubated at 34 or 37°C, respectively. The view that this irreversible, DTT-resistant binding is due to a process of virus-cell fusion is strengthened by the results showing that a high degree of hemolysis was induced by the virus at temperatures higher than 30°C (data not shown; see Table 2). Under the conditions used, hemolysis reflects a process of virus-cell fusion (3, 8). When DTT was added simultaneously with the virus particles to the HE suspension (Fig. 2B), it was more effective in preventing virus-cell binding and fusion than when it was added to already cell-associated virus particles (cf. Fig. 2A and B). Trypsinization of intact SV particles causes selective inactivation of the viral fusogenic factor without affecting its agglutinating ability (13). Intact 1251-SV particles were treated with trypsin essentially as described previously (12). Untreated and trypsinized 125I-SV particles (4.2 pug) were incubated with HE (0.5 ml, 2.5%) for 10 min at 4°C and then for 30 min at 37°C. At the end of the incubation period, unbound virus was removed by centrifugation and the pellets
A 5.0 I-,
0
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0 2.5F C) N
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10 25 B
20
i
C
A
-
5
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0
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z
aC
100
200
300
100
200
300
z
10 lo
cot
0
cn w 2.5F
D
(I)
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200
300 0
100
200
300
125I-SV ADDED (ug) FIG. 3. Use of DIT to differentiate between virus-cell binding and virus-cell fusion. Increasing amounts of '25I-SV incubated with HE (A and B) or HEG (C and D). After 30 min at 4°C (A and C) or 37°C (B and D), DTT and EDTA were added. 125I-SV particles attached before (0) and after (0) addition of DTT.
p
v0
10 125
I-RSVE
20
added (pg)
FIG. 4. DTT-induced dissociation of RSVE from HE. (A) Intact
l251_SV. (B) 125I-RSVE. Particles attached before (-) and after (0)
addition of DTT.
1012
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were resuspended in solution A. DTT and EDTA (50 mM and 2 mM, respectively) were added, and the various systems were transferred to 27°C for 30 min. At the end of the incubation period, the degree of cell-associated SV particles was estimated. Almost the same amount of control or trypsinized virus particles attached to HE at 37°C. Reduction of these virus-cell complexes with DTT resulted in the removal of about 88% of the trypsinized virus particles, but only 25% of the untreated virions. These results clearly indicate that DTT can release bound but not fused particles from HE. Differentiation between virus-cell binding and virus-cell fusion. HE ghosts (HEG) were prepared as previously described (16). Increasing amounts of 1251-SV were incubated with HE (7.3 x 107 cells) or HEG (2 x 108 cells) for 10 min at 4°C. The unattached virus was removed by washing, and the pellets were resuspended in solution A (HE) or solution K (135 mM KCI, 53 mM NaCl, 0.8 mM MgSO4 buffered with 20 mM Tricine, pH 7.8) (HEG). After 30 min at 4 or 370C, DTT and EDTA were added to give a final concentration of 50 mM and 2 mM, respectively, and the systems were further incubated for 15 min at 4°C. After an additional 30-min incubation at room temperature, released and cell-associated radioactive virus numbers were estimated. Incubation of HE with increasing concentrations of virus particles at 4°C resulted in a linear increase in the amount of cell-associated virus particles, reaching saturation at 6.6 ,ug of virus particles per 107 cells (Fig. 3A). As expected, the addition of DTT led to an almost complete removal of the cell-bound virus particles, leaving only 10 to 15% of the maximal number of bound virus at each point. Different results were obtained when DTT was added to virus-cell complexes incubated for 20 min at 37°C (Fig. 3B). As expected, very few or no virus particles were removed. A best-fit straight line for the data shown in Fig. 3B indicates that 7.3 and 7.2 ,ug of 1251-SV particles bound to 107 HE before and after treatment with DTT, respectively. Based on the SV molecular weight of 5 x 108 (16), this gives a maximum of approximately 1,000 to 1,200 bound and 600 to 800 fused viral particles for each erythrocyte. HEG behaved almost identically to intact HE in the number of SV particles that attached to and fused with their membranes (Fig. 3C and D). It is noteworthy that, as TABLE 2. Ability of DTT to remove unfused SV or RSVE
Degree of:
DTT
System
FITC-SV
addition (50 mM)
Hemolysis (% of total)
-
95 90 2-5 2-5 98 86 2-5 2-5
+
FITC-SV with TPCK and TLCK FITC-RSVE
+
+
Fusiona
Furs Fluores-b
+++ ++
+++ ++
-
+
+++
+++
++
++
FITC-RSVE with + TPCK and TLCK + a + + +, Polyerythrocytes or polyghosts of 5 to 15 cells fused with about 70 to 90% of the cells in the population; + +, polyerythrocytes of 5 to 15 cells fused with about 30 to 35% of the cells; -, no fusion apparent. b + + +, Fluorescence rings appeared in 80 to 90% of the erythrocytes or polyerythrocytes in the population; + +, fluorescence rings appeared in 50% of the cells in the population; +, fluorescence rings appeared in 10 to 20% of the cells in the population; -, no fluorescence rings appeared.
