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Inhibition of HIV-1, HIV-2 and SIV envelope glycoprotein-mediated cell fusion by calmodulin
Virus Research 50 (1997) 119 – 127
Inhibition of HIV-1, HIV-2 and SIV envelope glycoprotein-mediated cell fusion by calmodulin Etienne Malvoisin *, Fabian Wild Inserm Unit 404, Immunity and Vaccination, Institute Pasteur de Lyon, A6enue Tony Garnier, 69365 Lyon Cedex 07, France Received 8 January 1997; accepted 15 April 1997
Abstract Calmodulin, an EF-hand protein, inhibited the fusion between CD4 + human cells and cells stably expressing HIV-1 envelope proteins. Fusion was also inhibited when HIV-1, HIV-2 or SIV envelope glycoproteins were expressed by vaccinia virus (VV) recombinants, but calmodulin did not inhibit syncytia formation induced by measles virus glycoproteins. Calmodulin also inhibited fusion induced by vPE17, a VV-recombinant expressing a truncated form of HIV-1gp160 which lacks the two known calmodulin-binding sites located in the cytoplasmic domain of gp41. The inhibitory activity was specific to calmodulin among the EF-hand proteins. These observations may be important in understanding the mechanism of retroviral envelope glycoprotein-mediated cell fusion. Several possible mechanisms of action are discussed. © 1997 Elsevier Science B.V. Keywords: Calmodulin; HIV-1; EF-hand protein; Glycoprotein
1. Introduction The envelope glycoprotein of HIV-1 is composed of two subunits: a surface glycoprotein gp120 and a transmembrane glycoprotein gp41, which interact with each other in a nonconvalent manner. Glycoprotein gp120 is critical in attachment to the host cell CD4 receptor, while gp41 contains the fusion peptide sequence. The capac* Corresponding author. Tel.: + 33 472 722553; fax: + 33 472 722567; e-mail: [email protected]
ity of the HIV-1 envelope glycoproteins to induce cell fusion is an interesting property, since molecules which inhibit the fusion process are possible antivirals and may reveal important functional regions either on the viral glycoprotein or on cellular membranes. Calmodulin (CaM) is a Ca2 + -binding protein which belongs to the superfamily of EF-hand proteins, which includes troponin C, parvalbumin and S100 proteins (James et al., 1995). These proteins are characterized by a common structural motif, the EF hand, which selectively binds Ca2 + with a high affinity. CaM
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has four Ca2 + -binding sites, two that have a high affinity for Ca2 + /Mg2 + and two sites of low affinity for Ca2 + (Babu et al., 1985). Previous studies have demonstrated that the cytoplasmic tail of the gp41 subunit binds CaM with high affinity, and synthetic peptides corresponding to the two CaM-binding domains detected in the cytoplasmic tail have CaM-binding properties and inhibit CaM-dependent enzymatic processes (Miller et al., 1993; Srinivas et al., 1993; Burroughs Tencza et al., 1997). A role of CaM has been also demonstrated in HIV-potentiated Fasmediated apoptosis (Pan et al., 1996). In a recent paper Ebenbichler et al. (1996) described an EF-hand like structure within the extracellular region of gp41 and suggested it interacts with the putative second receptor molecules in a calcium-dependent manner. On the basis of this observation, it was proposed that a molecule which possesses an EF-hand structural motif which resembles that of gp41 may interfere with the binding with the putative co-receptor. In the present work we have tested the capacity of several EF-hand proteins to modify the fusion induced by retroviral glycoproteins.
