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Human immunodeficiency virus infection studied in CD4-expressing human-murine T-cell hybrids
VIROLOGY
168,267-273
(1989)
Human
M. TERSMETTE,*
*Central
Laboratory University
lmmunodeficiency Virus Infection Studied Human-Murine T-Cell Hybrids
J. J. M. VAN DONGEN,t P. R. CLAPHAM,+ A. GEURTS VAN KESSEL,t J. G. HUISMAN,*
in CD4-Expressing
R. E. Y. DE GOEDE,* I. L. M. WOLVERS-TEl-TERO,t R. A. WEISS,+ AND F. MIEDEMA**’
of the Netherlands Red Cross Blood Transfusion Service and Laboratory for Experimental and Clinical Immunology, of Amsterdam, Amsterdam; tDepartment of Cell Biology. Immunology, and Genetics, Erasmus University, Rotterdam. The Netherlands; and *Chester Beatty Laboratories, London, United Kingdom Received
July 6, 1988; accepted
October
14, 1988
Human immunodeficiency virus (HIV) infection was studied by means of CDCexpressing human-murine T-cell hybrids, containing a variable amount of human chromosomes. Fusion of the HPRT- murine cell line BW5147 with human T-cell acute lymphoblastic leukemia or normal human blood cells resulted in a panel of human-murine T-cell hybrids. For this study, we used four hybrids containing all or several human chromosomes, which all expressed the CD4 antigen, as assessed by different anti-CD4 monoclonal antibodies (e.g., OKT4A, Leu-3a, and MT1 51) and, in addition, a variable number of other human T-cell antigens. For infection, HTLV-IIIB-infected H9 cells, pretreated with mitomycin C, and cell-free concentrated supernatants from these cells were used. In cells. of inoculated cultures of the CD4+ T-cell hybrids, no viral antigen could be demonstrated. Culture supernatants of inoculated hybrids, except for an initial rise due to the virus inoculum, never showed reverse transcriptase activity above background. Cocultivation of these cell cultures with H9 cells did not result in detectable virus replication. Cocultivation of CD4-expressing hybrid cells with HIVinfected cells did not result in syncytium formation. Moreover, these hybrids were resistent to infection with vesicular stomatitis virus (VSV)-HIV pseudotypes. These findings imply that expression of the CD4 antigen on the cell surface is not sufficient for productive infection with HIV. The infectivity block observed in these hybrids seems to occur at the level of virus penetration, presumably at the stage of membrane fusion events. o 1989Academic Press, h.
INTRODUCTION
antibodies (Mab) directed against the CD4 molecule (Dalgleish et a/., 1984; Klatzmann et al., 1984) and it has been shown that the viral envelope glycoprotein gp120 binds to the CD4 molecule (McDougal et al., 1986). Apparently, expression of the CD4 molecule is essential for binding of HIV. Cellular factors influence other stages of the HIV replication cycle as well. For instance, it has been reported that among subclones of the T-cell line Molt-4, a significant variation in viral replication can be observed, independent of CD4 expression (Kikukawa eta/., 1986). Also, activation of the HIV promotor by cellular transcription factors has been described (Jones et al., 1986; Nabel and Baltimore, 1987). We used a set of four CD4-expressing human-murine T-cell hybrids differing in their human chromosome content as a model for the study of requirements for HIV infection. One of these hybrids contained all, and two others contained most, of the human chromosomes. Using these hybrids, we expected to identify and localize cellular molecules essential for HIV replication next to the CD4 molecule. However, it appeared that even hybrids containing most or all of the human chromosomes could not be infected by HIV, and that the infectivity block occurred at an early stage of infection.
The acquired immunodeficiency syndrome (AIDS)2 and related conditions are caused by a human retrovirus, now commonly called human immunodeficiency virus (HIV), variants of which have been described by several investigators (Barr&Sinoussi et a/., 1983; Gallo et a/., 1984; Levy et a/., 1984). This retrovirus is CD4tropic and has been shown to replicate in human CD4+ lymphocytes of T-cell origin (Klatzmann et al., 1984), but also of B-cell origin (Dalgleish et al., 1984; Montagnier et a/., 1984; Tersmette et al,, 1985; Levy et a/., 1985). In addition, virus replication in CD4-expressing human myeloid cells has been reported (Levy et al., 1985). Infection by HIV can be blocked by monoclonal ’ To whom requests for reprints should be addressed: c/o Publication Secretariat, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, P.O. Box 9406, 1006 AK Amsterdam, The Netherlands. ’ Abbreviations used: AIDS, acquired immune deficiency syndrome; HIV, human immune deficiency virus; Mab, monoclonal antibody; RT, reverse transcriptase; PEG, polyethylene glycol; FITC, fluorescein isothiocyanate; T-ALL, T-cell acute lymphoblastic leukemia; LFA-1, lymphocyte function-associated antigen; CPE. cytopathic effect; TCA. trichloroacetic acid; VSV, vesicular stomatitis virus.
267
0042.6822/89
$3.00
Copyrtght 0 1989 by Academic Press. Inc All rights of reproduction I” any form reserved.
268
TERSMETTE
MATERIALS
AND
METHODS
Cell lines t-l9 cells, uninfected, and HIV-infected (strain HTLVIIIB) cells (Popovic et al., 1984), kindly provided by Dr. R. C. Gallo, were cultured in Iscove’s modified Dulbecco’s medium, supplemented with 10% fetal calf serum and antibiotics. Human-murine
T-cell hybrids
Human-murine somatic cell hybrids of the BWSP series were constructed between the murine hypoxanthine phosphoribosyltransferase-deficient HPRT- AKR thymoma cell line BW5147 (Goldsby et al., 1977) and the leukemic T-cells of a patient suffering from a T-cell acute lymphoblastic leukemia (T-ALL). Clone BWBU3ElO was obtained from a fusion of the BW5147 cell line and T-cells of an individual with a deficiency of the lymphocyte function-associated antigen (LFA-1) described before (Miedema et a/., 1985). T-cells of this person had a normal T-cell phenotype except for a lack of LFA-1. Fusion and subsequent hybrid selection were performed according to standard procedures (Littlefield, 1964; Pontecorvo, 1975; van Dongen et al., 1986). Phenotypic
analysis
of hybrid clones
Surface-marker expression on the hybrids was evaluated with a panel of Mabs directed against human differentiation antigens, including CD3 (T3), CD4 (T4), CD5 (Tl), CD7 (Tp41), and others, as described in detail elsewhere (van Dongen et al., 1986). CD4 expression was investigated with a panel of different Mabs against CD4, including OKT4,OKT4A, MT1 51, Leu-3a, T4-RIV6 and CLB-T4-1, and CLB-T4-2. Mabs were used in an indirect immunofluorescence assay, using a fluorescein isothiocyanate (FITC)-conjugated goat antimouse immunoglobulin antiserum as a second step reagent. The immunofluorescence staining was evaluated either by fluorescence microscopy or on a Coulter Epics-C cytofluorometer (Coulter Electronics, Hialeah, FL). Chromosome
analysis
Air-dried chromosome spreads were R-banded with acridine orange after heat denaturation. At least 10 metaphases of each hybrid cell were analyzed. The same population of cells was used for chromosome and surface marker analyses. Cell-free
infection
with
HIV
Virus inocula with infectious titers >106 (as determined by quadruplicate titrations onto 5-ml cultures of
ET AL.