opposed to HE, HEG are not fused (cell-cell fusion) by SV particles unless they are loaded with bovine serum albumin or treated with sulfhydryl-blocking reagents (4, 5). Reconstituted Sendai virus envelopes (RSVE) are fusogenic vesicles containing only the two viral envelope glycoproteins (15). Increasing amounts of intact 1251-SV or 1251_ RSVE, prepared from intact 1251-SV particles as previously described (15), were incubated at 4°C with HE for 10 min, and after the cells were washed, DTT and EDTA were added to a final concentration of 50 mM and 2 mM, respectively. The virus-cell complexes were then incubated at 4°C for another 15 min and transferred to room temperature for an additional 30 min. At the end of the incubation period, free and cell-associated SV and RSVE numbers were estimated. The binding characteristics of RSVE to HE were almost the same as those of intact SV particles. Within a certain range of protein concentration, the number of cell-associated RSVE increased linearly, and the addition of DTT (50 mM) resulted in almost complete removal of the RSVE (Fig. 4), regardless of the degree of cell-associated RSVE. The same amount of RSVE was displaced by DTT when it was added to RSVE-cell complexes incubated at temperatures between 4 and 27°C (data not shown). In this temperature range, the amount of RSVE still cell-associated after incubation with DTT was about 15% of the maximal amount (data not shown). As do intact SV particles, RSVE induce a high degree of cell-cell fusion and hemolysis at 37°C, resulting in the formation of polyerythrocyte ghosts and sheared membrane fragments (15). However, because of the low density of the RSVE vesicles, it was impossible to separate quantitatively the displaced RSVE vesicles obtained after treatment with DTT from the erythrocyte membrane fragments. A way to overcome this difficulty and to study the fate of the cellassociated RSVE was to follow their localization by fluorescence microscopy. Incubation of fluorescein-isothiocyanate (FITC) with SV particles, under the conditions developed in our laboratory, led to the formation of highly fluorescent, fully active SV particles. SV particles were labeled with FITC as follows: a suspension of 1 ml of SV particles in solution A (about 3 to 5 mg of protein) was incubated with 1 mg of FITC in 140 mM NaCI-10 mM NaHCO3, pH 9.5, for 10 to 12 h at 40C with gentle shaking. At the end of the incubation period, excess FITC was removed by three sequential passages through a Sephadex G-25 column (5 ml, swollen and suspended in solution A). The eluted virus (FITC-SV) was centrifuged and resuspended in solution A to give 1.5 to 3 mg/ml. Fluorescently labeled RSVE were obtained after solubilizing FITClabeled intact SV particles with Triton X-100 as previously described (15). Both the fluorescently labeled intact virus particles and RSVE were biologically active, i.e., they were able to agglutinate, fuse, and hemolyse HE. Fluorescently labeled SV particles or RSVE (25 ,tg of protein) were added to 0.5 ml of HE (2.5% [vol/vol] in solution A), and the solution was incubated for 10 min at 4°C (for induction of agglutination) and then for 30 min at 37°C (for promotion of hemolysis and cell-cell fusion). L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK) (5 mM) and N-a-p-tosyl-L-lysine chloromethyl ketone (TLCK) (5 mM) were added directly to the reaction mixture containing SV or RSVE and HE (2). The degrees of fusion and fluorescence were then determined (Table 2). Gel electrophoresis analysis of the fluorescently labeled virus particles revealed that the main viral polypeptides became labeled (data not shown). The fluorescently labeled SV particles, or
NOTES 1013
VOL. 49, 1984
