A New View on Apoptosis in HIV Infection in Vitro

Virology 282, 48–55 (2001) doi:10.1006/viro.2000.0811, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE

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Syncytium Formation Amplifies Apoptotic Signals: A New View on Apoptosis in HIV Infection in Vitro

Carsten Scheller and Christian Jassoy1

Institute for Virology and Immunobiology, Julius Maximilians University, 97078 Wu¨rzburg, Germany Received September 19, 2000; returned to author for revision November 8, 2000; accepted December 13, 2000

Infection of CD4ϩ cells with HIV in vitro causes extensive cytopathicity. The mechanism that underlies this process is unclear and conflicting data exist regarding whether cytotoxicity is due to necrosis or apoptosis. It was previously reported and is shown here that the coculture of HIV glycoprotein-expressing cells with CD4ϩ cells results in apoptosis within several hours. This study demonstrates that apoptosis did not occur in single cells and was mediated neither by CD4 nor by coreceptor signaling, indicating that apoptosis was not induced by intra- or intercellular glycoprotein–receptor interaction. Detection of apoptosis required cell-to-cell fusion and undetectable levels of apoptotic cell death were substantially amplified upon syncytium formation. Similar results were obtained with syncytium-forming cultures of measles virus glycoprotein- expressing cells. These findings indicate that the apoptotic cell death observed in cultures of HIV and other syncytium- forming viruses is primarily due to amplification of background apoptosis in the wake of cell-to-cell fusion. © 2001 Academic Press Key Words: apoptosis; HIV; envelope glycoprotein; CD4; T20; syncytium; measles virus.

INTRODUCTION N-terminal hydrophobic helix in the ectodomain of gp41 bind together with high affinity and form a hairpin struc- Infection with the human immunodeficiency virus (HIV) ture which pulls the two membranes into close proximity. is characterized by the gradual depletion of CD4ϩ T This step allows fusion of the phospholipid bilayers of lymphocytes in vivo, a process that results in immune the cells. The prehairpin intermediate is relatively long- dysfunction and progression to AIDS (Levy, 1994). The lived and is susceptible to inhibition with synthetic pep- mechanism that causes the decline of helper T cells in tides that mimic the C-terminal or N-terminal hydropho- infected individuals is still poorly understood. In vitro, HIV bic helix of gp41 such as T20 or DP107, respectively infection causes syncytium formation and rapid cell (Chan and Kim, 1998; Chen et al., 1995; Wild et al., 1992; death. Although syncytium formation is not readily ob- Kilby et al., 1998). served in most affected tissues or blood from infected Syncytium formation can be observed in many other individuals, it is assumed that the cytopathicity observed viral infections in vitro, including infections with diverse in culture is relevant to the T cell depletion in vivo (Cao retroviruses and paramyxoviruses. The mechanism that et al., 1996). underlies cell membrane fusion in infection with these Infection with HIV or expression of functional virus viruses is related to that with HIV. For instance, syncy- glycoproteins in CD4ϩ cells in vitro leads to cell-to-cell fusion and syncytium formation (Lifson et al., 1986a,b; tium formation in measles virus infection involves the two Sodroski et al., 1986). This process first requires the virus glycoproteins, the fusion protein F, and hemagglu- interaction between the HIV glycoproteins and the recep- tinin H (Seya et al., 1997; Wild et al., 1991). tors. The HIV glycoproteins consist of two noncovalently The process of cytotoxicity in HIV infection in vitro has associated subunits, the surface glycoprotein gp120 and not been elucidated. On the one hand, it was reported the transmembrane glycoprotein gp41. Binding of gp120 that cells predominantly die by necrosis (Cao et al., 1996; to CD4 and one of several potential coreceptors, in Park et al., 1996). On the other hand, apoptotic cell death particular CXCR4 or CCR5, enables gp41 to undergo a has been observed in numerous other examinations conformational change and to form a prehairpin interme- (Corbeil and Richman, 1995; Biard-Piechaczyk et al., diate which penetrates the target membrane with its 1999, 2000; Blanco et al., 1999; Guillerm et al., 1998) and fusion domain. In a second step, a C-terminal and an the possible contribution of apoptosis to the T helper cell death in infected individuals has drawn considerable interest. It was demonstrated that apoptosis in HIV infection in vitro involves the interaction of cellularly ex- 1 To whom reprint requests should be addressed at Institute for Virology and Immunobiology, Versbacher Strasse 7, 97078 Wu¨rzburg, pressed HIV envelope glycoprotein with CD4 or the co- Germany. Fax ϩ49 (0)931 201-3934. E-mail: [email protected] receptor or both (Corbeil and Richman, 1995). In partic- wuerzburg.de. ular, it was reported that expression of the HIV envelope

