Original Papers Intervirology 11: 158-166(1979)
Reduction of 51Cr-Permeability of Tissue Culture Cells by Infection with Herpes Simplex Virus Type l 1 Jörg R. Schlehofer, Karl-Otto Habermehl, Wolfgang Diefenthal and Hartmut Hampl Institut für Klinische und Experimentelle Virologie, Freie Universität, Berlin
Key Words. Herpes simplex virus type 1 • 51Cr-permeability, cellular membranes Summary. Infection of different strains of tissue culture cells with herpes simplex virus type 1 (HSV-1) resulted in a reduced 51Cr-permeability. A stability of the cellular membrane to Triton X-100, toxic sera and HSV-specific complement-mediated immune-cytolysis could be observed simultaneously. The results differed with respect to the cell strain used in the experiments.
1 This study was supported by the ‘Deutsche For schungsgemeinschaft’. Address inquiries to: Dr. med. Jörg R. Schlehofer, Institut für Klinische und Experimentelle Virologie der W E04, Freie Universität Berlin, Hindenburgdamm 27, D-1000 Berlin 45 Received: April 3, 1978
In this communication we demonstrate that HSV-infected cells are protected not only from immunological injuries but also from other cytotoxic agents.
Materials and Methods Tissue Culture and Media The following cell strains were used: primary embryonic chick fibroblasts (ECF), HEp-2, HeLa, FL, L, Chang-liver, primary rabbit kidney (PRK), KB and BHK21. Growth medium consisted o f Eagle’s MEM (Gibco, Grand Island, N.Y.) supplemented with 1.7 g/1 bicarbonate, 5% newborn calf serum (Flow Labs, FRG; heat-inactivated at 56° for 30 min), and anti biotics (penicillin G, 100 U/ml; streptomycin sulfate, 50 (xg/ml). Mock inoculum consisted of Eagle’s MEM without serum. Viruses The following viruses were used in these experi ments: HSV-1 (a laboratory strain and the HFEM strain; courtesy o f Dr. Schneweis), vaccinia virus (strain
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Herpesviruses change the ‘social behavior’ of infected cells. Depending on the type and strain of the virus, clumping, rounding up, fusion or transformation of cells can be ob served [1,2]. Furthermore, evidence exists for the development of herpes simplex virus (HSV)-induced Fc receptors at the cellular membrane [3, 4], Westmoreland et al. [5] sug gested that these receptors may play a part in the protection of infected cells from cytotoxic antibodies, and that the protection may be mediated by coating the cells with immune complexes or IgG molecules attached by the Fc end.
Bern), poliomyelitis virus type 1 (strain Mahoney), vesicular stomatitis virus (strain Indiana), coxsackie virus B6, and ME (mouse elberfeld). The virus strains were propagated in HEp-2 cells and were assayed in HEp-2, ECF, or L cells by the plaque technique. Triton X-100 Triton X-IOO(Serva, Heidelberg, FRG) was diluted in phosphate-buffered saline (PBS), pH 7.0-7.2, before being added to the growth medium o f the monolayers. Sera Pooled human convalescent sera with antibodies against HSV were obtained courtesy o f Dr. EndersRuckle. The complement hemolytic titerso (indicating 50% hemolysis in the hemolytic system o f the comple ment fixation test) o f the anti-HSV serum was 1:200 (antiserum dilution); antigen dilution, 1:8. Hyperimmune serum against HEp-2 cells was ob tained by immunization o f rabbits with multiple intracutaneous and intramuscular injections o f homogen ized HEp-2 cells in Freund’s adjuvant (Difco, Detroit, Mich.; Miles, III.). The complement hemolytic titerr,o o f the anti-HEp-2 serum was 1:1,000 (serum dilution); antigen dilution, 1:8. Guinea pig serum was obtained by heart puncture of several animals. The complement hemolytic titerjo was 1:128. Toxic sera from guinea pigs were defined by the ability to induce 51Cr-release from cells even after heatinactivation o f the sera. Unless indicated otherwise, 1 ml of Eagle’s MEM was applied per Petri dish. Anti serum or complement were present in final concentra tions o f 1:8. bxCr-assay Confluent monolayer cell cultures in 35-mm Petri dishes containing about 1.2 x 106 cells were incubated for 1 h in 1 ml o f growth medium with 13 p.Ci 51Crsodium chromate (Radiochemical Centre Amersham, England). The supernatant fluid then was removed and the cells were washed 3 times with growth medium. Subsequently, the cells were inoculated with virus at the MOIs indicated in the figures or were mockinfected. The inoculum was removed after 1 h, and the monolayers were washed and incubated with growth medium at 37° in an atmosphere o f 5% COs. At the times indicated in the figures either Triton X-100 or the various sera were added. The release o f 51Cr was measured by counting the activity o f 20- or 50-jxl samples o f the supernatant
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tissue culture fluid in Instagel. The incorporation o f 5ICr into HEp-2 cells was measured by removing the inoculum or mock inoculum from the cells and pulsing for 30 min with 51Cr. The cells then were washed three times with Eagle’s MEM, solved in 0.2 ml formic acid/ dish, and then 0.8 ml aqua dest. and 10 ml Instagel were added. The radioactivity o f the samples was deter mined in the tritium channel o f a Packard Tri-Carb liquid scintillation counter. Equilibrium Dialysis Equilibrium dialysis experiments were performed according to the method described by Nierhaus and Nierhaus{ 6], using chambers containing 100 ¡j.I per side. 51Cr-Iabeled or unlabeled HEp-2 cells were infected
Fig. 1. Significant reduction o f 51Cr-release from HEp-2 cells after infection with HSV-1 and vaccinia virus was observed, in contrast to an enhanced release o f 51Cr after infection with ME, polio-1, coxsackie B6 and vesicular stomatitis viruses. Monolayer cells were labeled with 51Cr for 1 h and then inoculated with virus at the following MOIs (PFU/cell): polio-1, 71.7; cox sackie B 6 ,18.3; vesicular stomatitis virus, 0.5; vaccinia virus, 1.2; HSV-1, 5.0; ME, 23.3. Mock-infected cells served as controls. At the times indicated in the figure the activity o f 51Cr was determined by counting 50-,ul samples of the supernatant culture fluid.