RSVE prepared from them, agglutinated HE at 4°C and fused them at 37°C (Table 2). Based on the fluorescence microscopy observations, it appeared that reduction with DTT, as expected, caused only partial displacement of RSVE incubated with HE at 37°C (Table 2). To study whether the DTT-resistant, cell-associated RSVE were fused with the HE membranes, we made use of previous observations showing that inhibitors of proteolytic enzymes block viral fusogenic activity without affecting its binding ability (1). FITC-RSVE as well as FITC-SV treated with inhibitors such as TLCK and TPCK were able to attach to and agglutinate HE at 37°C but could neither promote cell-cell fusion nor induce hemolysis (Table 2). Reduction with DTT caused the complete removal of TLCKand TPCK-treated RSVE or intact SV from the HE even after the virus-cell complexes were incubated at 37°C. These results clearly show that DTT is also useful in removing erythrocyte-adsorbed RSVE and in discriminating between cell-adsorbed and cell-fused RSVE. In the present work we have used a new approach for the release of cell-bound SV particles. With some modifications, the method described here may be applicable also to other members of the paramyxo- or myxovirus groups. It has been well established that the structure of the SV envelope glycoproteins, i.e., the HN and F proteins, is preserved by intermolecular disulfide bonds (9). Incubation of SV particles with mild reducing agents, such as Pmercaptoethanol and DTT, leads to irreversible reduction of the viral HN glycoprotein. Evidently, DTT-reduced virus particles are unable to agglutinate or to infect or fuse eucaryotic cells (9). Indeed, a quantitative determination showed that SV particles treated with 7.5 to 10 mM DTT completely lost their binding ability (data not shown) (9). However, much higher concentrations of DTT were required to dissociate erythrocyte-bound virus particles. This, as shown in the present work, is due to the consumption of a high percentage of the reducing agent by the erythrocytes in the system. Under the conditions used in the present work, only treatment of virus-cell complexes with 50 mM DTT resulted in the release of 85 to 90% of the adsorbed virus particles. The remaining 10 to 15% were probably attached unspecifically to the erythrocytes by a mechanism other than specific virus receptors (16). It is conceivable that a large percentage of the adsorbed virus particles are able to fuse with the erythrocyte membrane. It appears that the main factor which limits fusion between the virus particles and the erythrocyte membranes is the number of virus particles adsorbed. Our results show that maximally about 1 x 103 to 1.2 x 103 virus particles can adsorb to each erythrocyte membrane, of which about 600 to 800 can fuse with it. It should be kept in mind that during incubation at 37°C, about 10 to 30% of the adsorbed virus particles are released from the cell surface due to hydrolysis of the membrane sialic acid residues by the viral neuraminidase. Hence, it appears that penetration of virus particles by a process of virus-cell fusion is controlled, as in endocytosis, mainly by the number of virus receptors available on the cell surface. The method developed in the present work allowed us to demonstrate that HEG can fuse with SV particles to the same extent as intact HE can. In spite of this, fusion of SV with HEG does not lead to promotion of cell-cell fusion (5). From the data in the present work, it seems that RSVE behave very similarly to intact virus particles in their ability to fuse with HE. Attempts are now being made in our
laboratory to study, by the method described here, the ability of SV and its reconstituted envelopes to fuse with plasma membranes of growing cells. It is also possible that most of the adsorbed virus particles, and especially the RSVE, will fuse with the recipient cell plasma membrane. This may pave the way for the use of RSVE as very efficient biological carriers. This work was supported by grant AZ:I/36 082-083 from Stiftung Volkswagenwerk and by grant 2481/81 from the United States-Israel Binational Science Foundation, Jerusalem. LITERATURE CITED 1. Ginsberg, D., D. J. Volsky, N. Zakai, Y. Laster, and A. Loyter.
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