H9 cells) were obtained by polyethylene glycol (PEG) 6000 (final concentration 9.4%) precipitation (1400 g, 30 min) or ultracentrifuge pelleting (120,000 g, 2 hr) of cell-free culture supernatants of HS/HTLV-IIIB cells. H9 cells, BW5147 cells, or hybrids (1 07/ml) were inoculated in the presence of polybrene (25 pglml), incubated for 1 hr at 37”, and cultured at cell densities of 0.5-l X 1 06/ml. PHA-stimulated mononuclear cells (5 x 1 06) of both human fusion partners were inoculated with cell-free HS/HTLV-IIIB supernatant (1 ml), incubated for 1 hr at 37”, washed, and cultured at 0.5-l X 1 O6 cells/ml. Transmission
of HIV by cocultivation
procedures
HTLV-IIIB-infected H9 cells were treated for 30 min with mitomycin C (25 pg/ml), washed extensively, and concentrated to 0.5 X lo7 cells/ml. This cell suspension was mixed in 1: 1 ratio with target cells (l-2 X 1 07/ ml) and incubated for 1 hr with polybrene (25 pg/ml). The cells were cultured at densities of 0.5-l X 1 O”/ml. Reverse
transcriptase
(RT) assay
For determination of RT activity, 5 ml of culture supernatant was precipitated with PEG 6000, final concentration 9.4% (1400 g, 30 min). The pellet was resuspended in 300 ~1 of a 50 mn/rTris-HCI buffer, pH 7.5, containing 0.25 M KCI, 20% (v/v) glycerol, and 0.25% Triton X-l 00. After three cycles of freeze-thawing, 10 ~1 of this suspension was added to 40 ~1 of a reaction mixture [lo0 mA# Tris-HCI, pH 7.5, containing 6.25 mM DTT, 7.5 mM MgCI,, [3H]lTP (sp act 1.59 TBq/ mmol, 37 kBq/assay) and poly(A) dT10 (0.625 U/ml; purchased from Pharmacia Fine Chemicals, Uppsala, Sweden)]. The mixture was incubated for 1 hr at 37”. Then the reaction was stopped by addition of 10 ~1 of yeast RNA (5 mg/ml) and 10% cold TCA containing 10 mM sodium pyrophosphate. Precipitated activity was collected on filters and counted by liquid scintillation. Detection of viral antigens preparations
in cytocentrifuge
Cells from cultures inoculated with cell-free virus were spun onto slides and air-dried for 3 hr without a fixation procedure. Viral membrane or cytoplasmic proteins were detected by a human anti-HIV serum, recognizing all major viral proteins on immunoblot, and, as a second step reagent, either FITC-conjugated goat antihuman IgG or biotinylated anti-human lg antibodies, followed by preformed avidin-biotin complex-coupled peroxidase and substrate (3 amino-9-ethylcarbazole, Sigma Chemical Co., St. Louis, MO).
CD4-EXPRESSING
T-CELL
HYBRIDS TABLE
SURFACE-MARKER
EXPRESSION AND HUMAN
INSUSCEPTIBLE
TO
1 2 3 4
clone
BWSP-BE10 BWSP-2F6 BWSP-lFl0 BWBU-3ElO
Human
chromosomes
CHROMOSOME
9, 12,20,x All, except, All All, except
6,7,
present
15, 18-20,
22
1, 2. 9, 15-20
CONTENT OF SELECTED HYBRID CLONES
induction
assay
Long-term HIV (HTLV-lIIB)-infected 24 cells (an Epstein-Barr virus (EBV)-transformed B-cell line) (Tersmette eta/., 1985) were cocultivated at a concentration of 2 X 105/ml with CD4-positive target cells (1 X 1 06/ ml). Cultures were observed for the presence of syncytia after 16, 40, and 64 hr. As positive indicator cells, H9 cells were used. Vesicular
stomatitis
virus (VSV) pseudotype
assay
VSV(HIV) and VSV(MuLV) pseudotypes were prepared as described before (Clapham el a/., 1984). Briefly, cells chronically infected with either HIV or MuLV are superinfected with VSV, Indiana serotype. A proportion of the resulting VSV progeny carries retroviral glycoproteins in its envelope. For these pseudotypes, the host range is restricted to cells carrying the viral receptor. Following penetration and uncoating, however, the VSV genome contained in the pseudotype particle replicates to produce normal VSV progeny. For titration of VSV pseudotypes, excess anti-VSV serum is added to neutralize normal VSV particles. VSV pseudotypes were titrated onto adherent or poly+lysne-attached target cells in 30-mm wells. After a 1-hr incubation at 37”, cells were washed and lung mink cells (CCL 64), susceptible to secondary VSV infection, were added as indicator cells. Subsequently, cultures were overlaid with agar medium. Plaques were counted 1 and 2 days after plating of pseudotypes. Specificity of VSV(HIV)-induced plaques was tested by adding a human serum with high-titer HIV-neutralizing antibodies. RESULTS Four human-murine CD4-expressing hybrids, resulting from fusions of the murine BW5147 cell line with human T-cells, were selected for infectivity experiments with HIV (van Dongen et al., 1986) (Table 1). In
of human
T-cell
antigens
CD3 (T3)
CD4 (T4)
CD5 (Tl)
CD7 (Tp41)
0 0 20 0
78” 71b 24a 57b
0 54 35 0
0 61 67 41
Note. The presence of human chromosomes was determined as described under Materials sion experiments are expressed as percentage positive cells. a CD4 Mab used: Leu-3A. OKT4, T4-RIV6, CLB T4-1, and CLB T4-2. ’ CD4 Mab used: Leu-BA, MT1 5l,OKT4,OKT4A, T4-RIV6, CLB T4-1, and CLB T4-2.
Syncytium
269
1
Expression Hybrid
HIV
and Methods.
Results
of surface-marker
expres-
some clones, next to CD4, other human T-cell differentiation antigens, such as CD3 (T3), CD5 (Tl), and CD7 (Tp41), were also expressed. In a previous report, it was shown that the genes encoding the CD4 antigen are located on chromosome 12 in man (van Dongen et a/., 1986). Chromosome analysis of the four selected hybrids revealed that all four hybrids contained chromosome 12 as well as other human chromosomes. Hybrid BWSP-1 FlO contained all human chromosomes. Peripheral mononuclear cells of donors Bu. and Sp., used to constitute these hybrids, were readily infected with cell-free HS/HTLV-IIIB supernatant as judged by the presence of syncytia and RT activity in the supernatant on Day 5 (25,000 and 167,000 cpm, respectively). In an initial experiment, cell-free infection was attempted of a CD4+ hybrid (BWSP-3El O), using H9 cells as a positive control, and a CD4- hybrid (BWSP-2C6) and the murine fusion partner as negative controls (Table 2). The inoculated cell lines or hybrid clones were TABLE
2
LACK OF VIRUS REPLICATION IN CULTURES OF CD4+ T-CELL AFTER INOCULATION WITH CELL-FREE HIV Reverse Cell line BW5147 BWSP-BE10 BWSP-2C6 H9
Day
transcriptase
activity
HYBRIDS
(cpm x 1 O-3)
6
9
13
19
27
1,1 0. 9 1.8 26, 6
1, 1 0,9 0,7 48, 6
1.0 0,7 0,6 nt
0,3 0,2 0,7 nt
0.4 0,3 0,2 nt
Note. On Day 0, cultures of a CD4+ hybrid (BWSP-BE1 O), a CD4hybrid (BWSP-2C6), the murine fusion partner BW5147, and H9 cells (1 07/ml) were inoculated with PEG-precipitated HS/HTLV-IIIB supernatant as described under Materials and Methods. Supernatants of inoculated cultures were repeatedly tested for RT activity. Results shown are the means of duplicate experiments.
