(~) INSTITUTPASTEUR/ELSEVIER Paris 1991
Res. Virol. 1991, 142, 183-188
Infection of monocytic cells by HI[VI: combined role of FcR and CD4 T. J o u a u l t o)(*)., F. Chapuis (1), E. Bahraoui (2) and J.-C. G l u c k m a n O) (l) Laboratoire de Biologie et Gdndtique des Dgficits Immunit¢ires, CER VL Groupe Hospitalier Pitid-Salpdtribre, 83 Bd de l'h6pital, 75651 Paris Cedex 13, and (2) C N R S URA 1179, Facultd de Mddecine Nord, 13326 Marseille Cedex 15 (France)
SUMMARY
Human immunodeficiency virus (HIV) complexed with human anti-HIV IgG can attach to FcT receptors (Fch) of mononuclear phagocytes. To determine whether the FcRmediated infection that results also requires interaction between H|V gp! 20 and cell membrane CD4, monocytic cells of the U977 line were transiently treated with phorbol 12,13-dibutyrate (PUB) so that they temporarily presented a CD4-FcR + phenotype at the time of HIV infection. HIV production was not abolished, but only significantly delayed after infection of these cells with free virus. Leu3a monoclonal antibody or soluble recombinant CD4 completely blocked this delayed infection. This indicates that enough CD4 still remained at the membrane to allow infection of a reduced cell number. Infection of PDB-treated cells with virus preincubated with high anti-HIV igG concentrations was inhibited, contrasting with what was Jbserved wit~. ~on*,,-'_ .;: ~ "--'%~.,,:~,~'~i=d under the same conditions. Inhibition of infection ,vas also observed when HIV became a__t~_chedto untreated U937 cells through the binding of CD4-1gG hybrid molecu,es to FcR. Thus, the binding of igG-coated virus to FcR is not sufficient in itself to elicit productive infection of monocytic cells, which still requires the interaction of viral gp120 and membrane CD4. Key-words: CD4, IgG, Monocyte, FcR, HIV; gp120, Antibody-mediated infection.
INTRODUCTION Membrane CD4 is the cellular receptor of human immunodeficiency virus (HIV) envelope glycoprotein gp 120 (Klatzmann et al., 1990) but monocytic cells may also be infected through the attachment of antibody-coated HIV particles to Fc-f-recepto~s (FcR). It is possible that such antibedydependent enhancement of infection (ADE) does not require the interaction of gp120 and membrane
(*) Corresponding author.
CD4. However, previous studies (Jouault et al., 1989; Matsuda et al., 1989; Perno et al., 1990; Zeira et aL, 1990; Takeda et al., 1990) have shown that monoclonal antibod,_'es (mAb) to CD4 that interfere with gp120 binding (Klatzmann et al., 1990), or soluble recombinant CD4 (srCD4), block infection of monocytic cells with immune-compined HIV. To confirm these observations, we used two different approaches to examine whether membrane CD4 is still required for ADE.
MATERIALS AND METHODS Cells and virus strain The U937 line (Sundstrom et al., 1976) was grown as described (Jouault et al., 1989). The LAVBRU strain of HIV was used. Phorbol 12,13-dibutyrate (PDB) (Sigma Chemicals, St Louis, MO, USA) was added to the cells at different concentrations for various periods at 37°C.
T. J O U A LILT E T A L .
184 Human Ig, srCD4 and h'ybrid CD4-1g
IgG from heat-inacti'~ated HlV-seropositive sera (ant:,.[ilV IgG) were purified as described (Jouault et al., 1989). F(ab') 2 fragments were obtained by pepsin digestion, srCD4 was a gift from David Klatzmann (T. Idziorek et al., 1991). The CD4-1gG preparation (CD4-H-~3) was a gift from Andr6 Traunecker (Basel Institute for Immunology, Basel, Switzerland) (Traunecker et al., 1989). Fluorescence analysis of membrane markers
Cell membrane markers were analysed with the "FACS Analyzer" (Becton-Dickinson, Mountain View, CA, USA) (Jouault et al., 1989).
Binding of 12Sl-gp160 to cells
Soluble recombinant gp 160 was a gift from Pasteur Vaccins (Marnes-la-Coquette, France) 0Oeny et al., 1988). Radiolabelling and investigation of binding of ~2Sl-gpl60 to the cells was conducted as described (Jouault et ai., 1989).