2. Results In our study we have used the human TF228.1.16 cells which is a BJAB cell line that stably expresses functionally active HIV-1 envelope protein (BH-10 clone of HIV-1LAI) (Jonak et al., 1993). It has been reported that TF228.1.16 cells fuse with CD4 + cells (SupT1 and Primary human lymphocytes) in a similar manner to HIV (Jonak et al., 1993). We found that TF228.1.16 cells fuse with 293/CD4 + cells (human embryonic kidney 293 cells overexpressing human CD4) forming large syncytia after overnight culture (Reil et al., 1994) (Fig. 1, panel C). To examine the effect of CaM on HIV-1 envelope glycoprotein-mediated fusion, 293/CD4 + cells were mixed with TF228.1.16 cells in the presence of CaM. As a positive control of fusion inhibition, we studied in parallel the effect on fusion of the mannose-specific lectin concanavalin A (Con A). Con A has been previously used to investigate the
role of the mannose residues of envelope glycoprotein on HIV infectivity and HIV- and measles virus-induced cell fusion (Hansen et al., 1989; Malvoisin and Wild, 1994). Fusion was examined by light microscopy after cocultivation for 24 h. We observed that CaM and Con A completely inhibited the fusion between TF228.1.16 and 293/ CD4 + (Fig. 1, panels D and E). CaM was not toxic for the cells under the concentration used, i.e. up to 0.5 mg/ml. The CaM used in this study was from bovine brain. However we found similar results with CaM from human erythrocytes (not shown). In Table 1, the inhibitory activity of CaM on fusion of 293/CD4 + cells with TF228.1.16 cells was determined as a function of the concentration and was compared with the inhibitory activity of several compounds which are known to interact with the HIV-envelope protein, i.e. dextran sulfate (DXSF), suramin and heparin (Baba et al., 1988). Five mg/ml of CaM completely inhibited the fusion. The concentration of CaM which inhibited the formation of syncytia was comparable to that of DXSF.500 000. To confirm these observations, we used a system in which fusion took place more rapidly i.e. SupT1 cells. These cells were mixed with TF228.1.16 cells in the presence of CaM and fusion was examined by light microscopy after 90 min co-cultivation. Fusion was inhibited by CaM but not by parvalbumin (from frog muscle) (not shown) or troponin (from rabbit muscle) at concentrations of 0.34 mg/ml (Fig. 1, panels F, G and H). The capacity of increasing concentrations of CaM to inhibit the fusion of SupT1 cells with TF228.1.16 cells was determined and the extent of syncytium formation was quantitated after different times after co-cultivation (Table 2). A concentration of CaM between 15 and 45 mg/ml was able to completely inhibit the fusion of SupT1 cells with TF228.1.16. When compared with the fusion of SupT1 cells with TF228.1.16 cells, the fusion of 293/CD4 + cells with TF228.1.16 cells was completely inhibited by a lower dose of CaM. CaM did not lyse the syncytia already formed but stopped the fusion reaction and the apparition of new syncytia: SupT1 cells were co-cultivated with TF228.1.16 cells in the absence of CaM for 2 h. CaM (20 mg/ml) was then added to
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Fig. 1. Formation of syncytia between TF228.1.16 and 293/CD4 + or SUPT1 cells: effect of CaM, Con A and troponin. Panels A–E, Cells were co-cultured for 24 h in the presence or absence of CaM (10 mg/ml) or Con A (0.8 mg/ml); panels F – H, Cells were co-cultured for 90 min in the presence or absence of CaM (10 mg/ml) or troponin (Tro) (0.34 mg/ml). CaM and other chemicals used in this study were from Sigma.
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Table 1 Effect of drugs on fusion of TF228.1.16 cells with 293/CD4+ cells Treatment
Fusiona
None CaM Con A Heparin Suramin DXSFb. 10 000 DXSF. 500 000
F F F F F F F
[0.60] [0.09] [2.70] [1.60] [0.02] [0.38]
F P [1.80] P [0.27] P [8.00] P [4.70] P [0.06] P [1.15]
I I I I I I
F [5.50] [0.80] [24.0] [14.0] [0.20] [3.45]
TF228.1.16 cells were mixed with 293/CD4+ cells (1:1 cell ratio) and transferred to a 96-well plate (105 cells per well in 200 ml of culture medium). Co-cultures of TF228.1.16 cells and 293/CD4+ cells were incubated in the presence or absence of the drug (the final concentration in mg/ml is indicated in parenthesis) for 18 h. Following co-cultivation, three random fields of cells were photographed and the percentage fusion was evaluated as previously described (Malvoisin and Wild, 1990). The percentage fusion is defined as the ratio of cell surface involved in syncytia compared to the total cell surface. Syncytia were defined as giant cells ]4 times the diameter of single cells. a F, 50-60% of the surface is covered by syncytia; P, partial inhibition of fusion: B10% of the surface is covered by syncytia; I, inhibition of syncytia formation. b DXSF, dextran sulfate.
