Vol. 24, No. 3 Printed in U.S.A.
JOURNAL
OF VIROLOGY, Dec. 1977, p. &36-845 Copyright i 1977 American Society for Microbiology
Replication of Epstein-Barr Virus: Ultrastructural and Immunofluorescent Studies of P3HR1-Superinfected Raji Cells J-M. SEIGNEURIN,* M. VUILLAUME, G. LENOIR, AND G. DE-THE International Agency for Research on Cancer, 69372 Lyon, France
Received for publication 1 July 1977
We have studied by means of electron microscopy and immunofluorescence the different steps of the replication of the P3HR1 strain of Epstein-Barr virus in Raji cells. The virus entered the cell by fusion of the viral envelope with the plasma membrane, followed by the disintegration of the capsid. In some cases, the migration of nucleocapsids toward the nuclear membrane was observed. The synthesis of new virions began as early as 7 h after infection (in the case of a high multiplicity of infection [MOI]-800 particles per cell) and took place in low-electron-density areas of the nucleus. A viral envelope was acquired by budding either through the nuclear membrane or more often through membranes of the Golgi apparatus or cytoplasmic vacuoles. Comparing immunofluorescence and electron microscopic data a good correlation was found between the presence of early antigen and ultrastructurally altered cells, as well as between the presence of viral capsid antigen and virus-producing cells. With different MOIs, different types of viral cycles were observed: at a low MOI (-50 particles per cell), a nonproducer cycle was induced, with early antigen synthesis only; at a higher MOI (100 particles per cell), a transient production of a small amount of virions was observed, and at a high MOI (a300 particles per cell), a productive cycle was the rule. The study of the replication cycle of EpsteinBarr virus (EBV) is difficult because it has no known fully permissive cell system. Therefore, the only means of effecting this study is either to infect the rare EBV-negative cell lines (16) (e.g., BJAB) or to use cord blood lymphocytes. In both cases one can observe the early phases of the viral cycle, but not the late phases, since the occurrence of transformation to a malignant state leads to very little production. In nasopharyngeal carcinoma (NPC) (5), epithelial tumor cells may be permissive, but nobody has yet succeeded in demonstrating the full cycle in vitro. In the spontaneous lymphoblastoid producer cell lines, it is possible to study the late events of EBV replication (8, 22), but not the early phases. We therefore chose the Raji cell system, which allows the study of early and late phases of EBV replication. The human lymphoblastoid cell line Raji, derived from a Burkitt's lymphoma, contains the EBV genome in a repressed state; no early antigens (EA) or viral capsid antigens (VCA) are produced, and it only synthesizes the EBV nuclear antigen (EBNA), detectable by anticomplement immunofluorescence (ACIF) (21). Superinfection of this cell line by the P3HR1 strain of EBV induces the synthesis of EA but usually not of VCA (12, 15). However, recently complete
replicative cycles were obtained when Raji cells were superinfected in phosphate-free minimum essential medium (24). MATERIALS AND METHODS Cell line. The Raji cell line was propagated in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 U of penicillin per ml, and 250 jig of streptomycin per ml at 37°C in a 5% C02-containing atmosphere. Source ofvirus. EBV was prepared from the tissue culture supernatant fluid of P3HR1 tissue cultures, concentrated 200-fold by ultracentrifugation, which was obtained by courtesy of J. Gruber, Virus Cancer Program of the National Cancer Institute. The same virus pool, distributed in 1-ml ampoules and stored at -80°C, was used for all experiments. The virus preparations were filtered before use through a 1.2,um membrane filter (Millipore Corp.) to remove the cell debris; particle counts were performed with a known concentration of latex spheres as reference (176-nm diameter, supplied by Dow Chemical) (18). Seventy-five percent of the viral particles were found to be enveloped. Infection by EBV. A total of 106 Raji cells, pelleted by low-speed centrifugation, were infected with P3HR1 virus at different multiplicities of infection (MOI; 50, 100, 300, 500, and 800 viral particles per cell) in 1 ml of medium. After gentle agitation of the mixture, the virus was left to adsorb at 37°C for 90 min. After removal of the unadsorbed virus by washing
836
REPLICATION OF EBV
VOL. 24, 1977