HIV-binding assay
U937 cells were incubated for 3 0 m i n at 37°C with heatinactivated HIV. Bound particles were revealed by anti-HIV F(ab')2 followed by staining with biotinylated sheep anti-human a:,_tibodies and phycoerythrin-streptavidin. Parallel experiments were performed with HIV that had been
ADE mAb .'~BD
previously complexed with antiHIV IgG.
PDB treatment modifies the susceptibility of U937 cells to HIV infection
tllV infection
Infection of PDB-treated cells by free virus was not abolished but virus replication was significantly delayed even after the addition of fresh u n t r e a t e d U937 cells (fig. 2a). This delay was similar to that observed when virus load (TCID) was reduced 10-fold in order to infect untreated cells (fig. 2b). Infection of PDB-treated cells was completely blocked in the presence of mAb Leu3a, or w~Lh virus preincubated with srCD4 (data not shown). Thus, transient membrane CD4 downregulation after PDB-treatment decreased the susceptibility of cells to HIV.
Virus (103 TCID) was incubated with or without different dilutions of anti-HIV IgG or CD4-Hy3 as described (Jouault et al., 1989). The complexed or free virus suspension was added to the cells. After 2 washes, cells were cultured without additional anti-HIV IgG. Viral production in cell-free supern a t a n t s was d e t e r m i n e d by antigen-capture e n ~ e - ! i n k e d immunosorbent assay (ELAVIA AgI Diagnostics Pasteur, Marnes-LaCoquette, France). RESULTS P D ~ treatment of U937 cells modifies the expression of membrane molecules and alters HIV binding to cells
Phorbol esters, which are known for down regulating CD4 membrane expression (Clapham et al., 1987, Hoxie et al., 1988), were used to obtain U937 cells that transiently presented a FcR+CD4 phehotype at the time of infection (fig. 1). The maximum loss of CD4 was obtained after a 5-h incubation with I lxM PDB, as determined using mAb Leu3a, which corresponded to the loss of HIV particle binding detected in flow cytometry. Attachment of antiHIVIgG-complexed 125i.gp 160 to these cells was still possible, and a comparable eiihancement of gpl60 was obtained whether they had been treated or not with PDB; membrane-bound radioactivity increased from 4 , 5 0 0 c p m to 25,000 cpm and to 23,000 cpm, respectively.
ffi antibody-dependentenhancement. = monoclonalantibody. = phorbol 12,13-dibutyrate.
Under these conditions, no increase in viral replication was observed when virus was preincubated with low anti-HIV igG concentrations as compared with free-virus-infected cells. When higher antibody amounts were used to complex virus, only limited virus production occurred (fig. 2c), contrasting with what was observed when untreated U937 cells were infected under the same cond':tion~ T h n ¢ l:?,-I~_ mediated binding of antibodycoated virus to monocytic cells does not enable infection by itself when few membrane CD4 are expressed. HIV binding to FcR through CD4-1gG hybrid molecules does not result in productive infection of U937 cells Whether productive infection occurs when HIV enters the cells vie, FcR while at the same time interacting with CD4 was then investigated using CD4-Ig constructs.
srCD4 = solublerecombinant CD4. TCID = tissueculture infective dose.
185
CD4 A N D F c R - M E D I A T E D H ! V I N F E C T I O N
The binding of CD4-HT3 to U937 FcR, determined by direct immunofluorescence, resulted in increased ~abe!!ing of CD4, directly revealed by phycoerythrin-labelled mAb Leu3a (fig. 3a,b), and increased the capacity of the cells to bind 125I-gp160, in the same range or greater than that obtained with anti-HIV IgG (fig. 3c). This indicates that the binding of CD4-H73 to U937 cell FcR actually enables HIV env glycoprotein and, most probably, virus particles to attach to the cells. When HiV was incubated with different doses of CD4-HT3 before addition to ceils, total inhibition of virus replication was observed even with the lowest dose of CD4-HT3. Though less complete, the same result was noted when CD4-HT3 was bound to FcR prior to addition of free virus (fig. 3d).