the culture medium and maintained for a further 8 h incubation: we observed that the formation of new syncytia was blocked and the size of the syncytia already formed remained unchanged (not shown). Table 2 Effect of CaM on fusion of TF228.1.16 cells with SupT1 cells Treatment
% fusion
CaM (mg/ml)
2.5 h
8h
32 h
0 1.6 5 15 45
65 99 63 9 11 20 9 8 0 0
859 12 849 9 33 9 8 B1 0
859 16 80 9 7 359 9 B1 0
TF228.1.16 cells were mixed with SupT1 cells (1:1 cell ratio) and transferred to a 96-well plate (105 cells per well in 200 ml of culture medium). After 2.5, 8 and 32 h of co-cultivation in the presence or absence of CaM (mg/ml), three random fields of cells were photographed, and the percentage fusion was evaluated as indicated in Table 1.
To determine if CaM can inhibit the fusion induced by recombinant vaccinia viruses (VV) expressing HIV-1 envelope, 293/CD4 + cells were infected with VVTG9.1 (HIV-1 LAI)(Kieny et al., 1988) or vPE17 (HIV-1 BH-8) and incubated in the presence of CaM. VV vPE17 expresses a truncated form of 747 aa (aa 748–851 deleted) of the env protein (Earl et al., 1991). In parallel, 293/CD4 + cells were infected with VVH/F, a recombinant vaccinia virus which expresses both glycoproteins of measles virus (MV), the haemagglutinin (HA) and fusion (F) proteins. Both glycoproteins are necessary to induce syncytia formation in susceptible cells (Wild et al., 1991). As shown in Fig. 2, CaM inhibited the fusion induced by the HIV-1 gp160 but not MV glycoproteins. Dextran sulfate, suramin and heparin are known to inhibit HIV-induced cell fusion by electrostatic interaction with the envelope protein (Callahan, 1994). We have examined the effect of these compounds on MV-induced cell fusion. Following treatment with the drug, Hela T4 cells were infected with VVH/F (5 pfu/cell) and 16 h later the effect on fusion was examined. Syncytium formation in HeLa T4 cells infected by the recombinant vaccinia virus expressing both MV glycoproteins (VVH/F) was almost completely (more than 90%) blocked by 9 mg/ml of DXSF. 10 000, 18 mg/ml of suramin and 30 mg/ml of heparin sodium salt (not shown). To extend our observation to other primate retroviruses, we used recombinant vaccinia viruses expressing SIVmac (VVSIV) and HIV-2 (VVHIV2) envelope glycoproteins (Otteken et al., 1993). SIV glycoproteins are similar in structure and function to the envelope protein of HIV-1 and HIV-2 and share approximately 75% amino acid sequence identity with the envelope glycoprotein of HIV-2 and approximately 40% with that of HIV-1. VVSIV and VVHIV-2 did not induce syncytia in 293/CD4 + cells (not shown) but did in SupT1 cells. Infection of SupT1 cells with the VV recombinants showed that syncytia appeared more rapidly with VVHIV-1 than the other two recombinants. In addition, the syncytia were larger and more widespread in VVHIV-1-infected SupT1 cells than those observed in VVHIV-2 and particularly VVSIV-infected SupT1 cells (Fig. 3).
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Fig. 2. Effect of CaM on syncytia formation in 293/CD4 + cells infected with recombinant vaccinia viruses expressing HIV-1 or measles virus envelope glycoproteins. The cells were infected with vaccinia recombinant viruses (5 pfu/cell) in the presence (right panels) or absence of CaM (10 mg/ml): VVH/F, expressing the measles virus haemagglutinin and fusion proteins (Wild et al., 1991), VVTG9.1 expressing the full-length HIV-1 LAI envelope protein (Kieny et al., 1988), vPE17 expressing an env protein aa 1 – 747 (HIV-1 IIIB, clone BH-8) (Earl et al., 1991). The cells were photographed 24 h after infection.