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HSV-Induced Reduction of 51Cr-Permeability
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with HSV-1 (MOI: 2 PFU/cell) or mock-infected as described above. 24 h postinfection (p.i.) the cells were removed with trypsin-EDTA (1 ml/Petri dish) at 37°. Trypsinization was stopped by adding 0.5 ml o f icecold Trasylol (Hoechst, Frankfurt/M., FRG) per Petri dish. After pelleting by low-speed centrifugation (4°) the cells were disrupted by freezing ( —80°) and thawing and were then homogenized by 20 strokes in a Dounce homogenizer (B-pestle) in 1 ml o f PBS. Equilibrium dialysis was performed either with debris o f labeled HSV-1 - or mock-infected cells against PBS or, inversely, with debris of unlabeled infected or uninfected cells against 51Cr-containing PBS. The dialysis chambers were continuously shaken at room temperature up to 24 h. Samples (8 ¡jl!) were taken after 0.3,1, 5, and 24 h. Equilibrium was reached within 1 h. Statistics Reduction o f 51Cr-release was considered positive when a statistically significant reduction (p=0.01 by Student’s t test) was observed.
. Schlehofer/Habermehl/Diefenthal/Hampl
Results Reduction of 51Cr-Release in HSV-1-Infected Cells The infection of HEp-2 cells with HSV or vaccinia virus led to a significant reduction of 51Cr-release (fig. 1). This effect began 6-8 h after inoculation. These findings were in con trast to the typical enhancement of slCrrelease after infection with ME, polio-1, coxsackie B6 and vesicular stomatitis viruses. The HSV-1-induced reduction of slCrrelease from HEp-2 cells also could be observed in six other cell strains (fig. 2): Chang-liver, FL, BHK 21, PRK, HeLa and KB. In ECF cells, however, HSV infection caused an enhance ment of 51Cr-release.
Reduction of slCr-Incorporation in HSV-1-Infected HEp-2 Cells
Support for the Validity of the Reduction of 51Cr-Permeability 61Cr-release was the same in HSV-1- and mock-infected cells during the inoculation period (1 h). This indicated that no radio activity was lost during events early in virus infection. Assuming the radioactivity of the
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Fig. 2. Reduction o f 51Cr-release from six different cell strains by infection with HSV. Contrasting effect in ECF cells. Statistically significant: p=0.01 for Chang-liver, HEp-2, FL, PRK, and HeLa. The results are expressed as percent 51Cr-release from mockinfected cells o f the corresponding cell strain (100% = ----- ). Monolayer cells were labeled with 51Cr for 1 h, washed three times, and subsequently inoculated for 1 h with HSV-1 at an MOI o f 44.3 PFU/cell. 24 h after inoculation the activity o f slCr was determined by counting 20-¡xl samples o f the supernatant culture fluid. The data represent the average o f triplicate experi ments.
The HSV-induced reduction of 51Cr-release indicated an altered permeability of the cel lular membrane. This assumption was con firmed by a reduced incorporation of slCr (fig.3). The effect began 8 h p.i. and corre sponded temporally with the onset of the re duction of 51Cr-release and with the logarith mic increase of HSV reproduction (compare fig. 1 and fig. 6A).
HSV-Induced Reduction of 51Cr-Permeability
Stability of the Membranes of HSV-1Infected Cells against Different Injuries Resistance o f HSV-l-Infected Cells to Triton X-100 The Triton-induced injury of eight cell strains was determined by the release of 51Cr (fig.4A). Using a low concentration of Triton X-100 (0.09 mM=0.005%), different sensi tivities of the cell strains could be observed.
Table I. Equilibrium dialysis: percentage o f the shift o f equilibrium toward the chamber containing cell debris1 Cells
Pretreated (51Cr) cells dialyzed against PBS %
Untreated cells dialyzed against PBS containing 51Cr %
Mock-Infected HSV-l-infected
21 7
19 5
1 Equilibrium =0% difference between both chantbers. For details see ‘Materials and Methods’.
F ig.3. Incorporation o f 51Cr into HSV-l-infected HEp-2 cells ( • ) . Beginning 8 h after infection, a reduc tion o f 31Cr-incorporation could be observed. Monolayer cells were infected with HSV-1 at an MOI o f 4.72 PFU/cell. Before the times indicated in the figure, the cells were pulsed for 30 min with 51Cr. After removal o f the pulse medium, the cells were washed three times with Eagle’s MEM, dissolved in 0.2 ml formic acid for 30 min, and counted for radioactivity. Mock-infected cells, treated in the same manner, served as controls
(o).
BHKei, PRK and ECF cells were resistant to the detergent; they showed the same amount of 51Cr-reIease as the nontreated control cells. HSV-1 infection of theTriton-sensitivecells (HEp-2, Chang-liver, KB, HeLa, FL) pre vented the Triton-induced enhancement of 51Cr-release (fig.4B). The findings suggested a virus-induced alteration of the cellular mem brane resulting in an increased stability against Triton X-100. The effect could not be demon strated in BHK 21, PRK and ECF cells, since these cell strains were not sensitive to Triton X-100 (fig.4A). The high level of activity ob tained from ECF cells could be explained by the enhancement of 51Cr-release after infection with HSV-1 (fig. 2). Experiments with a lower concentration of Triton X-100 (0.04 m M = 0.0025%) showed
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cells plus supernatant as total activity (100%), 51Cr-release in mock-infected cells was 21% (24 h p.i.) and in HSV-1-infected cells was 15%. Hence, 5lCr-release in HSV-1 infected cells was reduced by 29%. Equilibrium dialysis data revealed that fragmented HSV-l-infected HEp-2 cells had a lower affinity to 51Cr than mock-infected cells (table I). This lower affinity could be demon strated by dialyzing unlabeled cell debris against PBS containing 51Cr, as well as in the inverse test with debris of labeled cells dialyzed against PBS. These results excluded a binding of 51Cr to HSV-l-induced components in in fected HEp-2 cells.
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.Schlehofer/Habermehl/Diefcnthal/Hampl
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the same HSV-induced resistance in all tested cells. A representative experiment with HEp-2 cells is shown in figure 5. Resistance o f HSV-1-Infected HEp-2 Cells to Toxic Serum Toxic guinea pig serum (active or inactivat ed) caused an increased release of 5ICr in non infected HEp-2 or ECF cells. Infection with HSV-1 prevented this effect in HEp-2 cells but not in ECF cells (fig.6). The HSV-l-induced reduction of slCr-release from HEp-2 cells began 8 h p.i., corresponding to the logarith mic increase of the viral reproduction cycle (fig. 6A).