DISCUSSION We (Jouault et al., 1989) and others (Matsuda et al., 1989; Perno et ai., 1990; Zeira et al., 1990; Takeda et aL, i990) have shown that interaction with membrane CD4 appears necessary for FcR-mediated infection (ADE) of monocytic cells, as it is blocked by anti-CD4 mAb (known to interfere with gp 120 binding to CD4) or by srCD4. Because binding of antiCD4 mAb to CD4 may entail negative signalling to the target cells (Blue et al., 1988) or even compete for FcR binding of immune-complexed virus (Kurlander, 1983), we again raised the question, using different approaches, as to whether the interaction with membrane CD4 may. be circumvented for HIV infection when virions were bound to the cells through FcR. First, U937 cells were incubated for a short period with PDB, which elicited a temporary downregulation of membrane CD4 expression as previously reported (Clapham et al., 1987; Hoxie et
ai., 1988), associated with the apparent loss of the ability to bind HIV particles. This did not abolish infection by free virus, but only delayed virus replication, similar to what was observed with a 10-foldreduced virus load. Because this delayed infection of PDB-treated cells could be blocked by Leu3a or srCD4, there remained enough membrane CD4 to permit HIV infection. These conditions enabled an analysis of the consequences on infection of antibody-mediated HIV binding when few CD4 m o , ~ . , ~ were , ~ . , . . . D , ~ , ~ the c e i l membrane to be undertaken.
FcR-mediated binding to PDBtreated U937 cells of HIV opsonized by saturating antibody concentrations led to neutralizatior, of virus infectivity, contrasting with what occurs with untreated cells that express detectable CD4. Hence, there exists a relationship between susceptibility to FcR-mediated infection and the amount of membrane CD4. This suggests that when interaction be-
tween viral gpl20 and its CD4 receptor is impeded, opsonized HIV bound to FcR enters the endocytic/phagocytic pathway leading to virus destruction and not to productive infection. Such an hypothesis is reinforced by the results obtained using hybrid CD4-HT3, which binds to FcR, to complex HIV at the membrane of PDB-untreated U937 cells. HIV infection was prevented, even when CD4-HT3 were adsorbed to the cells prior to virus addition, and membrane CD4 remained accessible, a situation in which virus could gain access to the cells by using either the bivalent CD4-HT3 attached to FcR, or normal membrane CD4. The lack of ADE under these conditions argues against the possibility of efficient HIV e n t ~ into monocytic cells through the endo/phagocytic pathway. Therefore, it appears that at least in U937 cells, even when HIV attaches to the cells by means of FcR, the interaction of gpl20 with
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HIV binding
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without" HIV
Fig. 1. Flow cytometry analysis of membrane markers after PDB treatment of
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T. J O U A U L T E T A L .
186
its CD4 membrane receptor" cannot be bypassed for productive infection to occur.
Acknowledgements
We are grateful to A. Traunecker for the gift of hybrid CD4-1gG preparations and to D. Klatzmann and C. Parravicini for helpful discussions. This work was supported by the Fondation de l'Avenir and the Agence Natio. nale de Recherche sur le SIDA (Paris, France).
2 24 Infection
des mqnocytes
p a r le V I H I : r61e des F c R et de CD4 Le V I H (virus de l'immunod6ficience humaine) peut utiliser comme site d ' a t t a c h e m e n t les FcR port6s par les cellules monocytaires, lorsqu'il est complex6 aux anticorps. Afin de d6terminer si l'interaction entre la gpl20 de V I H et le CD4 membranaire est n6cessaire au cours de l'infection m6di6e par les anticorps, des cellules monocytaires U937 ont 6t6 trait6es transitoirement avec un ester de phorbol (le P D B ) afin d'induire la
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(a) Cells were treated (triangles) or not (squares) with l IzM PDB for 5 h, After washing, cells were incubated with HIV for 3 h and washed. At day 1 after infection, fresh PDBuntreated U937 cells were added (closed symbols) or not (open symbols). (b) Cells were infected with 10 ( B ), 100 (A) or 1,000 ( e ) TCID of HIV. The dashed lines indicate the time periods to obtain 50 °7o of maximum HIV production. (c) After PDB treatment, cells were infected with HIV preincubated without (D) or with 700 ( m ), 7 (&), or 0.07 ( e ) izg/ml of anti-HIV IgG.