To examine the effect of CaM on fusion, SupT1 cells were infected with recombinant vaccinia viruses VVTG9.1, vPE17, VVHIV-2 or VVSIV in the presence or absence of CaM. As a vaccinia control we used VVMVHA which expresses the haemagglutinin of measles virus. VVMVHA is unable by itself to induce syncytia formation (Wild et al., 1991). As shown in Fig. 3, CaM inhibited the fusion induced by HIV-1 (panel B),
HIV-2 (panel A) and SIV (panel C) envelope glycoproteins. In addition we found that CaM was less efficiently bound to its target than Con A. When the TF228.1.16 cells were incubated in the presence of either Con A (10 mg/ml) or CaM (0.34 mg/ml) for 18 h and after washing co-cultured with untreated 293/CD4 + , the CaM-pretreated cells underwent fusion but not the Con A-pretreated cells (not
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Fig. 3. Effect of CaM on syncytia formation in SupT1 cells infected with recombinant vaccinia viruses expressing HIV-1, HIV-2 or SIV envelope glycoproteins. Cells were infected with vaccinia recombinant viruses (5 pfu/cell) in the presence or absence of CaM (10 mg/ml). VVMVHA expressing the measles virus haemagglutinin (Wild et al., 1991), vv9002 (ADP270.1) expressing SIVmac env gp160 (SIVmac32H[pJ5] clone)(termed VVSIV) (obtained through the MRC AIDS Directed Programme), VVenv863 expressing the HIV-2ben envelope protein (termed VVHIV-2) (Otteken et al., 1993). In panels A and C, the cells were photographed 24 h after infection at 200× magnification; in panel B, 10 h after infection at 100× magnification.
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shown). This may indicate that CaM bound to its target with a low affinity.
3. Discussion In the present study, we have shown that CaM inhibits retroviral glycoprotein-induced fusion and not that of measles virus. BJAB and 293 cells stably transfected with human CD4 are highly susceptible to HIV-1 infection (Reil et al., 1994; Kru¨ger et al., 1996). CaM inhibited cell-cell and recombinant-induced fusion mediated by HIV env protein using different cell types as targets. The fusion between TF228.1.16 cells and HeLaT4 cells (HeLa cells stably expressing CD4) or H4/CD4 (CD4 positive glial cell line obtained by transfection of human H4 neuroglioma cells) (Volsky et al., 1992) was also inhibited by CaM (not shown). If the binding of CaM to its target protein requires the presence of Ca2 + , the fusion-inhibitory effect of CaM does not seem to be due to a deprivation of Ca2 + in the medium. Troponin C and parvalbumin also bind Ca2 + with high affinity (Scha¨fer and Heizmann, 1996). Nevertherless, it is possible that the MV-mediated cell fusion may be less Ca2 + dependent than the HIV-mediated cell fusion. The calcium-binding subunit of the troponin complex (troponin C) is highly similar in both amino acid sequence and three dimensional structure to CaM (Babu et al., 1985; Strynadka and James, 1989). Like CaM, the C-domain of troponin C has two high-affinity sites for Ca2 + that also bind Mg2 + , whereas the N-domain has two low-affinity, Ca2 + -specific sites (Grabarek et al., 1992). This suggests that the effect of CaM may be specific. In addition, despite sequence similarities between HIV gp41 and paramyxovirus fusion proteins (GonzalezScarano et al., 1987), CaM inhibited HIV-mediated cell fusion but not MV-mediated cell fusion, whereas DXSF.10 000, suramin and heparin inhibited both. Using [125I]CaM overlays of SDS-PAGE gels or NMR and CD spectroscopy, previous studies showed that the cytoplasmic domain of the HIV-1 or SIV glycoproteins but not the gp120 interacted with CaM (Srinivas et al., 1993; Yuan et al.,