products or in individual cells, A3.01 cells were infected overnight with an HIV glycoprotein-expressing vaccinia virus construct. To prevent stable cell-to-cell contacts during infection, cells were mixed by rotating the culture tube end-over-end. After infection, fresh uninfected A3.01 cells were added and cells split and were either incubated in a culture plate or spun for an additional 6 h. Cells in the culture plate formed syncytia and exhibited DNA fragmentation after only a few hours. In contrast, no apoptotic DNA fragmentation was observed in continu- ously rotating cells, indicating that HIV glycoprotein-related apoptosis is prevented if stable interaction between cells is avoided (Fig. 1). It was previously demonstrated that neutralizing monoclonal antibodies directed at the interface regions FIG. 1. Expression of HIV glycoproteins in CD4ϩ itself does not of gp120 and CD4, other syncytium-inhibiting mAbs, re- induce apoptosis in the absence of sustained cell contact. T lympho- combinant soluble CD4, and syncytium-inhibiting core- blasts were infected with vac/env and the cell culture was rotated ceptor antagonists block apoptotic cell death (Blanco et end-over-end for 16 h to inhibit cell-to-cell fusion. Fresh cells were al., 2000; Guillerm et al., 1998; Laurent-Crawford et al., added and spinning was continued (ϩ) or stopped (Ϫ) and cells were cultured for an additional 6 h. DNA was prepared and analyzed by 1993; Ohnimus et al., 1997; Terai et al., 1991). To differ- agarose gel electrophoresis. Lane M: DNA marker, 100-bp ladder. entiate between mechanisms of apoptosis induction by processes that involve glycoprotein–receptor interactions and the subsequent events of cell membrane fu- glycoproteins in CD4ϩ cells is sufficient for induction of sion, we performed coculture experiments in the pres- apoptosis (Laurent-Crawford et al., 1993) and that inhibi- ence of the peptide T20. This peptide does not interfere tion of the contact between the glycoprotein and CD4 or with binding of the glycoprotein to the receptor or to the the coreceptor prevents apoptotic cell death (Blanco et coreceptor but inhibits subsequent membrane fusion by al., 1999; Laurent-Crawford et al., 1993; Maldarelli et al., blocking a conformational change of gp41 (Chan and 1995; Ohnimus et al., 1997; Terai et al., 1991). This indi- Kim, 1998; Kilby et al., 1998). Moreover, T20 does not cates that apoptosis requires proper interaction between inhibit vaccinia virus infection or expression of the HIV HIV glycoproteins and their cellular receptors. Alterna- glycoproteins. The peptide T20 blocked syncytium for- tively, it was hypothesized that apoptosis in HIV-infected mation (data not shown) and apoptosis (Fig. 2), indicating cultures is triggered intracellularly by the interaction of that contact and interaction of gp120 with CD4 or the the glycoprotein with one of the receptors (Lu et al., coreceptor is not sufficient to initiate apoptosis but 1994). apoptosis induction requires cell membrane fusions. As The aim of this study was to reexamine the HIV gly- expected, the fusion-inhibiting anti-CD4 mAb SIM.2 and coprotein-associated cell death in vitro and to elucidate the caspase inhibitor zVAD similarly prevented apoptotic the molecular mechanism of apoptosis in CD4ϩ cells in DNA fragmentation. a model system for the HIV cytopathology. In particular, we tested whether apoptotic cell death occurs in single cells or syncytia and how apoptosis is generated in culture. Our data demonstrate that significant apoptosis takes place in syncytia but not in single cells. The results indicate that apoptosis induction by virus glycoprotein– receptor interactions is insignificant and suggest that the apoptosis observed in HIV infection in vitro is the result of an amplification of background apoptosis in syncytia.