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Res. Virol. 1991, 142, 183-188
Infection of monocytic cells by HI[VI: combined role of FcR and CD4 T. J o u a u l t o)(*)., F. Chapuis (1), E. Bahraoui (2) and J.-C. G l u c k m a n O) (l) Laboratoire de Biologie et Gdndtique des Dgficits Immunit¢ires, CER VL Groupe Hospitalier Pitid-Salpdtribre, 83 Bd de l'h6pital, 75651 Paris Cedex 13, and (2) C N R S URA 1179, Facultd de Mddecine Nord, 13326 Marseille Cedex 15 (France)
SUMMARY
Human immunodeficiency virus (HIV) complexed with human anti-HIV IgG can attach to FcT receptors (Fch) of mononuclear phagocytes. To determine whether the FcRmediated infection that results also requires interaction between H|V gp! 20 and cell membrane CD4, monocytic cells of the U977 line were transiently treated with phorbol 12,13-dibutyrate (PUB) so that they temporarily presented a CD4-FcR + phenotype at the time of HIV infection. HIV production was not abolished, but only significantly delayed after infection of these cells with free virus. Leu3a monoclonal antibody or soluble recombinant CD4 completely blocked this delayed infection. This indicates that enough CD4 still remained at the membrane to allow infection of a reduced cell number. Infection of PDB-treated cells with virus preincubated with high anti-HIV igG concentrations was inhibited, contrasting with what was Jbserved wit~. ~on*,,-'_ .;: ~ "--'%~.,,:~,~'~i=d under the same conditions. Inhibition of infection ,vas also observed when HIV became a__t~_chedto untreated U937 cells through the binding of CD4-1gG hybrid molecu,es to FcR. Thus, the binding of igG-coated virus to FcR is not sufficient in itself to elicit productive infection of monocytic cells, which still requires the interaction of viral gp120 and membrane CD4. Key-words: CD4, IgG, Monocyte, FcR, HIV; gp120, Antibody-mediated infection.
INTRODUCTION Membrane CD4 is the cellular receptor of human immunodeficiency virus (HIV) envelope glycoprotein gp 120 (Klatzmann et al., 1990) but monocytic cells may also be infected through the attachment of antibody-coated HIV particles to Fc-f-recepto~s (FcR). It is possible that such antibedydependent enhancement of infection (ADE) does not require the interaction of gp120 and membrane
(*) Corresponding author.
CD4. However, previous studies (Jouault et al., 1989; Matsuda et al., 1989; Perno et al., 1990; Zeira et aL, 1990; Takeda et al., 1990) have shown that monoclonal antibod,_'es (mAb) to CD4 that interfere with gp120 binding (Klatzmann et al., 1990), or soluble recombinant CD4 (srCD4), block infection of monocytic cells with immune-compined HIV. To confirm these observations, we used two different approaches to examine whether membrane CD4 is still required for ADE.
MATERIALS AND METHODS Cells and virus strain The U937 line (Sundstrom et al., 1976) was grown as described (Jouault et al., 1989). The LAVBRU strain of HIV was used. Phorbol 12,13-dibutyrate (PDB) (Sigma Chemicals, St Louis, MO, USA) was added to the cells at different concentrations for various periods at 37°C.
T. J O U A LILT E T A L .
184 Human Ig, srCD4 and h'ybrid CD4-1g
IgG from heat-inacti'~ated HlV-seropositive sera (ant:,.[ilV IgG) were purified as described (Jouault et al., 1989). F(ab') 2 fragments were obtained by pepsin digestion, srCD4 was a gift from David Klatzmann (T. Idziorek et al., 1991). The CD4-1gG preparation (CD4-H-~3) was a gift from Andr6 Traunecker (Basel Institute for Immunology, Basel, Switzerland) (Traunecker et al., 1989). Fluorescence analysis of membrane markers
Cell membrane markers were analysed with the "FACS Analyzer" (Becton-Dickinson, Mountain View, CA, USA) (Jouault et al., 1989).
Binding of 12Sl-gp160 to cells
Soluble recombinant gp 160 was a gift from Pasteur Vaccins (Marnes-la-Coquette, France) 0Oeny et al., 1988). Radiolabelling and investigation of binding of ~2Sl-gpl60 to the cells was conducted as described (Jouault et ai., 1989).
HIV-binding assay
U937 cells were incubated for 3 0 m i n at 37°C with heatinactivated HIV. Bound particles were revealed by anti-HIV F(ab')2 followed by staining with biotinylated sheep anti-human a:,_tibodies and phycoerythrin-streptavidin. Parallel experiments were performed with HIV that had been
ADE mAb .'~BD
previously complexed with antiHIV IgG.