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1995). Using synthetic peptide analogs, Srinivas et al., 1993 demonstrated the presence of two CaMbinding domains (768–788 and 826–854) in the intracellular carboxy-terminal segment of gp41. The truncated form expressed by vPE17 lacks these two CaM-binding domains. Miller et al., 1993 showed lack of CaM binding by a construct containing the first 185 residues of gp41. This construct was purified and then subjected to SDSPAGE followed by overlay in situ with [125I]CaM. However, during the purification procedures and SDS-PAGE the proteins may be denatured, undergo conformational changes, lose their affinity for CaM and, consequently, low-affinity CaMbinding sites may not be detected. Recently several groups have identified proteins that work in conjunction with cell-surface CD4 enabling HIV-1 to infect cells (Alkhatib et al., 1996; Deng et al., 1996; Drajic et al., 1996; Feng et al., 1996). These cell-surface receptors belong to the family of seven-transmembrane-spanning (7 tm), G-proteincoupled receptors. Several regions located in the extracellular domain of gp41 are conserved among HIV-1, HIV-2 and SIV isolates and possibly interacts with gp120 (Stoiber et al., 1994) or with the second-receptor molecule (Ebenbichler et al., 1996). It is tempting to suggest that CaM interferes with the binding of gp41 to gp120 or to the co-receptor. CaM may mask a site on a protein necessary for fusion and prevent it from interacting with another. However, CaM may also induce a structural change in the protein target or react specifically with regions involved in the fusion process, i.e. the fusion domain or the leucine zipper-like motif (Dubay et al., 1992; Wild et al., 1992). The leucine zipper-like motif has been demonstrated to bind to the host cell membrane bringing the viral and cellular membrane closer and facilitating fusion (Rabenstein and Shin, 1995). CaM is a small ubiquitous eukaryotic protein expressed extra- and intracellularly, which binds and activates many classes of proteins, including protein kinases, calcium pump, cytoskeletal proteins. An extracellular role for CaM has been demonstrated as it can act as a growth factor for a variety of cells in culture, including human leukemia lymphocytes (Gorbachevskaya et al.,
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1983; Crocker et al., 1988; Dalley et al., 1996). To our knowledge no CaM receptor has been identified. However, it cannot be excluded that the extracellular portion of a molecule necessary for fusion has a recognition site for CaM. It would be an attractive hypothesis to postulate that extracellular CaM may mediate Ca2 + entry into the cell; Ca2 + , as second messenger may control the biological process involved in HIV-mediated cell fusion. Similarly, SDF-1, a chemokine and a biological ligand for the HIV-1 entry cofactor LESTR (CXCR-4) has been demonstrated to induce an increase in intracellular free Ca2 + in CXCR-4-transfected cells (Bleul et al., 1996). Recently, molecules which inhibit the Na + /K + / 2Cl − cotransporter function by direct binding, have been shown to block the cytopathic effect of HIV-1 (Voss et al., 1996). CaM may regulate a Ca2 + activated ion channel via direct binding to the channel. However, the importance of our observation has to be determined. Further studies are necessary to determine if extracellular CaM can prevent cells from HIV infection or modulate the life-cycle of the virus in infected individuals.
Acknowledgements We thank Drs M.P. Kie´ny, B. Moss and A. Otteken for generously providing recombinant vaccinia viruses, Drs Z.L. Jonak, H. Hauser, D.J. Volsky for cell lines. We would like also to thank Dr H.C. Holmes of the ADP reagent programme for reagents and Bernadette Maret for editorial assistance. E.M. held a SIDACTION fellowship.