TERSMETTE
270 TABLE HIV
3
REPLICATION NOT DETECTABLE IN CD4+ T-CELL HYBRIDS COCULTIVATION WITH HS/HTLV-IIIB CELLS
Reverse activity
AFTER
Detection of infectious virus after
transcnptase (cpm X 1 Om3)
Method of transmission
Day 5
12
I5
20
cocultivation with H9 cells
BW5147
Cell-free Cocultivation
22,9 0, 9
1,3 0, 5
ND 0,9
ND l,o
-
BWBU-BE10
Ceil-free Cocultivation
46.8 0. 7
I,6 0. 7
1,8 0,7
0,7 0.7
-
Cell-free Cocultivation
46,l 1, 4
1,5 0, 7
1,2 1,50.5
2,2
-
Cell-free Cocultivation
50,6 80,5
Cell lines
BWSP-1 H9
FlO
48,5 32,7
Note. Target cells were cocultivated oculated with ultracentrifuge-pelleted with an infectious titer of lo9 on Day tested for RT activity. After 3 weeks, cocultivated with H9 cells to detect the
ND 207, 1
-
ND ND
+ +
with HS/HTLV-IIIB cells or inHS/HTLV-IIIB supernatant 0. Cultures were repeatedly cells of these cultures were presence of infectious virus.
followed up to 27 days after inoculation for the presence of cytopathic effect (CPE) and for RT activity in the supernatant (Table 2). A rapid infection of H9 cells with extensive syncytium formation was observed, but neither virus replication nor syncytium formation could be demonstrated in the hybrids or the BW5 147 cell line. In similar experiments, two other CD4+ hybrids (BWSP1 FlO and BWSP-2F6) also could not be infected with a cell-free HIV inoculum (not shown). Since the CD4+ T-cell hybrids could possibly be more sensitive to cell-to-cell transmission of the virus, it was investigated whether infection with HIV could be achieved by cocultivation techniques. Cultures of BW5147, BWBU-3E10, BWSP-1 Fl 0, and H9 were cocultivated with mitomycin-C-treated HS/HTLV-IIIB cells and, in parallel, inoculated with high-titer (10’) cell-free HIV. The cultures were repeatedly checked for virus replication by RT activity in the supernatants for 26 days. In the CD4+ T-cell hybrids, RT activity was never detectable above background values, whether they were infected by cell-free transmission or by cocultivation procedures (Table 3). The RT activity found on Day 5 after inoculation with high-titer HIV probably is due to a direct detection of the inoculum in the cultures that has disappeared on Day 12 (Table 3). H9 cell cultures infected either way showed persistent high RT activity in their supernatants. Inoculated hybrids were repeatedly evaluated for the expression of viral proteins in cytocentrifuge preparations. In none of these cells were viral proteins detectable, whereas up to 70% of HIV-
ET AL.
infected H9 cells were clearly positive for viral protein expression. To exclude that low levels of virus, undetectable by the techniques mentioned above, were produced, it was attempted to recovervirus from the HIV-inoculated T-cell hybrids by cocultivation with H9 cells. For these experiments, hybrids were used that were infected at least 3 weeks before to prevent isolation of the original inoculum. After cocultivation, cultures were kept for 3 weeks and were regularly checked for CPE and RT activity. In none of these experiments was virus production detected. The hybrids BWSP-2F6 and BWBU-3E10, and the murinefusion partnerwere also tested in the syncytium induction assay. Syncytium formation was not observed when these hybrids or BW5147 were cocultivated with HIV-infected 24 cells. In contrast, cocultivation of 24/HIV cells with H9 cells resulted in extensive syncytium formation within 24 hr. To investigate whether HIV could properly penetrate these CD4-expressing hybrids, BWSP-2F6 and BWBU3ElO were also tested in the VSV pseudotype assay with VSV(HIV) and VSV(MuLV) pseudotypes. The binding and penetration of these pseudotypes are determined by the retroviral glycoproteins. Once uncoated, the VSV pseudotype gives rise to normally coated VSV that infects the mink lung cells added as indicator cells, resulting in plaque formation. Infectivity titers of VSV (HIV) in these hybrids were similar to the background levels observed with BW5147, the murine fusion partner, and were not reduced by addition of an HIV-neutralizing human serum (Table 4). In the same experiment, high-titer infectivity with VSV(HIV) pseudotypes TABLE CD4+
HYBRIDS
4
ARE RESISTENTTO INFECTION PSEUDOIYPES
WITH
VSV(HIV)
Cells CCRF-CEM
BWBU-3ElO
BWSP-2F6
BW5147
vsv pseudotypes HTLV-III RF HTLV-III RF MuLV-eco
Antibody treatment (human anti-HIV antiserum) + -
HTLV-III RF HTLV-III RF MuLV-eco
-
HTLV-Ill RF HTLV-III RF MuLV-eco
-
HTLV-III RF HTLV-III RF MuLV-eco
-
+ + + -
Titer (PFU/ml) 60,000 160 200 180 160 16,000 320 140 13,000 340 140 8,000
CD4-EXPRESSING
T-CELL
HYBRIDS
was observed in the CD4-expressing human CCRFCEM cell line. This titer was diminished for over 99% by addition of an HIV-neutralizing serum. VSV(MuLV) pseudotypes readily infected both hybrids and BW5147, indicating that these hybrids are susceptible to a VSV pseudotype (Table 4). DISCUSSION In the present study, we investigated whether the susceptibility to HIV is determined solely by the expression of the CD4 molecule, or if additional requirements for HIV infection and replication exist. For this purpose, we used human-murine T-cell hybrid clones that expressed the CD4 antigen. Peripheral blood mononuclear cells of the two human fusion partners could be infected with HIV. The CD4 expression of the four hybrids well exceeded the levels of CD4 expression observed in some permissive T-cell lines (Levy et al., 1985) and B-cell lines (Klatzmann et a/., 1984; Levy et al., 1985). All hybrids expressed CD4 epitopes, detected by Mabs Leu-3A, MT1 51, or OKT4A (Table l), which have been shown to be critical for binding of HIV to the CD4 molecule (Sattentau eta/., 1986). Nevertheless, productive infection with HIV could not be achieved in these hybrids in several experiments, by cell-free transmission or by cocultivation. These experiments demonstrate that the expression of the CD4 molecule is not sufficient for productive infection with HIV. Levy et a/. (1986) bypassed requirements for receptor expression, penetration, virus uncoating, and synthesis of double-stranded viral DNA by directly transfecting a biologically active molecular clone of HIV(ARV2) into mouse and human cells. The transfected mouse cells were able to support virus replication, although at a low level compared to human nonlymphoid cells, suggesting transcriptional or post-transcriptional repression. Since in these CD4+ T-cell hybrids a total absence of replication is observed, it is likely that other blocking mechanisms at an earlier stage of infection are present. Syncytium formation between HIV-infected cells and uninfected CD4-expressing cells has been shown to be based on the interaction between gp120 and the CD4 molecule (Lifson et a/., 1986; Sodroski et al., 1986). The process is independent of virus replication in the target cell, since effector cells expressing only gpl20 in the absence of other viral proteins are sufficient, and since syncytium formation may occur within 4 hr after cocultivation (Lifson et al., 1986; Sodroski et a/., 1986). Infection with cell-free virions and syncytium formation are thought to be mediated through similar mechanisms (Lifson et a/., 1986).