PDB treatment modifies the susceptibility of U937 cells to HIV infection
tllV infection
Infection of PDB-treated cells by free virus was not abolished but virus replication was significantly delayed even after the addition of fresh u n t r e a t e d U937 cells (fig. 2a). This delay was similar to that observed when virus load (TCID) was reduced 10-fold in order to infect untreated cells (fig. 2b). Infection of PDB-treated cells was completely blocked in the presence of mAb Leu3a, or w~Lh virus preincubated with srCD4 (data not shown). Thus, transient membrane CD4 downregulation after PDB-treatment decreased the susceptibility of cells to HIV.
Virus (103 TCID) was incubated with or without different dilutions of anti-HIV IgG or CD4-Hy3 as described (Jouault et al., 1989). The complexed or free virus suspension was added to the cells. After 2 washes, cells were cultured without additional anti-HIV IgG. Viral production in cell-free supern a t a n t s was d e t e r m i n e d by antigen-capture e n ~ e - ! i n k e d immunosorbent assay (ELAVIA AgI Diagnostics Pasteur, Marnes-LaCoquette, France). RESULTS P D ~ treatment of U937 cells modifies the expression of membrane molecules and alters HIV binding to cells
Phorbol esters, which are known for down regulating CD4 membrane expression (Clapham et al., 1987, Hoxie et al., 1988), were used to obtain U937 cells that transiently presented a FcR+CD4 phehotype at the time of infection (fig. 1). The maximum loss of CD4 was obtained after a 5-h incubation with I lxM PDB, as determined using mAb Leu3a, which corresponded to the loss of HIV particle binding detected in flow cytometry. Attachment of antiHIVIgG-complexed 125i.gp 160 to these cells was still possible, and a comparable eiihancement of gpl60 was obtained whether they had been treated or not with PDB; membrane-bound radioactivity increased from 4 , 5 0 0 c p m to 25,000 cpm and to 23,000 cpm, respectively.
ffi antibody-dependentenhancement. = monoclonalantibody. = phorbol 12,13-dibutyrate.
Under these conditions, no increase in viral replication was observed when virus was preincubated with low anti-HIV igG concentrations as compared with free-virus-infected cells. When higher antibody amounts were used to complex virus, only limited virus production occurred (fig. 2c), contrasting with what was observed when untreated U937 cells were infected under the same cond':tion~ T h n ¢ l:?,-I~_ mediated binding of antibodycoated virus to monocytic cells does not enable infection by itself when few membrane CD4 are expressed. HIV binding to FcR through CD4-1gG hybrid molecules does not result in productive infection of U937 cells Whether productive infection occurs when HIV enters the cells vie, FcR while at the same time interacting with CD4 was then investigated using CD4-Ig constructs.
srCD4 = solublerecombinant CD4. TCID = tissueculture infective dose.
185
CD4 A N D F c R - M E D I A T E D H ! V I N F E C T I O N
The binding of CD4-HT3 to U937 FcR, determined by direct immunofluorescence, resulted in increased ~abe!!ing of CD4, directly revealed by phycoerythrin-labelled mAb Leu3a (fig. 3a,b), and increased the capacity of the cells to bind 125I-gp160, in the same range or greater than that obtained with anti-HIV IgG (fig. 3c). This indicates that the binding of CD4-H73 to U937 cell FcR actually enables HIV env glycoprotein and, most probably, virus particles to attach to the cells. When HiV was incubated with different doses of CD4-HT3 before addition to ceils, total inhibition of virus replication was observed even with the lowest dose of CD4-HT3. Though less complete, the same result was noted when CD4-HT3 was bound to FcR prior to addition of free virus (fig. 3d).