RESULTS Apoptosis in cultures of HIV glycoprotein-expressing CD4ϩ cells depends on cell-to-cell fusion Expression of the HIV envelope glycoprotein in CD4ϩ T lymphoblasts or coculture of glycoprotein-expressing FIG. 2. Inhibition of cell-to-cell fusion prevents apoptosis upon con- ϩ tact of HIV glycoprotein-expressing cells and CD4ϩ cells. Uninfected T cells with CD4 cells causes syncytium formation, cyto- lymphoblasts were cocultured with vac/env-infected T cells. DNA was toxicity, and apoptosis. To examine whether expression prepared and analyzed by agarose gel electrophoresis. Lane M: DNA of the HIV glycoprotein induces apoptosis in cell fusion marker, 100-bp ladder. 50 SCHELLER AND JASSOY

FIG. 3. Apoptosis is amplified by cell-to-cell fusion. (A) A3.01 T lymphoblasts were infected with vac/env for 9 h and cocultured for an additional 8 h with uninfected A3.01 cells with (ϩ) or without (Ϫ) 1% of cells rendered apoptotic by pretreatment with the anti-CD95 mAb 7C11. Cells were cultured in the presence (SIM.2) or absence (w/o) of an anti-CD4 mAb. (B) A3.01 T lymphoblasts were infected with vac/HF for 9 h and cocultured for an additional 8 h with uninfected cells with (ϩ) or without (Ϫ) 10% anti-CD95 mAb-treated apoptotic cells. Lymphoblasts were cultured in the presence (ϩ) or absence (Ϫ) of the anti-measles H mAb L77 and the caspase inhibitor zVAD. Lane M: DNA marker, 100-bp ladder.

Syncytium formation amplifies apoptotic signals additional 8 h. As expected, the coculture resulted in marked syncytium formation (data not shown). Despite Having demonstrated that apoptosis in HIV glycopro- that, no signs of apoptosis were observed, indicating that tein-expressing cultures depends on cell-to-cell fusion, we aimed to examine how apoptosis is generated in cell-to-cell fusion itself does not induce apoptosis (Fig. syncytia. Induction of apoptosis leads to activation of a 3A). Addition of a small fraction (1%) of cells that were cascade of cytoplasmic enzymes that mediate and exer- rendered apoptotic by prior incubation with an anti-CD95 cise the apoptotic process. We hypothesized that when antibody induced marked DNA fragmentation. When in apoptotic cells fuse with living cells, enzymes activated this situation cell-to-cell fusion was inhibited by the anti- during apoptosis might trigger an apoptotic signaling CD4 mAb, apoptotic DNA fragmentation was prevented cascade in the healthy cells and disseminate apoptosis demonstrating that (1) apoptosis of the anti-CD95-pre- in the syncytium, resulting in an amplification of apopto- treated cells was below the detection limit and (2) the sis in the culture. To test this assumption A3.01 T lym- undetectable level of apoptosis was amplified to detect- phoblasts were infected with vac/env for only 9 h to able signals by cell-to-cell fusion and syncytium forma- minimize any vaccinia-virus-associated cytopathicity and tion (Fig. 3A). cocultured with uninfected A3.01 T lymphoblasts for an To investigate whether cell-to-cell fusion in infections AMPLIFICATION OF APOPTOSIS IN SYNCYTIA 51 with other fusogenic viruses may similarly result in am- 1995; Nardelli et al., 1995), or glycoprotein gene-trans- plification of apoptosis, experiments analogous to those fected cell lines (Lu et al., 1994). Blockade of syncytium with vac/env were performed with measles virus glyco- formation by physical means, a neutralizing mAb that protein HF-expressing cells. Figure 3B shows that signs blocks gp120-CD4 binding or T20, prevented the devel- of apoptosis generated by treatment with anti-CD95 opment of apoptosis. In the absence of cell-to-cell fusion, were markedly augmented in the presence of syncytium even substantial expression of the glycoprotein