INSUSCEPTIBLE
TO
HIV
271
Previously, we found that HIV-infected cells of the EBV-transformed B-cell line, named 24, induced rapid syncytium formation upon cocultivation with CD4-expressing cells (Gruters et al., 1987). Although both BWSP-2F6 and BWBU-3ElO cells have a high level of CD4 expression, cocultivation of these hybrids with HIV-infected 24 cells did not result in detectable syncytia. In the VSV pseudotype assay the VSV(HIV) infection is dependent on the retroviral envelope only for the initial phase of infection and is VSV-dependent from the moment the viral nucleocapsid is uncoated. BWSP-2F6 and BWBU-3E10 hybrid cells appeared to be resistent to VSV(HIV) pseudotype infection; in contrast, like BW5147 cells, they were susceptible to VSV(MuLV) pseudotypes. The combined results of the syncytium induction assay and the VSV pseudotype assay therefore support the hypothesis that an early block of infection, that occurs after binding but before uncoating of the viral nucleocapsid, is present in these hybrids. Evidence has been obtained that indicates that the primary mechanism by which HIV enters the cell involves direct fusion of the virus to the cell membrane (Stein et a/., 1987). Our results indicate that in the hybrids this fusion event, thought to be mediated by gp41 (Kowalski et a/., 1987) does not take place. The precise cause of this defect remains uncertain. Maddon et a/. (1986) have demonstrated that T4+transformed mouse cells do bind HIV but cannot be infected by HIV or HIV pseudotypes, also indicating a block at the penetration level. In contrast, T4+-transformed human cells of lymphoid and nonlymphoid lineages could be infected with HIV(Maddon eta/., 1986). These results could imply that in the T4+-transformed mouse cells a human household gene product necessary for HIV penetration is missing, which may be expressed in the hybrids used in our study. Three of these hybrids contain all or most human chromosomes, and most human chromosomes are present in at least two of these hybrids. Somatic cell hybrids as a rule express the constitutive phenotypes of both partners (Davis and Adelberg, 1973). Gene extinction, described for intra- and interspecies somatic cell hybrids, seems to apply mainly to differentiated functions (Fougere and Weiss, 1978; Koeffler et al., 1981), as would be expected on theoretical grounds (Davis and Adelberg, 1973). Thus, although most, if not all, human household genes may be expected to be expressed in at least one of the hybrids, our experiments do not provide evidence for a specific human gene product next to CD4 necessary for HIV-cell fusion. Nevertheless, this possibility cannot be ruled out completely.
272
TERSMETTE
Recently, it has been shown that upon binding of HIV to the CD4 receptor, PKC-dependent phosphorylation of CD4 is required for penetration of the virus (Fields et a/., 1988). It may well be that human CD4 expressed on these hybrids is not linked to a signal-transducing pathway that results in PKC activation. An alternative explanation could be that, upon gpl20 binding, some conformational change of the CD4 molecule has to occur to allow the virus-to-cell fusion to proceed. It can be envisaged that due to steric hindrances or reduced mobility of the CD4 molecule (Maddon et a/., 1986), such a conformational change is prohibited in a cell membrane composed of human and murine constituents. Studies with somatic cell hybrids as described here may be of use for closer analysis of the requirements for the virus-to-cell fusion process. ACKNOWLEDGMENTS We are indebted to Dr. C. J. M. Melief and Prof. Dr. R. Benner for encouraging this work and for helpful discussions, and to Dr. R. A. W. van Lier for a gift of Mabs CLB-T4-1 and -2. This study was supported in part by a grant from the Netherlands Foundation for Preventive Medicine and by a grant from the Netherlands Cancer Organization (Koningin Wilhelmina Fonds). F. Miedema is a fellow of The Royal Dutch Academy of Arts and Sciences.
REFERENCES BARR~SINOUSSI, F., CHERMANN, J. C., REY, R., NUGEYRE, M. T., CHAMARET, S., GRUEST, J., DAUGUET, C., ROZEN~AUM, W., and MONTAGNIER, L. (1983). Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome. Science 220,868-871. CLAPHAM, P., NAGY, K., and WEISS, R. A. (1984). Pseudotypes of human T-cell leukemia virus types 1 and 2: Neutralization by patients’ sera. Proc. Nati. Acad. Sci. USA 81,2886-2889. DALGLEISH, A. G., BEVERLEY, P. C. L., CLAPHAM, P. R., CRAWFORD, D. H., GREAVES, M. F., and WEISS, R. A. (1984). The CD4 (l4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature (London) 312, 763-767. DAVIS, F. M., and ADELEERG, A. D. (1973). Use of somatic cell hybrids for analysis of the differentiated state. Bacterial. Rev. 37, 197-214. FIELDS, A. P., BEDNARIK, D. P., HESS, A., and SRATFORD MAY, W. (1988). Human immunodeficiency virus induces phosphorylation of its cell surface receptor. Nature (London) 333, 278-280. FOUG~RE. C., and WEISS, M. C. (1978). Phenotypic exclusion in mouse melanoma-rat hepatoma hybrid cells: Pigment and albumin production are not re-expressed simultaneously. Cell 15,843854. GALLO, R. C., SALAHUDDIN, S. Z., POPOVIC, M., SHEARER, G. M., KAPLAN, M., HAYNES, B. F.. PALKER, T. J., REDFIELD. R., OLESKO, J., SAFAI, B., WHITE, G., FOSTER, P., and MARKHAM, P. D. (1984). Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk of AIDS. Science 224, 500503. GOLDSBY, R. A., OSBORNE, B. A., SIMPSON, E., and HERZENBERG, L. A. (1977). Hybrid cell lines with T-cell characteristics. Nature (London) 267,707-708.
ET AL. GRUTERS, R. A., NEEFJES, J. J., TERSMET~E, M., GOEDE, R. E. Y. DE, TULP, A., HUISMAN, J. G., MIEDEMA, F., and PLOEGH. H. L. (1987). Interference with HIV-induced syncytium formation and viral infectivity by inhibitors of trimming glucosidase. Nature (London) 330, 74-77. JONES, K. A., KADONAGA, 1. T., LUCIW, P. A., and TJIAN. R. (1986). Activation of the AIDS retrovirus promoter by the cellular transcription factor, Spl. Science 232, 755-758. KIKUKAWA, R., KOYANAGI, Y., HARADA, S., KOBAYASHI, N.. HATANAKA, M., and YAMAMOTO, N. (1986). Differential susceptibility to the acquired immunodeficiency syndrome retrovirus in cloned cells of human leukemic T-cell line Molt-4. /. !/ire/. 57, 1159-l 162. KLATZMANN, D. F., BARR~SINOUSSI, F., NUGEYRE, M. T., DAUGUET. C.. VILMER, E., GRISCELLI, C., BRUN-VEZINET, F., ROUZIOUX, C.. GLUCKMAN, J. C., CHERMANN, J. C.. and MONTAGNIER, L. (1984). Selective tropism of lymphadenopathy-associated virus (LAV) for helper-inducerT lymphocytes. Science 225,59-62. KLATZMANN, D., CHAMPAGNE, E., CHAMARET, S., GRUEST, J., GUETARD, D., HERCEND, T., GLUCKMAN, J.