F ig.4. Prevention o f Triton-X-100-induced en hancement o f 51Cr-release by HSV-1 infection. A Dif ferent sensitivities of 8 noninfected cell strains to Triton X-100 (statistically significant: p=0.01 for HEp-2, Chang-liver, KB, HcLa and FL). Results are expressed as percent 5lCr-relcase from untreated, mock-infected cells (100% =-----). Cells were 5ICrlabeled, mock-infected, and Triton-treated as described below. B Prevention ofTriton-X-100-induced enhance ment o f 51Cr-release from HEp-2, Chang-liver, KB, HeLa and FL cells by infection with HSV-I (statisti cally significant). This effect could not be demonstrated in BHK21, PRK and ECF cells, since these cells were not sensitive to Triton X-100, as shown in A. The high level o f activity obtained with ECF cells could be ex plained by enhancement of 51Cr-release from this cell line after infection with HSV-1 (fig.2). Results are ex pressed as percent of 5lCr-release from Triton-treated, mock-infected cells (100% =----- ). Monolayer cells were labeled with 51Cr for 1 h, washed three times, and subsequently inoculated for 1 h with HSV-1 at an aver age MOI o f 44. 3 PFU/cell or mock-inoculated. 24 h after inoculation, the activity o f 51Cr was determined by counting 20-pt.l samples o f the supernatant culture fluid. Triton X-100 was added at a final concentration o f 0.09 mM 8 h after infection or mock-infection.
Fig. 5. Prevention o f Triton-X-100-induced en hancement of release o f 51Cr from HEp-2 cells by in fection with HSV-1. Mock-infected cells served as con trols. The arrow indicates the addition o f Triton X-100 8 h after inoculation (at concentrations of 0.09 or 0.04 mM ). Monolayer cells were labeled with 51Cr for 1 h, washed three times, and subsequently inoculated for 1 h with HSV-1 at an MOI o f 1.61 PFU/cell. At the times indicated in the figure, the activity o f 51Cr was determined by counting 20-|xl samples o f the super natant culture fluid.
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B
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Fig. 6. Effect of toxic serum on 51Cr-release in HSV-l-infcctcd cells. Active (S) or heat-inactivated (Sh) toxic guinea pig sera enhanced 51Cr-release in noninfected (CON TROL) cells (A, HEp-2; B, ECF). HSV infection prevented this toxic effect in HEp-2 cells (A, HSV + S, HSV + Sii) but not in ECF cells (B). Furthermore, the HSV-induced reduction o f 51Crrelease from HEp-2 cells started approximately 8 h after infection (A: noninfected cells, CONTROL; in fected cells, HSV). Monolayer cells were labeled with 51Cr for 1 h, washed three times, and subsequently in oculated for 1 h with HSV-1 at MOIs of 4.72 PFU/cell (A, HEp-2 cells) and 1.61 PFU/cell (B, ECF cells). 8 h after infection (indicated by the arrows), 100 ul/ml toxic guinea pig serum was added. The activity o f 5lCr was measured in 20-{a1 samples o f the supernatant cul ture fluid. Mock-infected cells served as controls. HSV-1 reproduction was assayed by inoculation of HEp-2 cells with an MOI o f 44.3 PFU/cell. After ad sorption for 1 h the cells were washed two times, eluted for 30 min, and washed two more times. At the times indicated in A, the culture fluid and the cells were col lected and the reproduction o f released virus (R.V.) and cell-associated virus (A.V.) was determined by plaque titration on ECF. Cells were sonicated before titration.
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HSV-Induced Reduction of 5ICr-Permeability
Schlehofer/Habermehl/Diefenthal/Hampl
Fig. 7. A Inhibition o f the HSV-specific comple ment-mediated immune-cytolysis in HSV-l-infected HEp-2 cells. N o enhancement o f 51Cr-release from HSV-l-infected HEp-2 cells (HSV) was observed after addition o f heat-inactivated HSV guinea pig antiserum (At) and active complement (C). B Control o f the HSVspecific immune-cytolytic system. HSV-infected ECF cells (HSV) showed the characteristic enhancement of s lCr-release after addition o f heat-inactivated HSV antiserum (Ai) and active complement (C). Monolayer cells were labeled with 6ICr for 1 h, washed three times,
and inoculated for 1 h with HSV-I at an MOI o f 0.18 PFU/cell. 12 h after inoculation (time zero in the figure) the cells were incubated with HSV-1 antiserum and guinea pig complement in the combinations indicated in the figure. Aj=Heat-inactivated HSV-antiserum; C =active guinea pig complement; Ci = heat-inacti vated guinea pig complement; NCSi = heat-inactivated newborn calf serum. Mock-infected cells served as controls (CONTROL). 51Cr activity was measured by counting 20-jxI samples o f the supernatant culture fluid.
Resistance o f HSV-l-Infected HEp-2 Cells to HSV-Specific Complement-Mediated Immune-Cytolysis HEp-2 or HeLa cells infected with HSV-1 showed no cytolysis after exposure to human or guinea pig HSV antiserum and guinea pig complement (fig.7A). Cytolysis did not occur, even with high titers of HSV antisera and complement (see ‘Materials and Methods’). The failure of the immune-cytolysis depended on the virus-cell system. Using another cell system (ECF) (fig. 7B) with the same HSV anti
serum and complement, HSV-specific immune-cytolosis could be demonstrated by the characteristic enhancement of 51Cr-release. To determine if HEp-2 cells were insensitive to immune-cytolysis in a general sense, noninfected HEp-2 cells were treated with HEp-2 antiserum and complement. Under these con ditions a significant cytolysis could be demon strated. In contrast to the experiments men tioned above, this cell-specific immune-cyto lysis could not be prevented by infection with HSV-1 (fig. 8).