DISCUSSION We (Jouault et al., 1989) and others (Matsuda et al., 1989; Perno et ai., 1990; Zeira et al., 1990; Takeda et aL, i990) have shown that interaction with membrane CD4 appears necessary for FcR-mediated infection (ADE) of monocytic cells, as it is blocked by anti-CD4 mAb (known to interfere with gp 120 binding to CD4) or by srCD4. Because binding of antiCD4 mAb to CD4 may entail negative signalling to the target cells (Blue et al., 1988) or even compete for FcR binding of immune-complexed virus (Kurlander, 1983), we again raised the question, using different approaches, as to whether the interaction with membrane CD4 may. be circumvented for HIV infection when virions were bound to the cells through FcR. First, U937 cells were incubated for a short period with PDB, which elicited a temporary downregulation of membrane CD4 expression as previously reported (Clapham et al., 1987; Hoxie et
ai., 1988), associated with the apparent loss of the ability to bind HIV particles. This did not abolish infection by free virus, but only delayed virus replication, similar to what was observed with a 10-foldreduced virus load. Because this delayed infection of PDB-treated cells could be blocked by Leu3a or srCD4, there remained enough membrane CD4 to permit HIV infection. These conditions enabled an analysis of the consequences on infection of antibody-mediated HIV binding when few CD4 m o , ~ . , ~ were , ~ . , . . . D , ~ , ~ the c e i l membrane to be undertaken.
FcR-mediated binding to PDBtreated U937 cells of HIV opsonized by saturating antibody concentrations led to neutralizatior, of virus infectivity, contrasting with what occurs with untreated cells that express detectable CD4. Hence, there exists a relationship between susceptibility to FcR-mediated infection and the amount of membrane CD4. This suggests that when interaction be-
tween viral gpl20 and its CD4 receptor is impeded, opsonized HIV bound to FcR enters the endocytic/phagocytic pathway leading to virus destruction and not to productive infection. Such an hypothesis is reinforced by the results obtained using hybrid CD4-HT3, which binds to FcR, to complex HIV at the membrane of PDB-untreated U937 cells. HIV infection was prevented, even when CD4-HT3 were adsorbed to the cells prior to virus addition, and membrane CD4 remained accessible, a situation in which virus could gain access to the cells by using either the bivalent CD4-HT3 attached to FcR, or normal membrane CD4. The lack of ADE under these conditions argues against the possibility of efficient HIV e n t ~ into monocytic cells through the endo/phagocytic pathway. Therefore, it appears that at least in U937 cells, even when HIV attaches to the cells by means of FcR, the interaction of gpl20 with
i
>,8O
o o c o o m o o
"-
membrane
HIV binding
markers
411
L_
¢ 20 t~ o
.
fi
E 0
CD8
CD4
CR3
FcR II
with HiV
without" HIV
Fig. 1. Flow cytometry analysis of membrane markers after PDB treatment of
U937 cells. Cells were treated without (medium-hatched column), with 0.01 ?.M (lighthatched column) or I ~M (dark-hatched column) of PDB. In parallel, cells were incubated with 2 l~g of heat-inactivated HIV t,nd re!!obound particles were revealed with anti-HiV F(ab')2. Control (blank column), in the absence of virus.
T. J O U A U L T E T A L .
186
its CD4 membrane receptor" cannot be bypassed for productive infection to occur.
Acknowledgements
We are grateful to A. Traunecker for the gift of hybrid CD4-1gG preparations and to D. Klatzmann and C. Parravicini for helpful discussions. This work was supported by the Fondation de l'Avenir and the Agence Natio. nale de Recherche sur le SIDA (Paris, France).
2 24 Infection
des mqnocytes
p a r le V I H I : r61e des F c R et de CD4 Le V I H (virus de l'immunod6ficience humaine) peut utiliser comme site d ' a t t a c h e m e n t les FcR port6s par les cellules monocytaires, lorsqu'il est complex6 aux anticorps. Afin de d6terminer si l'interaction entre la gpl20 de V I H et le CD4 membranaire est n6cessaire au cours de l'infection m6di6e par les anticorps, des cellules monocytaires U937 ont 6t6 trait6es transitoirement avec un ester de phorbol (le P D B ) afin d'induire la
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Fig. 2. Effect of PDB treatment on HIV infection.
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(a) Cells were treated (triangles) or not (squares) with l IzM PDB for 5 h, After washing, cells were incubated with HIV for 3 h and washed. At day 1 after infection, fresh PDBuntreated U937 cells were added (closed symbols) or not (open symbols). (b) Cells were infected with 10 ( B ), 100 (A) or 1,000 ( e ) TCID of HIV. The dashed lines indicate the time periods to obtain 50 °7o of maximum HIV production. (c) After PDB treatment, cells were infected with HIV preincubated without (D) or with 700 ( m ), 7 (&), or 0.07 ( e ) izg/ml of anti-HIV IgG.
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