-C., and MONTAGNIER. L. (1984). Tlymphocyte T4 molecule behaves as the receptor for human retrovirus LAV. Nature (London) 312,767-768. KOEFFLER, P. H., SPARKES, R. S., BILLING, R., and KLEIN, G. (1981). Somatic cell hybrid analyses of hematopoietic differentiation. Blood 58,1159-1163. KOWALSKI, &I., POTZ, J., BASIRIPOUR, L., DORFMAN, T., GOH, W. C., TERWILLIGER, E., DAYTON, A., ROSEN, G., HASELTINE, W., and SODROSKI, J. (1987). Functional regions of the envelope glycoprotein of human immunodeficiency virus type I. Science 237, 1351-l 355. LEVY, 1. A., CHENG-MAYERS, C.. DINA, D., and LUCIW. P. A. (1986). AIDS retrovirus (ARV-2) clone replicates in transfected human and animal fibroblast. Science 232, 998-l 001. LEVY, J. A., HOFFMAN, A. D., KRAMER, S. H., LANDIS, J. A., SHIMABUKURO, J. M., and OSHIRO. L. S. (1984). Isolation of lymphocytopathic retroviruses from San Francisco patients with AIDS. Science 225, 840-842. LEVY, J. A., SHIMABUKURO, J., MCHUGH, T., CASAVANT, C., SITES, D., and OSHIRO, L. (1985). AIDS-associated retroviruses (ARV) can productively infect other cells besides human T helper cells. W-o/ogy 147,441-448. LIFSON, J. D., FEINBERG, M. B., REYES. G. R., RABIN, L., BANAPOUR, B., CHAKRABANTI, S.. Moss, B., WONG STAAL, F., STEINER. K. S.. and ENGLEMAN, E. G. (1986). Induction of CD4-dependent cell fusion by the HTLVIII/LAV envelope glycoprotein. Nature (London) 323, 725-728. LITTLEFIELD, J. W. (1964). Selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants. Science 145, 709-710. MADDON, J. P., DALGEISH. A. G., MCDOUGAL, J. S., CLAPHAM, P. R.. WEISS, R. A., and AXEL, R. (1986). The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain. Cell47, 333-348. MCDOUGAL, 1. S.. KENNEDY, M. S., SLIGH, J. M., CORT, S. P., MAROLE, A., and NICHOLSON, J. K. A. (1986). Binding of HTLV-III/LAV to T4+ T cells by a complex of the 1 1 O-K viral protein and theT4 molecule. Science 231,382-385. MIEDEMA. F., TE~TEROO. P. A. T., TERPSTRA, F. G., KEIZER, G., Roos, M., WEENING, R. S., WEEMAES, C. M. R., Roos, D., and MELIEF, C. J. M. (1985). Immunological studies with LFA-l- and Mo-l-deficient lymphocytes from a patient with recurrent bacterial infections. 1. Immunol. 134,3075-308 1. MONIAGNIER. L., GRUEST, J., CHAMARET, S.. DAUGUET, C., AXLER, C., GUETARD, C.. NUGEYRE, M. T., BARRE-SINOUSSI, F., CHERMANN, 1. C., BRUNT, J. B., KLATZMANN, D., and GLUCKMAN, J. C. (1984). Adaption
CD4-EXPRESSING
T-CELL
HYBRIDS
of lymphadenopathy-associated virus (LAV) to replicate in EBVtransformed B lymphoblastoid cell lines. Science 225, 63-66. NABEL. G., and BALTIMORE. D. (1987). An inducible transcription factor activates expression of human immunodeficiency virus in T cells. Nature (London) 326, 71 l-71 3. PONTECORVO, G. (1975). Production of mamalian somatic cell hybrids by means of polyethylene glycol treatment. Somatic Cell. Genet. 1,397-400. POPOVIC, M.. SARNGADHARAN, (1984). Detection, isolation pathic retroviruses (HTLV-III) AIDS. Science 224,497-500. SAT~ENTAU, Q. J., DALGLEISH, P. C. L. (1986). Epitopes of Science 234, 1120-l 123.
M. G., READ, E., and GALLO, R. C. and continuous production of cytofrom patients with AIDS and preA. G.. WEISS, R. A., and BEVERLEY, the CD4 antigen and HIV Infection.
INSUSCEPTIBLE
TO
HIV
273
SODROSKI, J.. GOH, W. C., ROSEN, C., CAMPBELL, K., and HASELTINE, W. A. (1986). Role of the HTLVIII/LAV envelope in syncytium formation and cytopathicity. Nature (London) 322, 470-474. STEIN, B. S., GOWDA, S. D., LIFSON, J. D., PENHALLOW, R. C., BENSCH, K. G., and ENGLEMAN, E. G. (1987). pHindependent HIV entry into CD4-positive T cells via virus envelope fusion to the plasma membrane. Ce/l49,659-668. TERSMETTE, M., MIEDEMA, F.. HUISMAN, J. G., GOUDSMIT, J., and MELIEF, C. J. M. (1985). Productive HTLV-III infection of human B cell lines. Lancet 1,815-816. VAN DONGEN, J. J. M., GEURTSVAN KESSEL, A. H. M., WOLVERS-TET~ERO, J. L. M., VERZNET, M. A., OUDENAREN, A. VAN, SCHOENMAKER, E., and HAGEMEYER, A. (1986). Construction of human-mouse T-cell hybrids that express human T-cell-associated surface antigens and allow chromosomal localisation of these antigens. J. lmmunol. 137,1047-1053.
168,267-273
(1989)
Human
M. TERSMETTE,*
*Central
Laboratory University
lmmunodeficiency Virus Infection Studied Human-Murine T-Cell Hybrids
J. J. M. VAN DONGEN,t P. R. CLAPHAM,+ A. GEURTS VAN KESSEL,t J. G. HUISMAN,*
in CD4-Expressing
R. E. Y. DE GOEDE,* I. L. M. WOLVERS-TEl-TERO,t R. A. WEISS,+ AND F. MIEDEMA**’
of the Netherlands Red Cross Blood Transfusion Service and Laboratory for Experimental and Clinical Immunology, of Amsterdam, Amsterdam; tDepartment of Cell Biology. Immunology, and Genetics, Erasmus University, Rotterdam. The Netherlands; and *Chester Beatty Laboratories, London, United Kingdom Received
July 6, 1988; accepted
October
14, 1988
Human immunodeficiency virus (HIV) infection was studied by means of CDCexpressing human-murine T-cell hybrids, containing a variable amount of human chromosomes. Fusion of the HPRT- murine cell line BW5147 with human T-cell acute lymphoblastic leukemia or normal human blood cells resulted in a panel of human-murine T-cell hybrids. For this study, we used four hybrids containing all or several human chromosomes, which all expressed the CD4 antigen, as assessed by different anti-CD4 monoclonal antibodies (e.g., OKT4A, Leu-3a, and MT1 51) and, in addition, a variable number of other human T-cell antigens. For infection, HTLV-IIIB-infected H9 cells, pretreated with mitomycin C, and cell-free concentrated supernatants from these cells were used. In cells. of inoculated cultures of the CD4+ T-cell hybrids, no viral antigen could be demonstrated. Culture supernatants of inoculated hybrids, except for an initial rise due to the virus inoculum, never showed reverse transcriptase activity above background. Cocultivation of these cell cultures with H9 cells did not result in detectable virus replication. Cocultivation of CD4-expressing hybrid cells with HIVinfected cells did not result in syncytium formation. Moreover, these hybrids were resistent to infection with vesicular stomatitis virus (VSV)-HIV pseudotypes. These findings imply that expression of the CD4 antigen on the cell surface is not sufficient for productive infection with HIV. The infectivity block observed in these hybrids seems to occur at the level of virus penetration, presumably at the stage of membrane fusion events. o 1989Academic Press, h.
INTRODUCTION
antibodies (Mab) directed against the CD4 molecule (Dalgleish et a/., 1984; Klatzmann et al., 1984) and it has been shown that the viral envelope glycoprotein gp120 binds to the CD4 molecule (McDougal et al., 1986). Apparently, expression of the CD4 molecule is essential for binding of HIV. Cellular factors influence other stages of the HIV replication cycle as well. For instance, it has been reported that among subclones of the T-cell line Molt-4, a significant variation in viral replication can be observed, independent of CD4 expression (Kikukawa eta/., 1986). Also, activation of the HIV promotor by cellular transcription factors has been described (Jones et al., 1986; Nabel and Baltimore, 1987). We used a set of four CD4-expressing human-murine T-cell hybrids differing in their human chromosome content as a model for the study of requirements for HIV infection. One of these hybrids contained all, and two others contained most, of the human chromosomes. Using these hybrids, we expected to identify and localize cellular molecules essential for HIV replication next to the CD4 molecule. However, it appeared that even hybrids containing most or all of the human chromosomes could not be infected by HIV, and that the infectivity block occurred at an early stage of infection.