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Fig. 8. Demonstration o f a cell-specific comple ment-mediated immune-cytolysis in HEp-2 cells (A) and enhancement o f this effect by HSV-1 infection (A). Monolayer cells were labeled with 51Cr for 1 h, washed three times, and inoculated for 1 h with HSV-1 at an MOI of0.18 PFU./cell. 12 h after inoculation (time zero in the figure), the cells were incubated with rabbit HEp-2 antiserum in the presence of guinea pig comple ment in the combinations indicated in the figure. A i= Heat-inactivated HEp-2 antiserum; C =active comple ment. Mock-infcctcd cells served as controls (0 ). 51Crrelease was determined by counting the activity o f 20-p.l samples o f the supernatant culture fluid.
Discussion Infection with HSV-1 induces a significant change in the stability of the cellular mem branes. Determination of membrane fragility or cell lysis was performed by the 51Cr-release assay [7,8]. In contrast to other cytocidal viruses, which cause a characteristic enhancement of 51Crrelease from HEp-2 cells, HSV-1 infection leads to a reduction of the release and uptake of 51Cr. As demonstrated by 51Cr-release in the inocu lation period, this effect is not induced by a
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loss of 51Cr during HSV penetration or other events in the early stages of infection. Further more, it could be excluded by equilibrium dia lysis that 51Cr was preferentially bound to HSV-specified components of infected cells. The lower binding of 51Cr to HSV-1-infected cells than to uninfected control cells must be considered as evidence for the HSV-induced reduction of cell membrane permeability. This finding of a lower binding of 51Cr to debris of HSV-infected cells will be further investigated. Vaccinia virus, characterized by CPE simi lar to the HSV strains used in these experi ments, demonstrated a similar reduction of 51Cr-release. With the exception of ECF cells, this effect could be observed in permanent as well as in primary or transformed cell systems, and was independent of the MOI. The reduction of the release and uptake of 51Cr began 8 h after infection with HSV-1, corresponding to the logarithmic increase of virus reproduction. At this time, under our experimental conditions large amounts of HSV-1 antigen could be demonstrated by immunofluorescence. The discrepancy of our results with the findings of others concerning immune-cytolysis [9-11] may depend on the different behavior of the various cell-virus systems. In order to study if the observed reduction of permeability was correlated with a stabili zation of the membrane of the infected cell, the sensitivity of cells toward Triton X-100 and complement-mediated immune-cytolysis was tested. With the exception of ECF cells, HSV1 induced an increased stability of the cells toward the detergent. Furthermore, the effect of toxic sera on HEp-2 cells was prevented by HSV-1 infection, and immune-cytolysis, using HSV-1 antibodies, did not occur in infected HEp-2 cells. The lack of complement-medi ated immune-cytolysis and the stability toward
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HSV-Induced Reduction of 51Cr-Permeability
Triton X-100 and toxic sera indicated a de crease in fragility of the cells [12]. The results give no explanation of the mechanism of the HSV-l-induced stability of the cellular membrane. One might assume that newly synthesized viral antigens or virus-in duced Fc-receptors [13] on the cell surface were involved in the alteration of the membrane, resulting in a resistance toward immunologic and other cytolytic agents. Graham el al. [12] stated that ‘a thickening or folding of the plas ma membrane’ might be responsible for the decrease in cell fragility. This explanation must be judged in comparison with the observation that the reaction with other antigenic determi nants (treatment with anti-HEp-2 antibodies instead of anti-HSV-1 antibodies) is followed by an enhanced immune cytolysis. This result suggests a different location of the antigens in the cells and/or a modification of the receptor sites by HSV-1. It cannot be excluded that this effect could be due to differences in the species of origin of the antibodies. The HSV-induced stability of cellular mem branes may play a part in maintaining latent infections. Acknowledgments We are grateful to Miss Hanna Voss for her excellent assistance.
References 1 Keller, J.M .; Spear, P.G., and Roizman, B.: Pro teins specified by herpes simplex virus. III. Viruses differing in their effects on the social behavior of infected cells specify different membrane glyco proteins. Proc.natn.Acad.Sci. USA 65: 865-871 (1970).
2 Duff, R. and Rapp, F.: Properties o f hamster embryo fibroblasts transformed in vitro after ex posure to ultraviolet-irradiated herpes simplex virus type 2. J. Virol. 8: 469-477 (1971). 3 Watkins, J. F .: Adsorption o f sensitized sheep ery throcytes to HeLa cells infected with herpes simplex virus. Nature, Lond. 202: 1364-1365 (1964). 4 Westmoreland, D. and Watkins, J.F.: The IgG receptor induced by herpes simplex virus: studies using radioiodinated IgG. J.gen. Virol. 24: 167-178 (1974) . 5 Westmoreland, D.; Watkins, J.F., and Rapp, F.: Demonstration o f a receptor for IgG in Syrian hamster cells transformed with herpes simplex virus. J. gen. Virol. 25: 167-170(1974). 6 Nierhaus, D. and Nierhaus, K .H .: Identification o f the chloramphenicol-binding protein in Esche richia coli ribosomes by partial reconstitution. Proc. natn. Acad. Sci. USA 70: 2224-2228 (1973). 7 Wigzell, H.: Quantitative titrations o f mouse H-2 antibodies using Cr51-Iabelled target cells. Trans plantation 3: 423-431 (1965). 8 Hersey, P .; Edwards, J .; Edwards, A .; Adams, E.; Kearney, R., and Milton, G.W .: Comparison of 51Cr release and microcytotoxicity assays against human melanoma cells. Int.J. Cancer 16: 164-172 (1975) . 9 Brier,A. M .; Wohlenberg, C .; Rosenthal, J .; Mage, M., and Notkins, A. L .: Inhibition or enhancement o f immunological injury o f virus-infected cells. Proc.natn.Acad.Sci. USA 68: 3073-3077 (1971). 10 Snyderman, R.; Wohlenberg, C., and Notkins, A.L.: Inflammation and viral infection: chemotactic activity resulting from the interaction of anti viral antibody and complement with cells infected with herpes simplex virus. J. infect. Dis. 126: 207209 (1972). 11 Plummer, G.: A review of the identification and titration o f antibodies to herpes simplex viruses type 1 and type 2 in human sera. Cancer Res. 33: 1469-1476(1973). 12 Graham, J.M .; Sumner, M .C .B.; Curtis, D .H ., and Pasternak, C. A .: Sequence o f events in plasma membrane assembly during the cell cycle. Nature, Lond. 246: 291-295 (1973). 13 Kerbel, R.S.: Herpes virus induction o f Fc recep tors. Nature, Lond. 263: 192 (1976).