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Inhibition of HIV-1, HIV-2 and SIV envelope glycoprotein-mediated cell fusion by calmodulin Etienne Malvoisin *, Fabian Wild Inserm Unit 404, Immunity and Vaccination, Institute Pasteur de Lyon, A6enue Tony Garnier, 69365 Lyon Cedex 07, France Received 8 January 1997; accepted 15 April 1997
Abstract Calmodulin, an EF-hand protein, inhibited the fusion between CD4 + human cells and cells stably expressing HIV-1 envelope proteins. Fusion was also inhibited when HIV-1, HIV-2 or SIV envelope glycoproteins were expressed by vaccinia virus (VV) recombinants, but calmodulin did not inhibit syncytia formation induced by measles virus glycoproteins. Calmodulin also inhibited fusion induced by vPE17, a VV-recombinant expressing a truncated form of HIV-1gp160 which lacks the two known calmodulin-binding sites located in the cytoplasmic domain of gp41. The inhibitory activity was specific to calmodulin among the EF-hand proteins. These observations may be important in understanding the mechanism of retroviral envelope glycoprotein-mediated cell fusion. Several possible mechanisms of action are discussed. © 1997 Elsevier Science B.V. Keywords: Calmodulin; HIV-1; EF-hand protein; Glycoprotein
1. Introduction The envelope glycoprotein of HIV-1 is composed of two subunits: a surface glycoprotein gp120 and a transmembrane glycoprotein gp41, which interact with each other in a nonconvalent manner. Glycoprotein gp120 is critical in attachment to the host cell CD4 receptor, while gp41 contains the fusion peptide sequence. The capac* Corresponding author. Tel.: + 33 472 722553; fax: + 33 472 722567; e-mail: [email protected]
ity of the HIV-1 envelope glycoproteins to induce cell fusion is an interesting property, since molecules which inhibit the fusion process are possible antivirals and may reveal important functional regions either on the viral glycoprotein or on cellular membranes. Calmodulin (CaM) is a Ca2 + -binding protein which belongs to the superfamily of EF-hand proteins, which includes troponin C, parvalbumin and S100 proteins (James et al., 1995). These proteins are characterized by a common structural motif, the EF hand, which selectively binds Ca2 + with a high affinity. CaM
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has four Ca2 + -binding sites, two that have a high affinity for Ca2 + /Mg2 + and two sites of low affinity for Ca2 + (Babu et al., 1985). Previous studies have demonstrated that the cytoplasmic tail of the gp41 subunit binds CaM with high affinity, and synthetic peptides corresponding to the two CaM-binding domains detected in the cytoplasmic tail have CaM-binding properties and inhibit CaM-dependent enzymatic processes (Miller et al., 1993; Srinivas et al., 1993; Burroughs Tencza et al., 1997). A role of CaM has been also demonstrated in HIV-potentiated Fasmediated apoptosis (Pan et al., 1996). In a recent paper Ebenbichler et al. (1996) described an EF-hand like structure within the extracellular region of gp41 and suggested it interacts with the putative second receptor molecules in a calcium-dependent manner. On the basis of this observation, it was proposed that a molecule which possesses an EF-hand structural motif which resembles that of gp41 may interfere with the binding with the putative co-receptor. In the present work we have tested the capacity of several EF-hand proteins to modify the fusion induced by retroviral glycoproteins.