The acquired immunodeficiency syndrome (AIDS)2 and related conditions are caused by a human retrovirus, now commonly called human immunodeficiency virus (HIV), variants of which have been described by several investigators (Barr&Sinoussi et a/., 1983; Gallo et a/., 1984; Levy et a/., 1984). This retrovirus is CD4tropic and has been shown to replicate in human CD4+ lymphocytes of T-cell origin (Klatzmann et al., 1984), but also of B-cell origin (Dalgleish et al., 1984; Montagnier et a/., 1984; Tersmette et al,, 1985; Levy et a/., 1985). In addition, virus replication in CD4-expressing human myeloid cells has been reported (Levy et al., 1985). Infection by HIV can be blocked by monoclonal ’ To whom requests for reprints should be addressed: c/o Publication Secretariat, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, P.O. Box 9406, 1006 AK Amsterdam, The Netherlands. ’ Abbreviations used: AIDS, acquired immune deficiency syndrome; HIV, human immune deficiency virus; Mab, monoclonal antibody; RT, reverse transcriptase; PEG, polyethylene glycol; FITC, fluorescein isothiocyanate; T-ALL, T-cell acute lymphoblastic leukemia; LFA-1, lymphocyte function-associated antigen; CPE. cytopathic effect; TCA. trichloroacetic acid; VSV, vesicular stomatitis virus.
267
0042.6822/89
$3.00
Copyrtght 0 1989 by Academic Press. Inc All rights of reproduction I” any form reserved.
268
TERSMETTE
MATERIALS
AND
METHODS
Cell lines t-l9 cells, uninfected, and HIV-infected (strain HTLVIIIB) cells (Popovic et al., 1984), kindly provided by Dr. R. C. Gallo, were cultured in Iscove’s modified Dulbecco’s medium, supplemented with 10% fetal calf serum and antibiotics. Human-murine
T-cell hybrids
Human-murine somatic cell hybrids of the BWSP series were constructed between the murine hypoxanthine phosphoribosyltransferase-deficient HPRT- AKR thymoma cell line BW5147 (Goldsby et al., 1977) and the leukemic T-cells of a patient suffering from a T-cell acute lymphoblastic leukemia (T-ALL). Clone BWBU3ElO was obtained from a fusion of the BW5147 cell line and T-cells of an individual with a deficiency of the lymphocyte function-associated antigen (LFA-1) described before (Miedema et a/., 1985). T-cells of this person had a normal T-cell phenotype except for a lack of LFA-1. Fusion and subsequent hybrid selection were performed according to standard procedures (Littlefield, 1964; Pontecorvo, 1975; van Dongen et al., 1986). Phenotypic
analysis
of hybrid clones
Surface-marker expression on the hybrids was evaluated with a panel of Mabs directed against human differentiation antigens, including CD3 (T3), CD4 (T4), CD5 (Tl), CD7 (Tp41), and others, as described in detail elsewhere (van Dongen et al., 1986). CD4 expression was investigated with a panel of different Mabs against CD4, including OKT4,OKT4A, MT1 51, Leu-3a, T4-RIV6 and CLB-T4-1, and CLB-T4-2. Mabs were used in an indirect immunofluorescence assay, using a fluorescein isothiocyanate (FITC)-conjugated goat antimouse immunoglobulin antiserum as a second step reagent. The immunofluorescence staining was evaluated either by fluorescence microscopy or on a Coulter Epics-C cytofluorometer (Coulter Electronics, Hialeah, FL). Chromosome
analysis
Air-dried chromosome spreads were R-banded with acridine orange after heat denaturation. At least 10 metaphases of each hybrid cell were analyzed. The same population of cells was used for chromosome and surface marker analyses. Cell-free
infection
with
HIV
Virus inocula with infectious titers >106 (as determined by quadruplicate titrations onto 5-ml cultures of
ET AL.
H9 cells) were obtained by polyethylene glycol (PEG) 6000 (final concentration 9.4%) precipitation (1400 g, 30 min) or ultracentrifuge pelleting (120,000 g, 2 hr) of cell-free culture supernatants of HS/HTLV-IIIB cells. H9 cells, BW5147 cells, or hybrids (1 07/ml) were inoculated in the presence of polybrene (25 pglml), incubated for 1 hr at 37”, and cultured at cell densities of 0.5-l X 1 06/ml. PHA-stimulated mononuclear cells (5 x 1 06) of both human fusion partners were inoculated with cell-free HS/HTLV-IIIB supernatant (1 ml), incubated for 1 hr at 37”, washed, and cultured at 0.5-l X 1 O6 cells/ml. Transmission
of HIV by cocultivation
procedures
HTLV-IIIB-infected H9 cells were treated for 30 min with mitomycin C (25 pg/ml), washed extensively, and concentrated to 0.5 X lo7 cells/ml. This cell suspension was mixed in 1: 1 ratio with target cells (l-2 X 1 07/ ml) and incubated for 1 hr with polybrene (25 pg/ml). The cells were cultured at densities of 0.5-l X 1 O”/ml. Reverse
transcriptase
(RT) assay
For determination of RT activity, 5 ml of culture supernatant was precipitated with PEG 6000, final concentration 9.4% (1400 g, 30 min). The pellet was resuspended in 300 ~1 of a 50 mn/rTris-HCI buffer, pH 7.5, containing 0.25 M KCI, 20% (v/v) glycerol, and 0.25% Triton X-l 00. After three cycles of freeze-thawing, 10 ~1 of this suspension was added to 40 ~1 of a reaction mixture [lo0 mA# Tris-HCI, pH 7.5, containing 6.25 mM DTT, 7.5 mM MgCI,, [3H]lTP (sp act 1.59 TBq/ mmol, 37 kBq/assay) and poly(A) dT10 (0.625 U/ml; purchased from Pharmacia Fine Chemicals, Uppsala, Sweden)]. The mixture was incubated for 1 hr at 37”. Then the reaction was stopped by addition of 10 ~1 of yeast RNA (5 mg/ml) and 10% cold TCA containing 10 mM sodium pyrophosphate. Precipitated activity was collected on filters and counted by liquid scintillation. Detection of viral antigens preparations
in cytocentrifuge
Cells from cultures inoculated with cell-free virus were spun onto slides and air-dried for 3 hr without a fixation procedure. Viral membrane or cytoplasmic proteins were detected by a human anti-HIV serum, recognizing all major viral proteins on immunoblot, and, as a second step reagent, either FITC-conjugated goat antihuman IgG or biotinylated anti-human lg antibodies, followed by preformed avidin-biotin complex-coupled peroxidase and substrate (3 amino-9-ethylcarbazole, Sigma Chemical Co., St. Louis, MO).