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Reduction of 51Cr-Permeability of Tissue Culture Cells by Infection with Herpes Simplex Virus Type l 1 Jörg R. Schlehofer, Karl-Otto Habermehl, Wolfgang Diefenthal and Hartmut Hampl Institut für Klinische und Experimentelle Virologie, Freie Universität, Berlin
Key Words. Herpes simplex virus type 1 • 51Cr-permeability, cellular membranes Summary. Infection of different strains of tissue culture cells with herpes simplex virus type 1 (HSV-1) resulted in a reduced 51Cr-permeability. A stability of the cellular membrane to Triton X-100, toxic sera and HSV-specific complement-mediated immune-cytolysis could be observed simultaneously. The results differed with respect to the cell strain used in the experiments.
1 This study was supported by the ‘Deutsche For schungsgemeinschaft’. Address inquiries to: Dr. med. Jörg R. Schlehofer, Institut für Klinische und Experimentelle Virologie der W E04, Freie Universität Berlin, Hindenburgdamm 27, D-1000 Berlin 45 Received: April 3, 1978
In this communication we demonstrate that HSV-infected cells are protected not only from immunological injuries but also from other cytotoxic agents.
Materials and Methods Tissue Culture and Media The following cell strains were used: primary embryonic chick fibroblasts (ECF), HEp-2, HeLa, FL, L, Chang-liver, primary rabbit kidney (PRK), KB and BHK21. Growth medium consisted o f Eagle’s MEM (Gibco, Grand Island, N.Y.) supplemented with 1.7 g/1 bicarbonate, 5% newborn calf serum (Flow Labs, FRG; heat-inactivated at 56° for 30 min), and anti biotics (penicillin G, 100 U/ml; streptomycin sulfate, 50 (xg/ml). Mock inoculum consisted of Eagle’s MEM without serum. Viruses The following viruses were used in these experi ments: HSV-1 (a laboratory strain and the HFEM strain; courtesy o f Dr. Schneweis), vaccinia virus (strain
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Herpesviruses change the ‘social behavior’ of infected cells. Depending on the type and strain of the virus, clumping, rounding up, fusion or transformation of cells can be ob served [1,2]. Furthermore, evidence exists for the development of herpes simplex virus (HSV)-induced Fc receptors at the cellular membrane [3, 4], Westmoreland et al. [5] sug gested that these receptors may play a part in the protection of infected cells from cytotoxic antibodies, and that the protection may be mediated by coating the cells with immune complexes or IgG molecules attached by the Fc end.
Bern), poliomyelitis virus type 1 (strain Mahoney), vesicular stomatitis virus (strain Indiana), coxsackie virus B6, and ME (mouse elberfeld). The virus strains were propagated in HEp-2 cells and were assayed in HEp-2, ECF, or L cells by the plaque technique. Triton X-100 Triton X-IOO(Serva, Heidelberg, FRG) was diluted in phosphate-buffered saline (PBS), pH 7.0-7.2, before being added to the growth medium o f the monolayers. Sera Pooled human convalescent sera with antibodies against HSV were obtained courtesy o f Dr. EndersRuckle. The complement hemolytic titerso (indicating 50% hemolysis in the hemolytic system o f the comple ment fixation test) o f the anti-HSV serum was 1:200 (antiserum dilution); antigen dilution, 1:8. Hyperimmune serum against HEp-2 cells was ob tained by immunization o f rabbits with multiple intracutaneous and intramuscular injections o f homogen ized HEp-2 cells in Freund’s adjuvant (Difco, Detroit, Mich.; Miles, III.). The complement hemolytic titerr,o o f the anti-HEp-2 serum was 1:1,000 (serum dilution); antigen dilution, 1:8. Guinea pig serum was obtained by heart puncture of several animals. The complement hemolytic titerjo was 1:128. Toxic sera from guinea pigs were defined by the ability to induce 51Cr-release from cells even after heatinactivation o f the sera. Unless indicated otherwise, 1 ml of Eagle’s MEM was applied per Petri dish. Anti serum or complement were present in final concentra tions o f 1:8. bxCr-assay Confluent monolayer cell cultures in 35-mm Petri dishes containing about 1.2 x 106 cells were incubated for 1 h in 1 ml o f growth medium with 13 p.Ci 51Crsodium chromate (Radiochemical Centre Amersham, England). The supernatant fluid then was removed and the cells were washed 3 times with growth medium. Subsequently, the cells were inoculated with virus at the MOIs indicated in the figures or were mockinfected. The inoculum was removed after 1 h, and the monolayers were washed and incubated with growth medium at 37° in an atmosphere o f 5% COs. At the times indicated in the figures either Triton X-100 or the various sera were added. The release o f 51Cr was measured by counting the activity o f 20- or 50-jxl samples o f the supernatant
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tissue culture fluid in Instagel. The incorporation o f 5ICr into HEp-2 cells was measured by removing the inoculum or mock inoculum from the cells and pulsing for 30 min with 51Cr. The cells then were washed three times with Eagle’s MEM, solved in 0.2 ml formic acid/ dish, and then 0.8 ml aqua dest. and 10 ml Instagel were added. The radioactivity o f the samples was deter mined in the tritium channel o f a Packard Tri-Carb liquid scintillation counter. Equilibrium Dialysis Equilibrium dialysis experiments were performed according to the method described by Nierhaus and Nierhaus{ 6], using chambers containing 100 ¡j.I per side. 51Cr-Iabeled or unlabeled HEp-2 cells were infected
Fig. 1. Significant reduction o f 51Cr-release from HEp-2 cells after infection with HSV-1 and vaccinia virus was observed, in contrast to an enhanced release o f 51Cr after infection with ME, polio-1, coxsackie B6 and vesicular stomatitis viruses. Monolayer cells were labeled with 51Cr for 1 h and then inoculated with virus at the following MOIs (PFU/cell): polio-1, 71.7; cox sackie B 6 ,18.3; vesicular stomatitis virus, 0.5; vaccinia virus, 1.2; HSV-1, 5.0; ME, 23.3. Mock-infected cells served as controls. At the times indicated in the figure the activity o f 51Cr was determined by counting 50-,ul samples of the supernatant culture fluid.