itself formation but blocked by the caspase inhibitor zVAD. was insufficient to induce detectable levels of apoptotic cell death in single cells in the time period examined. Microscopic analysis of apoptosis in HIV Thus, apoptosis was not induced by intracellular inter- glycoprotein-expressing cell cultures actions of the HIV glycoprotein and CD4. Apoptosis in cell culture of HIV glycoprotein-express- Contact of the viral glycoproteins with the receptors ing cells with uninfected T lymphoblasts was further was not sufficient to induce apoptosis because the pep- examined by fluorescence microscopy using annexin V tide T20 that inhibits cell-to-cell fusion but does not affect and propidium iodide staining. In addition, caspase ac- binding of HIV gp120 to CD4 or the coreceptor prevented tivation, a characteristic feature of cells undergoing apoptosis. This is in contrast to what has been hypoth- apoptosis, was monitored using a fluorogenic caspase esized previously. In particular, it was postulated that substrate (Hug et al., 1999). Figure 4 shows the simulta- apoptosis might be triggered by the interaction of HIV neous presence of both apoptotic and nonapoptotic syn- gp120 with CXCR4 (Blanco et al., 2000). This assumption cytia in the culture. Moreover, green fluorescence in was based on the observation that agents that inhibit some syncytia was inhomogeneous, indicating that syncytium formation by blocking the contact of the HIV apoptosis was not evenly distributed over the whole glycoprotein to the coreceptor prevented apoptosis cellular complex. These structures may represent differ- (Blanco et al., 2000). However, since the inhibitors simul- ent stages in the development and spread of apoptosis taneously blocked both gp120–CXCR4 binding and cell- in syncytia. to-cell fusion, this experimental approach did not dis- criminate the role of either process in the induction of apoptosis. DISCUSSION It was suggested that a CD4-dependent signaling via The cause of the T helper cell depletion that occurs in the p56lck kinase induces apoptosis based upon the HIV-infected individuals has not been determined. Com- observation that cells that overexpress p56lck showed prehension of the mechanism that underlies T cell death increased apoptosis and the finding that a truncation of in infected cell cultures may help to solve this problem. the cytoplasmic domain of the CD4 receptor that pre- It was reported that infection with HIV and expression of vented association of the kinase to CD4 blocked HIV functional virus glycoproteins in vitro induce cell death. glycoprotein-associated apoptosis (Corbeil et al., 1996). Cell death in culture may occur in syncytia (Sodroski et This view has been challenged by other reports that al., 1986) or in single cells (Cao et al., 1996; Koga et al., describe apoptosis in cells expressing truncated CD4 1994; Somasundaran et al., 1987). Cytotoxicity in this receptors incapable of kinase binding (Biard-Piechaczyk situation may be due to necrosis (Cao et al., 1996; Kole- et al., 2000; Guillerm et al., 1998). Based on this discrep- snitchenko et al., 1997). Alternatively, numerous groups ancy and from the point of view of our observations, one have observed that HIV infection and expression of the may speculate that the enhancement of apoptosis by viral glycoproteins or coculture of HIV glycoprotein-ex- p56lck is related to its ability to enhance syncytium for- pressing cells with CD4ϩ cells result in typical signs of mation in culture (Briand et al., 1997). apoptosis (Glynn et al., 1996; Laurent-Crawford et al., Transmission of apoptosis upon cell-to-cell fusion has 1991, 1993; Terai et al., 1991; Ohnimus et al., 1997; Mal- recently been observed