CD4-EXPRESSING
T-CELL
HYBRIDS TABLE
SURFACE-MARKER
EXPRESSION AND HUMAN
INSUSCEPTIBLE
TO
1 2 3 4
clone
BWSP-BE10 BWSP-2F6 BWSP-lFl0 BWBU-3ElO
Human
chromosomes
CHROMOSOME
9, 12,20,x All, except, All All, except
6,7,
present
15, 18-20,
22
1, 2. 9, 15-20
CONTENT OF SELECTED HYBRID CLONES
induction
assay
Long-term HIV (HTLV-lIIB)-infected 24 cells (an Epstein-Barr virus (EBV)-transformed B-cell line) (Tersmette eta/., 1985) were cocultivated at a concentration of 2 X 105/ml with CD4-positive target cells (1 X 1 06/ ml). Cultures were observed for the presence of syncytia after 16, 40, and 64 hr. As positive indicator cells, H9 cells were used. Vesicular
stomatitis
virus (VSV) pseudotype
assay
VSV(HIV) and VSV(MuLV) pseudotypes were prepared as described before (Clapham el a/., 1984). Briefly, cells chronically infected with either HIV or MuLV are superinfected with VSV, Indiana serotype. A proportion of the resulting VSV progeny carries retroviral glycoproteins in its envelope. For these pseudotypes, the host range is restricted to cells carrying the viral receptor. Following penetration and uncoating, however, the VSV genome contained in the pseudotype particle replicates to produce normal VSV progeny. For titration of VSV pseudotypes, excess anti-VSV serum is added to neutralize normal VSV particles. VSV pseudotypes were titrated onto adherent or poly+lysne-attached target cells in 30-mm wells. After a 1-hr incubation at 37”, cells were washed and lung mink cells (CCL 64), susceptible to secondary VSV infection, were added as indicator cells. Subsequently, cultures were overlaid with agar medium. Plaques were counted 1 and 2 days after plating of pseudotypes. Specificity of VSV(HIV)-induced plaques was tested by adding a human serum with high-titer HIV-neutralizing antibodies. RESULTS Four human-murine CD4-expressing hybrids, resulting from fusions of the murine BW5147 cell line with human T-cells, were selected for infectivity experiments with HIV (van Dongen et al., 1986) (Table 1). In
of human
T-cell
antigens
CD3 (T3)
CD4 (T4)
CD5 (Tl)
CD7 (Tp41)
0 0 20 0
78” 71b 24a 57b
0 54 35 0
0 61 67 41
Note. The presence of human chromosomes was determined as described under Materials sion experiments are expressed as percentage positive cells. a CD4 Mab used: Leu-3A. OKT4, T4-RIV6, CLB T4-1, and CLB T4-2. ’ CD4 Mab used: Leu-BA, MT1 5l,OKT4,OKT4A, T4-RIV6, CLB T4-1, and CLB T4-2.
Syncytium
269
1
Expression Hybrid
HIV
and Methods.
Results
of surface-marker
expres-
some clones, next to CD4, other human T-cell differentiation antigens, such as CD3 (T3), CD5 (Tl), and CD7 (Tp41), were also expressed. In a previous report, it was shown that the genes encoding the CD4 antigen are located on chromosome 12 in man (van Dongen et a/., 1986). Chromosome analysis of the four selected hybrids revealed that all four hybrids contained chromosome 12 as well as other human chromosomes. Hybrid BWSP-1 FlO contained all human chromosomes. Peripheral mononuclear cells of donors Bu. and Sp., used to constitute these hybrids, were readily infected with cell-free HS/HTLV-IIIB supernatant as judged by the presence of syncytia and RT activity in the supernatant on Day 5 (25,000 and 167,000 cpm, respectively). In an initial experiment, cell-free infection was attempted of a CD4+ hybrid (BWSP-3El O), using H9 cells as a positive control, and a CD4- hybrid (BWSP-2C6) and the murine fusion partner as negative controls (Table 2). The inoculated cell lines or hybrid clones were TABLE
2
LACK OF VIRUS REPLICATION IN CULTURES OF CD4+ T-CELL AFTER INOCULATION WITH CELL-FREE HIV Reverse Cell line BW5147 BWSP-BE10 BWSP-2C6 H9
Day
transcriptase
activity
HYBRIDS
(cpm x 1 O-3)
6
9
13
19
27
1,1 0. 9 1.8 26, 6
1, 1 0,9 0,7 48, 6
1.0 0,7 0,6 nt
0,3 0,2 0,7 nt
0.4 0,3 0,2 nt
Note. On Day 0, cultures of a CD4+ hybrid (BWSP-BE1 O), a CD4hybrid (BWSP-2C6), the murine fusion partner BW5147, and H9 cells (1 07/ml) were inoculated with PEG-precipitated HS/HTLV-IIIB supernatant as described under Materials and Methods. Supernatants of inoculated cultures were repeatedly tested for RT activity. Results shown are the means of duplicate experiments.
TERSMETTE
270 TABLE HIV
3
REPLICATION NOT DETECTABLE IN CD4+ T-CELL HYBRIDS COCULTIVATION WITH HS/HTLV-IIIB CELLS
Reverse activity
AFTER
Detection of infectious virus after
transcnptase (cpm X 1 Om3)
Method of transmission
Day 5
12
I5
20
cocultivation with H9 cells
BW5147
Cell-free Cocultivation
22,9 0, 9
1,3 0, 5
ND 0,9
ND l,o
-
BWBU-BE10
Ceil-free Cocultivation
46.8 0. 7
I,6 0. 7
1,8 0,7
0,7 0.7
-
Cell-free Cocultivation
46,l 1, 4
1,5 0, 7
1,2 1,50.5
2,2
-
Cell-free Cocultivation
50,6 80,5
Cell lines
BWSP-1 H9
FlO
48,5 32,7
Note. Target cells were cocultivated oculated with ultracentrifuge-pelleted with an infectious titer of lo9 on Day tested for RT activity. After 3 weeks, cocultivated with H9 cells to detect the
ND 207, 1
-
ND ND
+ +
with HS/HTLV-IIIB cells or inHS/HTLV-IIIB supernatant 0. Cultures were repeatedly cells of these cultures were presence of infectious virus.
followed up to 27 days after inoculation for the presence of cytopathic effect (CPE) and for RT activity in the supernatant (Table 2). A rapid infection of H9 cells with extensive syncytium formation was observed, but neither virus replication nor syncytium formation could be demonstrated in the hybrids or the BW5 147 cell line. In similar experiments, two other CD4+ hybrids (BWSP1 FlO and BWSP-2F6) also could not be infected with a cell-free HIV inoculum (not shown). Since the CD4+ T-cell hybrids could possibly be more sensitive to cell-to-cell transmission of the virus, it was investigated whether infection with HIV could be achieved by cocultivation techniques. Cultures of BW5147, BWBU-3E10, BWSP-1 Fl 0, and H9 were cocultivated with mitomycin-C-treated HS/HTLV-IIIB cells and, in parallel, inoculated with high-titer (10’) cell-free HIV. The cultures were repeatedly checked for virus replication by RT activity in the supernatants for 26 days. In the CD4+ T-cell hybrids, RT activity was never detectable above background values, whether they were infected by cell-free transmission or by cocultivation procedures (Table 3). The RT activity found on Day 5 after inoculation with high-titer HIV probably is due to a direct detection of the inoculum in the cultures that has disappeared on Day 12 (Table 3). H9 cell cultures infected either way showed persistent high RT activity in their supernatants. Inoculated hybrids were repeatedly evaluated for the expression of viral proteins in cytocentrifuge preparations. In none of these cells were viral proteins detectable, whereas up to 70% of HIV-
ET AL.