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with HSV-1 (MOI: 2 PFU/cell) or mock-infected as described above. 24 h postinfection (p.i.) the cells were removed with trypsin-EDTA (1 ml/Petri dish) at 37°. Trypsinization was stopped by adding 0.5 ml o f icecold Trasylol (Hoechst, Frankfurt/M., FRG) per Petri dish. After pelleting by low-speed centrifugation (4°) the cells were disrupted by freezing ( —80°) and thawing and were then homogenized by 20 strokes in a Dounce homogenizer (B-pestle) in 1 ml o f PBS. Equilibrium dialysis was performed either with debris o f labeled HSV-1 - or mock-infected cells against PBS or, inversely, with debris of unlabeled infected or uninfected cells against 51Cr-containing PBS. The dialysis chambers were continuously shaken at room temperature up to 24 h. Samples (8 ¡jl!) were taken after 0.3,1, 5, and 24 h. Equilibrium was reached within 1 h. Statistics Reduction o f 51Cr-release was considered positive when a statistically significant reduction (p=0.01 by Student’s t test) was observed.
. Schlehofer/Habermehl/Diefenthal/Hampl
Results Reduction of 51Cr-Release in HSV-1-Infected Cells The infection of HEp-2 cells with HSV or vaccinia virus led to a significant reduction of 51Cr-release (fig. 1). This effect began 6-8 h after inoculation. These findings were in con trast to the typical enhancement of slCrrelease after infection with ME, polio-1, coxsackie B6 and vesicular stomatitis viruses. The HSV-1-induced reduction of slCrrelease from HEp-2 cells also could be observed in six other cell strains (fig. 2): Chang-liver, FL, BHK 21, PRK, HeLa and KB. In ECF cells, however, HSV infection caused an enhance ment of 51Cr-release.
Reduction of slCr-Incorporation in HSV-1-Infected HEp-2 Cells
Support for the Validity of the Reduction of 51Cr-Permeability 61Cr-release was the same in HSV-1- and mock-infected cells during the inoculation period (1 h). This indicated that no radio activity was lost during events early in virus infection. Assuming the radioactivity of the
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Fig. 2. Reduction o f 51Cr-release from six different cell strains by infection with HSV. Contrasting effect in ECF cells. Statistically significant: p=0.01 for Chang-liver, HEp-2, FL, PRK, and HeLa. The results are expressed as percent 51Cr-release from mockinfected cells o f the corresponding cell strain (100% = ----- ). Monolayer cells were labeled with 51Cr for 1 h, washed three times, and subsequently inoculated for 1 h with HSV-1 at an MOI o f 44.3 PFU/cell. 24 h after inoculation the activity o f slCr was determined by counting 20-¡xl samples o f the supernatant culture fluid. The data represent the average o f triplicate experi ments.
The HSV-induced reduction of 51Cr-release indicated an altered permeability of the cel lular membrane. This assumption was con firmed by a reduced incorporation of slCr (fig.3). The effect began 8 h p.i. and corre sponded temporally with the onset of the re duction of 51Cr-release and with the logarith mic increase of HSV reproduction (compare fig. 1 and fig. 6A).
HSV-Induced Reduction of 51Cr-Permeability
Stability of the Membranes of HSV-1Infected Cells against Different Injuries Resistance o f HSV-l-Infected Cells to Triton X-100 The Triton-induced injury of eight cell strains was determined by the release of 51Cr (fig.4A). Using a low concentration of Triton X-100 (0.09 mM=0.005%), different sensi tivities of the cell strains could be observed.
Table I. Equilibrium dialysis: percentage o f the shift o f equilibrium toward the chamber containing cell debris1 Cells
Pretreated (51Cr) cells dialyzed against PBS %
Untreated cells dialyzed against PBS containing 51Cr %
Mock-Infected HSV-l-infected
21 7
19 5
1 Equilibrium =0% difference between both chantbers. For details see ‘Materials and Methods’.
F ig.3. Incorporation o f 51Cr into HSV-l-infected HEp-2 cells ( • ) . Beginning 8 h after infection, a reduc tion o f 31Cr-incorporation could be observed. Monolayer cells were infected with HSV-1 at an MOI o f 4.72 PFU/cell. Before the times indicated in the figure, the cells were pulsed for 30 min with 51Cr. After removal o f the pulse medium, the cells were washed three times with Eagle’s MEM, dissolved in 0.2 ml formic acid for 30 min, and counted for radioactivity. Mock-infected cells, treated in the same manner, served as controls
(o).
BHKei, PRK and ECF cells were resistant to the detergent; they showed the same amount of 51Cr-reIease as the nontreated control cells. HSV-1 infection of theTriton-sensitivecells (HEp-2, Chang-liver, KB, HeLa, FL) pre vented the Triton-induced enhancement of 51Cr-release (fig.4B). The findings suggested a virus-induced alteration of the cellular mem brane resulting in an increased stability against Triton X-100. The effect could not be demon strated in BHK 21, PRK and ECF cells, since these cell strains were not sensitive to Triton X-100 (fig.4A). The high level of activity ob tained from ECF cells could be explained by the enhancement of 51Cr-release after infection with HSV-1 (fig. 2). Experiments with a lower concentration of Triton X-100 (0.04 m M = 0.0025%) showed
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cells plus supernatant as total activity (100%), 51Cr-release in mock-infected cells was 21% (24 h p.i.) and in HSV-1-infected cells was 15%. Hence, 5lCr-release in HSV-1 infected cells was reduced by 29%. Equilibrium dialysis data revealed that fragmented HSV-l-infected HEp-2 cells had a lower affinity to 51Cr than mock-infected cells (table I). This lower affinity could be demon strated by dialyzing unlabeled cell debris against PBS containing 51Cr, as well as in the inverse test with debris of labeled cells dialyzed against PBS. These results excluded a binding of 51Cr to HSV-l-induced components in in fected HEp-2 cells.
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the same HSV-induced resistance in all tested cells. A representative experiment with HEp-2 cells is shown in figure 5. Resistance o f HSV-1-Infected HEp-2 Cells to Toxic Serum Toxic guinea pig serum (active or inactivat ed) caused an increased release of 5ICr in non infected HEp-2 or ECF cells. Infection with HSV-1 prevented this effect in HEp-2 cells but not in ECF cells (fig.6). The HSV-l-induced reduction of slCr-release from HEp-2 cells began 8 h p.i., corresponding to the logarith mic increase of the viral reproduction cycle (fig. 6A).