in cells fused by polyethylene darelli et al., 1995; Nardelli et al., 1995; Lu et al., 1994). glycol (Huang et al., 1997). Therefore, we hypothesized Thus, the true mode of cell death in this situation and the that syncytium formation in viral infections may similarly factors that determine the way and process of cell killing be able to amplify preexistent apoptosis. To test this have not yet been clarified. assumption, uninfected and HIV glycoprotein-expressing In this study we observed significant levels of apopto- or measles virus H- and F-expressing cells were cocul- tic cell death within several hours in cultures of HIV tured. Some of the uninfected CD4ϩ cells were pre- glycoprotein-expressing CD4ϩ cells in the wake of cell- treated with an apoptosis-inducing mAb. In this situation, to-cell fusion. This observation compares favorably with a small fraction of apoptotic CD4ϩ cells caused vigorous those made previously by many groups that investigated DNA fragmentation. Agents that blocked syncytium for- apoptosis using vaccinia HIV glycoprotein-infected cells mation prevented DNA degradation. These findings dem- (Laurent-Crawford et al., 1993; Ohnimus et al., 1997), onstrate that apoptosis is a dominant process that is HIV-infected lymphoblasts (Corbeil and Richman, 1995; transferred from apoptotic to nonapoptotic cells upon Ohnimus et al., 1997; Laurent-Crawford et al., 1993; Mal- cell-to-cell fusion. Thus, low and undetectable numbers darelli et al., 1995), primary cells (Corbeil and Richman, of apoptotic cells in culture may represent the source of FIG. 4. Microscopic analysis of apoptosis in HIV glycoprotein-expressing cell cultures: (A) T lymphoblasts were cocultured with vac/env-infected B-LCL and stained with FITC-labeled annexin V and propidium iodide. The photograph shows apoptosis in syncytia in different stages of development: a nonapoptotic syncytium (1), an apoptotic cell adjacent to a nonapoptotic syncytium (2), a syncytium consisting of apoptotic and nonapoptotic cells (3), a syncytium with homogeneous distribution of apoptosis (4), and a late apoptotic or necrotic syncytium (5). (B) Light microscopic representation of the same area as in A. (C) A3.01 T lymphoblasts were cocultured with vac/env-infected A3.01 cells and the caspase substrate D2R was added. Caspase activity was monitored by fluorescence microscopy. The photograph shows a superimposition of a light- and fluorescence-microscopic view of syncytia after6hofculture. Caspase activity in apoptotic structures appears with yellowish–green fluorescence. AMPLIFICATION OF APOPTOSIS IN SYNCYTIA 53 high levels of apoptosis upon cell fusion in vitro. A low apoptosis in HIV glycoprotein-expressing cells in vitro is frequency of apoptotic cells may be present in normal mainly caused by syncytium formation, which amplifies cell cultures, reflects the end of the natural life span of small amounts of spontaneously occurring apoptosis to the cells, and constitutes the base of apoptosis in model marked signals. Since syncytium formation similarly oc- systems of fusion-inducing viruses. curs in cell cultures infected with HIV, our findings sug- Moreover, infection with virus vectors such as vaccinia gest that amplification of apoptosis is the principal ele- may generate apoptosis (Baixeras et al., 1998; Kibler et ment among the factors that underlie apoptosis in HIV al., 1997; Ramsey-Ewing and Moss, 1998) and this may infection in vitro. contribute to the phenomenon observed. This view is supported by the observation that infections for less than MATERIALS AND METHODS 10 h in contrast to infections for 16 h rarely caused Cell lines and recombinant vaccinia viruses detectable apoptosis (Fig. 3), despite