twice in phosphate-buffered saline, the cells were resuspended in 5 ml of RPMI 1640 medium and incubated at 37"C. The cell suspensions were harvested at different times after initiation of infection: 90 min, 3 h, 5 h, 7 h, 9 h, 11 h, 15 h, 24 h, 3 days, and 5 days. IF assays. Two types of immunofluorescence (IF) tests were performed, the ACIF technique (21) and the indirect IF test. The ACIF test, classically used to detect EBNA and much more sensitive than the indirect IF test, was also used here to detect VCA; this was possibly due to the fact that the serum Valentin was VCA positive and EBNA negative (Table 1). The smears were air dried and fixed in a 1:1 mixture of cold acetone and methanol. The reference sera used were Tu 367, Valentin, and RK003 (Table 1). Human complement from an EBV-negative donor (7028) was used at a 1:10 dilution; after washing, the smears were stained with a fluorescein isothiocyanate-conjugated goat anti-human complement C3 at a dilution of 1:20 (Hyland Laboratories). Indirect staining was performed by the procedure of Henle and Henle (11). Briefly, the smears were air dried and fixed with acetone for 10 min at room temperature. The sera Tu 110, Tu 367, and RK003 were used at dilutions of 1:20 for the positive ones and 1:10 for the negative one; after washing, the smears were stained with goat antihuman immunoglobulin conjugated with fluorescein. All smears were counterstained with Evans blue (0.005%) for 10 min at room temperature and examined under UV light with a Leitz Orthoplan microscope equipped with epi-illumination. Electron microscopy. Infected cells were fixed in 4% glutaraldehyde for 1 h, washed with 0.1 M sodium cacodylate buffer, postfixed for 45 min in 2% osmium tetroxide, dehydrated, and embedded in Epon 812. Thin sections were cut with an LKB ultramicrotome, double stained with uranyl acetate and lead citrate, and examined in a Siemens Elmiskop 101. For the preparation for particle counts, carbon-coated grids were allowed to float on a drop of virus-latex mixture (1:1, by volume) for 1 min; after draining, they were stained with 2.5% sodium tungsto-silicate and examined at a magnification of x30,000. RESULTS
Attachment and penetration. Viral attachwas not dependent on the temperature since a similar number of viral particles were found adsorbed at the surface of cells when incubated at 37 or 40C for 3 h. At 90 min after massive infection (1,000 viral particles per cell), ment
837
around 50% of the Raji cells were seen to have adsorbed EBV particles. We tried to evaluate the number of adsorbed virus particles per cell by counting in electron microscopy the number of adsorbed virus particles on 200 cell sections as well as by evaluating the difference between the input particles and those left over in the supernatant at 90 min after infection. An average of 50 to 100 adsorbed viral particles per cell were found at an MOI of 600 per cell; all of the particles were adsorbed within 90 min, and no further adsorption occurred when incubation lasted up to 8 h. EBV almost exclusively entered the cells by a fusion process. After fixation of the viral envelope on the cell coat, both the viral envelope and adjacent plasma membrane underwent morphological changes, whereas the viral coat displayed polarization of surface material at the opposite site of attachment (Fig. 1A); the envelope of the virus melted with the plasmalemma (Fig. 1B), and the viral outline became loose, probably due to virus lysis (Fig. 10). As mentioned above, fusion was the prevalent mechanism after EBV attachment; nevertheless, the presence of virus close to cellular invaginations was observed in exceptional cases (Fig. iD), suggesting a process of viropexis. Virus particles were never found within pinocytotic vesicles or in vacuoles near the cell surface. The binding sites were usually located at areas of microvilli, where small particulate debris abounded. In contrast, contiguous smooth segments of cell membrane were generally devoid of adsorbed virus. Even when EBV inoculum contained up to 25% of unenveloped virus, no naked nucleocapsids were ever observed in the process of virus adsorption by the cells. At 24 h after infection, numerous virus particles were still present at the cell surface, despite careful intermediate washing. After adsorption, the penetration of the virus did not present clear-cut images since the particles disintegrated just beneath the cell membrane, probably quickly after attachment. Further steps could, however, be observed due to rare cases where EBV nucleocapsids were ob-
TABLE 1. Reference sera used in this study for the detection of EBV antigens by IF" Titer
Serum"
Origin
NPC (Tunis) Tu 110 NPC (Tunis) Tu 367 Early infectious mononucleosis Valentin Normal donor RK 003 a Titers expressed as the reciprocal of serum dilution. b Sera are free of antinuclear factors.