infected H9 cells were clearly positive for viral protein expression. To exclude that low levels of virus, undetectable by the techniques mentioned above, were produced, it was attempted to recovervirus from the HIV-inoculated T-cell hybrids by cocultivation with H9 cells. For these experiments, hybrids were used that were infected at least 3 weeks before to prevent isolation of the original inoculum. After cocultivation, cultures were kept for 3 weeks and were regularly checked for CPE and RT activity. In none of these experiments was virus production detected. The hybrids BWSP-2F6 and BWBU-3E10, and the murinefusion partnerwere also tested in the syncytium induction assay. Syncytium formation was not observed when these hybrids or BW5147 were cocultivated with HIV-infected 24 cells. In contrast, cocultivation of 24/HIV cells with H9 cells resulted in extensive syncytium formation within 24 hr. To investigate whether HIV could properly penetrate these CD4-expressing hybrids, BWSP-2F6 and BWBU3ElO were also tested in the VSV pseudotype assay with VSV(HIV) and VSV(MuLV) pseudotypes. The binding and penetration of these pseudotypes are determined by the retroviral glycoproteins. Once uncoated, the VSV pseudotype gives rise to normally coated VSV that infects the mink lung cells added as indicator cells, resulting in plaque formation. Infectivity titers of VSV (HIV) in these hybrids were similar to the background levels observed with BW5147, the murine fusion partner, and were not reduced by addition of an HIV-neutralizing human serum (Table 4). In the same experiment, high-titer infectivity with VSV(HIV) pseudotypes TABLE CD4+
HYBRIDS
4
ARE RESISTENTTO INFECTION PSEUDOIYPES
WITH
VSV(HIV)
Cells CCRF-CEM
BWBU-3ElO
BWSP-2F6
BW5147
vsv pseudotypes HTLV-III RF HTLV-III RF MuLV-eco
Antibody treatment (human anti-HIV antiserum) + -
HTLV-III RF HTLV-III RF MuLV-eco
-
HTLV-Ill RF HTLV-III RF MuLV-eco
-
HTLV-III RF HTLV-III RF MuLV-eco
-
+ + + -
Titer (PFU/ml) 60,000 160 200 180 160 16,000 320 140 13,000 340 140 8,000
CD4-EXPRESSING
T-CELL
HYBRIDS
was observed in the CD4-expressing human CCRFCEM cell line. This titer was diminished for over 99% by addition of an HIV-neutralizing serum. VSV(MuLV) pseudotypes readily infected both hybrids and BW5147, indicating that these hybrids are susceptible to a VSV pseudotype (Table 4). DISCUSSION In the present study, we investigated whether the susceptibility to HIV is determined solely by the expression of the CD4 molecule, or if additional requirements for HIV infection and replication exist. For this purpose, we used human-murine T-cell hybrid clones that expressed the CD4 antigen. Peripheral blood mononuclear cells of the two human fusion partners could be infected with HIV. The CD4 expression of the four hybrids well exceeded the levels of CD4 expression observed in some permissive T-cell lines (Levy et al., 1985) and B-cell lines (Klatzmann et a/., 1984; Levy et al., 1985). All hybrids expressed CD4 epitopes, detected by Mabs Leu-3A, MT1 51, or OKT4A (Table l), which have been shown to be critical for binding of HIV to the CD4 molecule (Sattentau eta/., 1986). Nevertheless, productive infection with HIV could not be achieved in these hybrids in several experiments, by cell-free transmission or by cocultivation. These experiments demonstrate that the expression of the CD4 molecule is not sufficient for productive infection with HIV. Levy et a/. (1986) bypassed requirements for receptor expression, penetration, virus uncoating, and synthesis of double-stranded viral DNA by directly transfecting a biologically active molecular clone of HIV(ARV2) into mouse and human cells. The transfected mouse cells were able to support virus replication, although at a low level compared to human nonlymphoid cells, suggesting transcriptional or post-transcriptional repression. Since in these CD4+ T-cell hybrids a total absence of replication is observed, it is likely that other blocking mechanisms at an earlier stage of infection are present. Syncytium formation between HIV-infected cells and uninfected CD4-expressing cells has been shown to be based on the interaction between gp120 and the CD4 molecule (Lifson et a/., 1986; Sodroski et al., 1986). The process is independent of virus replication in the target cell, since effector cells expressing only gpl20 in the absence of other viral proteins are sufficient, and since syncytium formation may occur within 4 hr after cocultivation (Lifson et al., 1986; Sodroski et a/., 1986). Infection with cell-free virions and syncytium formation are thought to be mediated through similar mechanisms (Lifson et a/., 1986).
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Previously, we found that HIV-infected cells of the EBV-transformed B-cell line, named 24, induced rapid syncytium formation upon cocultivation with CD4-expressing cells (Gruters et al., 1987). Although both BWSP-2F6 and BWBU-3ElO cells have a high level of CD4 expression, cocultivation of these hybrids with HIV-infected 24 cells did not result in detectable syncytia. In the VSV pseudotype assay the VSV(HIV) infection is dependent on the retroviral envelope only for the initial phase of infection and is VSV-dependent from the moment the viral nucleocapsid is uncoated. BWSP-2F6 and BWBU-3E10 hybrid cells appeared to be resistent to VSV(HIV) pseudotype infection; in contrast, like BW5147 cells, they were susceptible to VSV(MuLV) pseudotypes. The combined results of the syncytium induction assay and the VSV pseudotype assay therefore support the hypothesis that an early block of infection, that occurs after binding but before uncoating of the viral nucleocapsid, is present in these hybrids. Evidence has been obtained that indicates that the primary mechanism by which HIV enters the cell involves direct fusion of the virus to the cell membrane (Stein et a/., 1987). Our results indicate that in the hybrids this fusion event, thought to be mediated by gp41 (Kowalski et a/., 1987) does not take place. The precise cause of this defect remains uncertain. Maddon et a/. (1986) have demonstrated that T4+transformed mouse cells do bind HIV but cannot be infected by HIV or HIV pseudotypes, also indicating a block at the penetration level. In contrast, T4+-transformed human cells of lymphoid and nonlymphoid lineages could be infected with HIV(Maddon eta/., 1986). These results could imply that in the T4+-transformed mouse cells a human household gene product necessary for HIV penetration is missing, which may be expressed in the hybrids used in our study. Three of these hybrids contain all or most human chromosomes, and most human chromosomes are present in at least two of these hybrids. Somatic cell hybrids as a rule express the constitutive phenotypes of both partners (Davis and Adelberg, 1973). Gene extinction, described for intra- and interspecies somatic cell hybrids, seems to apply mainly to differentiated functions (Fougere and Weiss, 1978; Koeffler et al., 1981), as would be expected on theoretical grounds (Davis and Adelberg, 1973). Thus, although most, if not all, human household genes may be expected to be expressed in at least one of the hybrids, our experiments do not provide evidence for a specific human gene product next to CD4 necessary for HIV-cell fusion. Nevertheless, this possibility cannot be ruled out completely.
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TERSMETTE
Recently, it has been shown that upon binding of HIV to the CD4 receptor, PKC-dependent phosphorylation of CD4 is required for penetration of the virus (Fields et a/., 1988). It may well be that human CD4 expressed on these hybrids is not linked to a signal-transducing pathway that results in PKC activation. An alternative explanation could be that, upon gpl20 binding, some conformational change of the CD4 molecule has to occur to allow the virus-to-cell fusion to proceed. It can be envisaged that due to steric hindrances or reduced mobility of the CD4 molecule (Maddon et a/., 1986), such a conformational change is prohibited in a cell membrane composed of human and murine constituents. Studies with somatic cell hybrids as described here may be of use for closer analysis of the requirements for the virus-to-cell fusion process. ACKNOWLEDGMENTS We are indebted to Dr. C. J. M. Melief and Prof. Dr. R. Benner for encouraging this work and for helpful discussions, and to Dr. R. A. W. van Lier for a gift of Mabs CLB-T4-1 and -2. This study was supported in part by a grant from the Netherlands Foundation for Preventive Medicine and by a grant from the Netherlands Cancer Organization (Koningin Wilhelmina Fonds). F. Miedema is a fellow of The Royal Dutch Academy of Arts and Sciences.
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