high levels of glycoprotein expression and syncytium formation. There- Human A3.01 T lymphoblasts (Folks et al., 1985) ex- fore, apoptosis in syncytium-forming systems might be press the CD4 molecule. These cells were maintained in more rapidly detected with vaccinia-virus-based expres- RPMI 1640 medium supplemented with 10% FCS, 10 mM sion systems (Laurent-Crawford et al., 1993; Ohnimus et HEPES, and antibiotics (Life Technologies, Karlsruhe, al., 1997) than with model systems that use transfected Germany). The EBV-transformed B lymphoblastoid cell cells (Biard-Piechaczyk et al., 2000; Lu et al., 1994). Since line (B-LCL) 010-035i was cultured in RPMI 1640 medium, syncytia may consist of large numbers of cells, incorpo- supplemented with 15% FCS. The recombinant vaccinia ration of apoptotic cells into syncytia may lead to rapid virus construct vPE17 (vac/env) encodes the HIV enve- and extensive amplification of the apoptotic signals. This lope glycoproteins derived from strain HIV-1IIIB and con- process may be operative in in vitro models of HIV tains a truncation in the cytoplasmic region. This leads to infection and deceive our perspective of the mechanism maximum cell surface expression (Earl et al., 1991). of HIV-induced cytopathology. Since this vaccinia virus construct was originally derived Although the experiments were performed with recom- from the vaccinia virus strain Western Reserve, which is binant vaccinia virus constructs, the findings may, in an able to inhibit apoptosis due to the presence of the B13R analogous fashion, relate to infection with HIV because gene product (Heinkelein et al., 1996; Kettle et al., 1997), HIV infection induces a comparable level of glycoprotein a recombinant vaccinia virus based on strain Copenha- expression and a similar degree of syncytium formation gen, which has a deletion in the B13R gene, was gener- (Lifson et al., 1986b; Sodroski et al., 1986) and apoptosis ated by homologous recombination using the pPE17 (Corbeil and Richman, 1995; Laurent-Crawford et al., plasmid and used in the study. The recombinant vaccinia 1991; Maldarelli et al., 1995; Nardelli et al., 1995; Ohni- virus (strain Copenhagen) vac/HF expresses the mea- mus et al., 1997) in vitro. However, cytopathic processes sles virus glycoproteins H and F (Wild et al., 1991). mediated by viral proteins other than the glycoproteins were not analyzed and their potential contribution still Coculture of cells has to be evaluated (Purvis et al., 1995; Li et al., 1995; B-LCL and A3.01 T lymphoblasts were infected with Okada et al., 1997). vac/env or vac/HF at a multiplicity of infection of 10 Amplification of apoptosis was not restricted to HIV plaque-forming units/cell and incubated for 16 h at 37°C. glycoprotein-induced syncytium formation but similarly To inhibit syncytium formation during infection with the occurred in the wake of cell-to-cell fusion induced by the HIV glycoprotein construct, lymphoblasts were infected measles virus glycoproteins. This indicates that amplifi- in the presence of the anti-CD4 mAb SIM.2 (65 ␮g/ml). cation of apoptosis in syncytia is not unique to HIV but Infected cells were cocultured with uninfected A3.01 T plays a role in other virus infections as well. This may be lymphoblasts at a ratio of 1:1 in a 24-well plate for8hin relevant. For instance, it was reported that the cytopathic the presence or absence of the anti-CD4 mAb SIM.2 (65 effect in measles-virus-infected cell cultures was asso- ␮g/ml) or the neutralizing anti-measles hemagglutinin ciated with apoptosis and there was a correlation of the mAb L77 (150 ␮g/ml), respectively. Alternatively, cells degree of measles-virus-induced syncytia and the num- were cocultured