VCA
2,560 640 320
JOURNAL
OF VIROLOGY, Dec. 1977, p. &36-845 Copyright i 1977 American Society for Microbiology
Replication of Epstein-Barr Virus: Ultrastructural and Immunofluorescent Studies of P3HR1-Superinfected Raji Cells J-M. SEIGNEURIN,* M. VUILLAUME, G. LENOIR, AND G. DE-THE International Agency for Research on Cancer, 69372 Lyon, France
Received for publication 1 July 1977
We have studied by means of electron microscopy and immunofluorescence the different steps of the replication of the P3HR1 strain of Epstein-Barr virus in Raji cells. The virus entered the cell by fusion of the viral envelope with the plasma membrane, followed by the disintegration of the capsid. In some cases, the migration of nucleocapsids toward the nuclear membrane was observed. The synthesis of new virions began as early as 7 h after infection (in the case of a high multiplicity of infection [MOI]-800 particles per cell) and took place in low-electron-density areas of the nucleus. A viral envelope was acquired by budding either through the nuclear membrane or more often through membranes of the Golgi apparatus or cytoplasmic vacuoles. Comparing immunofluorescence and electron microscopic data a good correlation was found between the presence of early antigen and ultrastructurally altered cells, as well as between the presence of viral capsid antigen and virus-producing cells. With different MOIs, different types of viral cycles were observed: at a low MOI (-50 particles per cell), a nonproducer cycle was induced, with early antigen synthesis only; at a higher MOI (100 particles per cell), a transient production of a small amount of virions was observed, and at a high MOI (a300 particles per cell), a productive cycle was the rule. The study of the replication cycle of EpsteinBarr virus (EBV) is difficult because it has no known fully permissive cell system. Therefore, the only means of effecting this study is either to infect the rare EBV-negative cell lines (16) (e.g., BJAB) or to use cord blood lymphocytes. In both cases one can observe the early phases of the viral cycle, but not the late phases, since the occurrence of transformation to a malignant state leads to very little production. In nasopharyngeal carcinoma (NPC) (5), epithelial tumor cells may be permissive, but nobody has yet succeeded in demonstrating the full cycle in vitro. In the spontaneous lymphoblastoid producer cell lines, it is possible to study the late events of EBV replication (8, 22), but not the early phases. We therefore chose the Raji cell system, which allows the study of early and late phases of EBV replication. The human lymphoblastoid cell line Raji, derived from a Burkitt's lymphoma, contains the EBV genome in a repressed state; no early antigens (EA) or viral capsid antigens (VCA) are produced, and it only synthesizes the EBV nuclear antigen (EBNA), detectable by anticomplement immunofluorescence (ACIF) (21). Superinfection of this cell line by the P3HR1 strain of EBV induces the synthesis of EA but usually not of VCA (12, 15). However, recently complete
replicative cycles were obtained when Raji cells were superinfected in phosphate-free minimum essential medium (24). MATERIALS AND METHODS Cell line. The Raji cell line was propagated in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 U of penicillin per ml, and 250 jig of streptomycin per ml at 37°C in a 5% C02-containing atmosphere. Source ofvirus. EBV was prepared from the tissue culture supernatant fluid of P3HR1 tissue cultures, concentrated 200-fold by ultracentrifugation, which was obtained by courtesy of J. Gruber, Virus Cancer Program of the National Cancer Institute. The same virus pool, distributed in 1-ml ampoules and stored at -80°C, was used for all experiments. The virus preparations were filtered before use through a 1.2,um membrane filter (Millipore Corp.) to remove the cell debris; particle counts were performed with a known concentration of latex spheres as reference (176-nm diameter, supplied by Dow Chemical) (18). Seventy-five percent of the viral particles were found to be enveloped. Infection by EBV. A total of 106 Raji cells, pelleted by low-speed centrifugation, were infected with P3HR1 virus at different multiplicities of infection (MOI; 50, 100, 300, 500, and 800 viral particles per cell) in 1 ml of medium. After gentle agitation of the mixture, the virus was left to adsorb at 37°C for 90 min. After removal of the unadsorbed virus by washing
836
REPLICATION OF EBV
VOL. 24, 1977