F ig.4. Prevention o f Triton-X-100-induced en hancement o f 51Cr-release by HSV-1 infection. A Dif ferent sensitivities of 8 noninfected cell strains to Triton X-100 (statistically significant: p=0.01 for HEp-2, Chang-liver, KB, HcLa and FL). Results are expressed as percent 5lCr-relcase from untreated, mock-infected cells (100% =-----). Cells were 5ICrlabeled, mock-infected, and Triton-treated as described below. B Prevention ofTriton-X-100-induced enhance ment o f 51Cr-release from HEp-2, Chang-liver, KB, HeLa and FL cells by infection with HSV-I (statisti cally significant). This effect could not be demonstrated in BHK21, PRK and ECF cells, since these cells were not sensitive to Triton X-100, as shown in A. The high level o f activity obtained with ECF cells could be ex plained by enhancement of 51Cr-release from this cell line after infection with HSV-1 (fig.2). Results are ex pressed as percent of 5lCr-release from Triton-treated, mock-infected cells (100% =----- ). Monolayer cells were labeled with 51Cr for 1 h, washed three times, and subsequently inoculated for 1 h with HSV-1 at an aver age MOI o f 44. 3 PFU/cell or mock-inoculated. 24 h after inoculation, the activity o f 51Cr was determined by counting 20-pt.l samples o f the supernatant culture fluid. Triton X-100 was added at a final concentration o f 0.09 mM 8 h after infection or mock-infection.
Fig. 5. Prevention o f Triton-X-100-induced en hancement of release o f 51Cr from HEp-2 cells by in fection with HSV-1. Mock-infected cells served as con trols. The arrow indicates the addition o f Triton X-100 8 h after inoculation (at concentrations of 0.09 or 0.04 mM ). Monolayer cells were labeled with 51Cr for 1 h, washed three times, and subsequently inoculated for 1 h with HSV-1 at an MOI o f 1.61 PFU/cell. At the times indicated in the figure, the activity o f 51Cr was determined by counting 20-|xl samples o f the super natant culture fluid.
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B
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Fig. 6. Effect of toxic serum on 51Cr-release in HSV-l-infcctcd cells. Active (S) or heat-inactivated (Sh) toxic guinea pig sera enhanced 51Cr-release in noninfected (CON TROL) cells (A, HEp-2; B, ECF). HSV infection prevented this toxic effect in HEp-2 cells (A, HSV + S, HSV + Sii) but not in ECF cells (B). Furthermore, the HSV-induced reduction o f 51Crrelease from HEp-2 cells started approximately 8 h after infection (A: noninfected cells, CONTROL; in fected cells, HSV). Monolayer cells were labeled with 51Cr for 1 h, washed three times, and subsequently in oculated for 1 h with HSV-1 at MOIs of 4.72 PFU/cell (A, HEp-2 cells) and 1.61 PFU/cell (B, ECF cells). 8 h after infection (indicated by the arrows), 100 ul/ml toxic guinea pig serum was added. The activity o f 5lCr was measured in 20-{a1 samples o f the supernatant cul ture fluid. Mock-infected cells served as controls. HSV-1 reproduction was assayed by inoculation of HEp-2 cells with an MOI o f 44.3 PFU/cell. After ad sorption for 1 h the cells were washed two times, eluted for 30 min, and washed two more times. At the times indicated in A, the culture fluid and the cells were col lected and the reproduction o f released virus (R.V.) and cell-associated virus (A.V.) was determined by plaque titration on ECF. Cells were sonicated before titration.
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Fig. 7. A Inhibition o f the HSV-specific comple ment-mediated immune-cytolysis in HSV-l-infected HEp-2 cells. N o enhancement o f 51Cr-release from HSV-l-infected HEp-2 cells (HSV) was observed after addition o f heat-inactivated HSV guinea pig antiserum (At) and active complement (C). B Control o f the HSVspecific immune-cytolytic system. HSV-infected ECF cells (HSV) showed the characteristic enhancement of s lCr-release after addition o f heat-inactivated HSV antiserum (Ai) and active complement (C). Monolayer cells were labeled with 6ICr for 1 h, washed three times,
and inoculated for 1 h with HSV-I at an MOI o f 0.18 PFU/cell. 12 h after inoculation (time zero in the figure) the cells were incubated with HSV-1 antiserum and guinea pig complement in the combinations indicated in the figure. Aj=Heat-inactivated HSV-antiserum; C =active guinea pig complement; Ci = heat-inacti vated guinea pig complement; NCSi = heat-inactivated newborn calf serum. Mock-infected cells served as controls (CONTROL). 51Cr activity was measured by counting 20-jxI samples o f the supernatant culture fluid.
Resistance o f HSV-l-Infected HEp-2 Cells to HSV-Specific Complement-Mediated Immune-Cytolysis HEp-2 or HeLa cells infected with HSV-1 showed no cytolysis after exposure to human or guinea pig HSV antiserum and guinea pig complement (fig.7A). Cytolysis did not occur, even with high titers of HSV antisera and complement (see ‘Materials and Methods’). The failure of the immune-cytolysis depended on the virus-cell system. Using another cell system (ECF) (fig. 7B) with the same HSV anti
serum and complement, HSV-specific immune-cytolosis could be demonstrated by the characteristic enhancement of 51Cr-release. To determine if HEp-2 cells were insensitive to immune-cytolysis in a general sense, noninfected HEp-2 cells were treated with HEp-2 antiserum and complement. Under these con ditions a significant cytolysis could be demon strated. In contrast to the experiments men tioned above, this cell-specific immune-cyto lysis could not be prevented by infection with HSV-1 (fig. 8).
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Fig. 8. Demonstration o f a cell-specific comple ment-mediated immune-cytolysis in HEp-2 cells (A) and enhancement o f this effect by HSV-1 infection (A). Monolayer cells were labeled with 51Cr for 1 h, washed three times, and inoculated for 1 h with HSV-1 at an MOI of0.18 PFU./cell. 12 h after inoculation (time zero in the figure), the cells were incubated with rabbit HEp-2 antiserum in the presence of guinea pig comple ment in the combinations indicated in the figure. A i= Heat-inactivated HEp-2 antiserum; C =active comple ment. Mock-infcctcd cells served as controls (0 ). 51Crrelease was determined by counting the activity o f 20-p.l samples o f the supernatant culture fluid.