with T20 (1 ␮g/ml) or zVAD (100 ␮M). ber of nuclei showing apoptotic DNA degradation (Es- T20 is a synthetic peptide that corresponds to amino olen et al., 1995). Furthermore, apoptosis was observed acids 643–678 of HIV gp41 (Wild et al., 1992; Kilby et al., in cultures infected with syncytium-inducing but not with 1998). zVAD (Bachem Biochemica, Heidelberg, Germany) noninducing strains of feline syncytial virus and apopto- is a broad-range caspase inhibitor and was used in sis occurred predominantly in syncytia (Ikeda et al., selected experiments to specifically identify the process 1997). Thus, in addition to initiation of apoptosis, ampli- of apoptosis. fication of apoptotic cell death should be taken into In particular experiments, T lymphoblasts were in- consideration when the cytopathicity of syncytium-induc- fected with vaccinia viruses for 9 h and cocultured with ing viruses is being studied. uninfected T cells. In the case of vac/env, the mAb SIM.2 In conclusion, the results of this study indicate that was added to the infection. Uninfected A3.01 cells were 54 SCHELLER AND JASSOY pretreated with the anti-CD95 mAb 7C11 (0.2 ␮g/ml) for ACKNOWLEDGMENTS 15 min at 37°C to induce apoptosis, washed twice with 5 The authors thank I. Euler-Ko¨nig for technical assistance and E. ml of medium, and added at the start of the coculture. Koutsilieri, S. Sopper, and M. Heinkelein for critical review of the 6 Cocultures (2 ϫ 10 cells) infected with vac/env con- manuscript. The B-LCL 010-35i was obtained from B. D. Walker, Mas- tained 1% and cocultures infected with vac/HF contained sachusetts General Hospital (Boston, MA). The vaccinia virus construct 10% of anti-CD95 mAb pretreated cells. Expression of the vPE17 (strain WR) and the corresponding plasmid pPE17 were obtained HIV glycoprotein in vac/env-infected cells was regularly from B. Moss, Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health (Bethesda, MD). monitored by flow cytometric analysis using the FITC- The vaccinia virus construct vac/HF from T. F. Wild, Immunovirologie labeled mouse mAb 902 (Chesebro and Wehrly, 1988) Moleculaire et Cellulaire, Lyon, France, was obtained through J. directed at gp120. Similarly, expression of the measles Schneider-Schaulies, Wu¨rzburg, Germany. The anti-H mAb L77 was virus glycoprotein was determined by indirect immuno- provided by J. Schneider-Schaulies. T. Matthews, Department of Sur- fluorescence using the mouse mAb L77 and a FITC- gery, Duke University Medical Center (Durham, NC), provided T20. The caspase substrate D2R was provided by H. Hug, Universita¨ts Kinder- labeled goat anti-mouse antiserum (Dako, Hamburg, klinik Ulm, Germany. The following reagents were obtained through the Germany). In the experiments presented in this study, AIDS Research and Reference Reagent Program, Division of AIDS, Na- infection rates were 70–90%. Formation of syncytia was tional Institutes of Health: the anti-gp120 hybridoma 902 from B. Chesebro, regularly monitored by light microscopy. the anti-CD4 hybridoma SIM.2 from J. Hildreth, and the T lymphoblast cell line A3.01 from T. Folks. This study was supported by the Deutsche Forschungsgemeinschaft, a BMBF collaborative research project on SIV End-over-end rotation of cultured cells pathogenesis, and EU Contract Number BMH4-CT97-2055. A3.01 T lymphoblasts (3 ϫ 10 6) were infected with vac/env for2hatamultiplicity of infection of 10 plaque- REFERENCES forming units (pfu)/cell. Cells were cultured in a 2-ml tube in a rotating rack (8–10 rotations/min) at 37°C for 16 h. A Baixeras, E., Cebrian, A., Albar, J. P., Salas, J., Martinez-A, C., Vinuela, E., ϫ 6 and Revilla, Y. (1998). 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