twice in phosphate-buffered saline, the cells were resuspended in 5 ml of RPMI 1640 medium and incubated at 37"C. The cell suspensions were harvested at different times after initiation of infection: 90 min, 3 h, 5 h, 7 h, 9 h, 11 h, 15 h, 24 h, 3 days, and 5 days. IF assays. Two types of immunofluorescence (IF) tests were performed, the ACIF technique (21) and the indirect IF test. The ACIF test, classically used to detect EBNA and much more sensitive than the indirect IF test, was also used here to detect VCA; this was possibly due to the fact that the serum Valentin was VCA positive and EBNA negative (Table 1). The smears were air dried and fixed in a 1:1 mixture of cold acetone and methanol. The reference sera used were Tu 367, Valentin, and RK003 (Table 1). Human complement from an EBV-negative donor (7028) was used at a 1:10 dilution; after washing, the smears were stained with a fluorescein isothiocyanate-conjugated goat anti-human complement C3 at a dilution of 1:20 (Hyland Laboratories). Indirect staining was performed by the procedure of Henle and Henle (11). Briefly, the smears were air dried and fixed with acetone for 10 min at room temperature. The sera Tu 110, Tu 367, and RK003 were used at dilutions of 1:20 for the positive ones and 1:10 for the negative one; after washing, the smears were stained with goat antihuman immunoglobulin conjugated with fluorescein. All smears were counterstained with Evans blue (0.005%) for 10 min at room temperature and examined under UV light with a Leitz Orthoplan microscope equipped with epi-illumination. Electron microscopy. Infected cells were fixed in 4% glutaraldehyde for 1 h, washed with 0.1 M sodium cacodylate buffer, postfixed for 45 min in 2% osmium tetroxide, dehydrated, and embedded in Epon 812. Thin sections were cut with an LKB ultramicrotome, double stained with uranyl acetate and lead citrate, and examined in a Siemens Elmiskop 101. For the preparation for particle counts, carbon-coated grids were allowed to float on a drop of virus-latex mixture (1:1, by volume) for 1 min; after draining, they were stained with 2.5% sodium tungsto-silicate and examined at a magnification of x30,000. RESULTS
Attachment and penetration. Viral attachwas not dependent on the temperature since a similar number of viral particles were found adsorbed at the surface of cells when incubated at 37 or 40C for 3 h. At 90 min after massive infection (1,000 viral particles per cell), ment
837
around 50% of the Raji cells were seen to have adsorbed EBV particles. We tried to evaluate the number of adsorbed virus particles per cell by counting in electron microscopy the number of adsorbed virus particles on 200 cell sections as well as by evaluating the difference between the input particles and those left over in the supernatant at 90 min after infection. An average of 50 to 100 adsorbed viral particles per cell were found at an MOI of 600 per cell; all of the particles were adsorbed within 90 min, and no further adsorption occurred when incubation lasted up to 8 h. EBV almost exclusively entered the cells by a fusion process. After fixation of the viral envelope on the cell coat, both the viral envelope and adjacent plasma membrane underwent morphological changes, whereas the viral coat displayed polarization of surface material at the opposite site of attachment (Fig. 1A); the envelope of the virus melted with the plasmalemma (Fig. 1B), and the viral outline became loose, probably due to virus lysis (Fig. 10). As mentioned above, fusion was the prevalent mechanism after EBV attachment; nevertheless, the presence of virus close to cellular invaginations was observed in exceptional cases (Fig. iD), suggesting a process of viropexis. Virus particles were never found within pinocytotic vesicles or in vacuoles near the cell surface. The binding sites were usually located at areas of microvilli, where small particulate debris abounded. In contrast, contiguous smooth segments of cell membrane were generally devoid of adsorbed virus. Even when EBV inoculum contained up to 25% of unenveloped virus, no naked nucleocapsids were ever observed in the process of virus adsorption by the cells. At 24 h after infection, numerous virus particles were still present at the cell surface, despite careful intermediate washing. After adsorption, the penetration of the virus did not present clear-cut images since the particles disintegrated just beneath the cell membrane, probably quickly after attachment. Further steps could, however, be observed due to rare cases where EBV nucleocapsids were ob-
TABLE 1. Reference sera used in this study for the detection of EBV antigens by IF" Titer
Serum"
Origin
NPC (Tunis) Tu 110 NPC (Tunis) Tu 367 Early infectious mononucleosis Valentin Normal donor RK 003 a Titers expressed as the reciprocal of serum dilution. b Sera are free of antinuclear factors.
VCA
2,560 640 320