Discussion Infection with HSV-1 induces a significant change in the stability of the cellular mem branes. Determination of membrane fragility or cell lysis was performed by the 51Cr-release assay [7,8]. In contrast to other cytocidal viruses, which cause a characteristic enhancement of 51Crrelease from HEp-2 cells, HSV-1 infection leads to a reduction of the release and uptake of 51Cr. As demonstrated by 51Cr-release in the inocu lation period, this effect is not induced by a
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loss of 51Cr during HSV penetration or other events in the early stages of infection. Further more, it could be excluded by equilibrium dia lysis that 51Cr was preferentially bound to HSV-specified components of infected cells. The lower binding of 51Cr to HSV-1-infected cells than to uninfected control cells must be considered as evidence for the HSV-induced reduction of cell membrane permeability. This finding of a lower binding of 51Cr to debris of HSV-infected cells will be further investigated. Vaccinia virus, characterized by CPE simi lar to the HSV strains used in these experi ments, demonstrated a similar reduction of 51Cr-release. With the exception of ECF cells, this effect could be observed in permanent as well as in primary or transformed cell systems, and was independent of the MOI. The reduction of the release and uptake of 51Cr began 8 h after infection with HSV-1, corresponding to the logarithmic increase of virus reproduction. At this time, under our experimental conditions large amounts of HSV-1 antigen could be demonstrated by immunofluorescence. The discrepancy of our results with the findings of others concerning immune-cytolysis [9-11] may depend on the different behavior of the various cell-virus systems. In order to study if the observed reduction of permeability was correlated with a stabili zation of the membrane of the infected cell, the sensitivity of cells toward Triton X-100 and complement-mediated immune-cytolysis was tested. With the exception of ECF cells, HSV1 induced an increased stability of the cells toward the detergent. Furthermore, the effect of toxic sera on HEp-2 cells was prevented by HSV-1 infection, and immune-cytolysis, using HSV-1 antibodies, did not occur in infected HEp-2 cells. The lack of complement-medi ated immune-cytolysis and the stability toward
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HSV-Induced Reduction of 51Cr-Permeability
Triton X-100 and toxic sera indicated a de crease in fragility of the cells [12]. The results give no explanation of the mechanism of the HSV-l-induced stability of the cellular membrane. One might assume that newly synthesized viral antigens or virus-in duced Fc-receptors [13] on the cell surface were involved in the alteration of the membrane, resulting in a resistance toward immunologic and other cytolytic agents. Graham el al. [12] stated that ‘a thickening or folding of the plas ma membrane’ might be responsible for the decrease in cell fragility. This explanation must be judged in comparison with the observation that the reaction with other antigenic determi nants (treatment with anti-HEp-2 antibodies instead of anti-HSV-1 antibodies) is followed by an enhanced immune cytolysis. This result suggests a different location of the antigens in the cells and/or a modification of the receptor sites by HSV-1. It cannot be excluded that this effect could be due to differences in the species of origin of the antibodies. The HSV-induced stability of cellular mem branes may play a part in maintaining latent infections. Acknowledgments We are grateful to Miss Hanna Voss for her excellent assistance.
References 1 Keller, J.M .; Spear, P.G., and Roizman, B.: Pro teins specified by herpes simplex virus. III. Viruses differing in their effects on the social behavior of infected cells specify different membrane glyco proteins. Proc.natn.Acad.Sci. USA 65: 865-871 (1970).
2 Duff, R. and Rapp, F.: Properties o f hamster embryo fibroblasts transformed in vitro after ex posure to ultraviolet-irradiated herpes simplex virus type 2. J. Virol. 8: 469-477 (1971). 3 Watkins, J. F .: Adsorption o f sensitized sheep ery throcytes to HeLa cells infected with herpes simplex virus. Nature, Lond. 202: 1364-1365 (1964). 4 Westmoreland, D. and Watkins, J.F.: The IgG receptor induced by herpes simplex virus: studies using radioiodinated IgG. J.gen. Virol. 24: 167-178 (1974) . 5 Westmoreland, D.; Watkins, J.F., and Rapp, F.: Demonstration o f a receptor for IgG in Syrian hamster cells transformed with herpes simplex virus. J. gen. Virol. 25: 167-170(1974). 6 Nierhaus, D. and Nierhaus, K .H .: Identification o f the chloramphenicol-binding protein in Esche richia coli ribosomes by partial reconstitution. Proc. natn. Acad. Sci. USA 70: 2224-2228 (1973). 7 Wigzell, H.: Quantitative titrations o f mouse H-2 antibodies using Cr51-Iabelled target cells. Trans plantation 3: 423-431 (1965). 8 Hersey, P .; Edwards, J .; Edwards, A .; Adams, E.; Kearney, R., and Milton, G.W .: Comparison of 51Cr release and microcytotoxicity assays against human melanoma cells. Int.J. Cancer 16: 164-172 (1975) . 9 Brier,A. M .; Wohlenberg, C .; Rosenthal, J .; Mage, M., and Notkins, A. L .: Inhibition or enhancement o f immunological injury o f virus-infected cells. Proc.natn.Acad.Sci. USA 68: 3073-3077 (1971). 10 Snyderman, R.; Wohlenberg, C., and Notkins, A.L.: Inflammation and viral infection: chemotactic activity resulting from the interaction of anti viral antibody and complement with cells infected with herpes simplex virus. J. infect. Dis. 126: 207209 (1972). 11 Plummer, G.: A review of the identification and titration o f antibodies to herpes simplex viruses type 1 and type 2 in human sera. Cancer Res. 33: 1469-1476(1973). 12 Graham, J.M .; Sumner, M .C .B.; Curtis, D .H ., and Pasternak, C. A .: Sequence o f events in plasma membrane assembly during the cell cycle. Nature, Lond. 246: 291-295 (1973). 13 Kerbel, R.S.: Herpes virus induction o f Fc recep tors. Nature, Lond. 263: 192 (1976).
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