2. Results In our study we have used the human TF228.1.16 cells which is a BJAB cell line that stably expresses functionally active HIV-1 envelope protein (BH-10 clone of HIV-1LAI) (Jonak et al., 1993). It has been reported that TF228.1.16 cells fuse with CD4 + cells (SupT1 and Primary human lymphocytes) in a similar manner to HIV (Jonak et al., 1993). We found that TF228.1.16 cells fuse with 293/CD4 + cells (human embryonic kidney 293 cells overexpressing human CD4) forming large syncytia after overnight culture (Reil et al., 1994) (Fig. 1, panel C). To examine the effect of CaM on HIV-1 envelope glycoprotein-mediated fusion, 293/CD4 + cells were mixed with TF228.1.16 cells in the presence of CaM. As a positive control of fusion inhibition, we studied in parallel the effect on fusion of the mannose-specific lectin concanavalin A (Con A). Con A has been previously used to investigate the
role of the mannose residues of envelope glycoprotein on HIV infectivity and HIV- and measles virus-induced cell fusion (Hansen et al., 1989; Malvoisin and Wild, 1994). Fusion was examined by light microscopy after cocultivation for 24 h. We observed that CaM and Con A completely inhibited the fusion between TF228.1.16 and 293/ CD4 + (Fig. 1, panels D and E). CaM was not toxic for the cells under the concentration used, i.e. up to 0.5 mg/ml. The CaM used in this study was from bovine brain. However we found similar results with CaM from human erythrocytes (not shown). In Table 1, the inhibitory activity of CaM on fusion of 293/CD4 + cells with TF228.1.16 cells was determined as a function of the concentration and was compared with the inhibitory activity of several compounds which are known to interact with the HIV-envelope protein, i.e. dextran sulfate (DXSF), suramin and heparin (Baba et al., 1988). Five mg/ml of CaM completely inhibited the fusion. The concentration of CaM which inhibited the formation of syncytia was comparable to that of DXSF.500 000. To confirm these observations, we used a system in which fusion took place more rapidly i.e. SupT1 cells. These cells were mixed with TF228.1.16 cells in the presence of CaM and fusion was examined by light microscopy after 90 min co-cultivation. Fusion was inhibited by CaM but not by parvalbumin (from frog muscle) (not shown) or troponin (from rabbit muscle) at concentrations of 0.34 mg/ml (Fig. 1, panels F, G and H). The capacity of increasing concentrations of CaM to inhibit the fusion of SupT1 cells with TF228.1.16 cells was determined and the extent of syncytium formation was quantitated after different times after co-cultivation (Table 2). A concentration of CaM between 15 and 45 mg/ml was able to completely inhibit the fusion of SupT1 cells with TF228.1.16. When compared with the fusion of SupT1 cells with TF228.1.16 cells, the fusion of 293/CD4 + cells with TF228.1.16 cells was completely inhibited by a lower dose of CaM. CaM did not lyse the syncytia already formed but stopped the fusion reaction and the apparition of new syncytia: SupT1 cells were co-cultivated with TF228.1.16 cells in the absence of CaM for 2 h. CaM (20 mg/ml) was then added to
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Fig. 1. Formation of syncytia between TF228.1.16 and 293/CD4 + or SUPT1 cells: effect of CaM, Con A and troponin. Panels A–E, Cells were co-cultured for 24 h in the presence or absence of CaM (10 mg/ml) or Con A (0.8 mg/ml); panels F – H, Cells were co-cultured for 90 min in the presence or absence of CaM (10 mg/ml) or troponin (Tro) (0.34 mg/ml). CaM and other chemicals used in this study were from Sigma.
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Table 1 Effect of drugs on fusion of TF228.1.16 cells with 293/CD4+ cells Treatment
Fusiona
None CaM Con A Heparin Suramin DXSFb. 10 000 DXSF. 500 000
F F F F F F F
[0.60] [0.09] [2.70] [1.60] [0.02] [0.38]
F P [1.80] P [0.27] P [8.00] P [4.70] P [0.06] P [1.15]
I I I I I I
F [5.50] [0.80] [24.0] [14.0] [0.20] [3.45]
TF228.1.16 cells were mixed with 293/CD4+ cells (1:1 cell ratio) and transferred to a 96-well plate (105 cells per well in 200 ml of culture medium). Co-cultures of TF228.1.16 cells and 293/CD4+ cells were incubated in the presence or absence of the drug (the final concentration in mg/ml is indicated in parenthesis) for 18 h. Following co-cultivation, three random fields of cells were photographed and the percentage fusion was evaluated as previously described (Malvoisin and Wild, 1990). The percentage fusion is defined as the ratio of cell surface involved in syncytia compared to the total cell surface. Syncytia were defined as giant cells ]4 times the diameter of single cells. a F, 50-60% of the surface is covered by syncytia; P, partial inhibition of fusion: B10% of the surface is covered by syncytia; I, inhibition of syncytia formation. b DXSF, dextran sulfate.
the culture medium and maintained for a further 8 h incubation: we observed that the formation of new syncytia was blocked and the size of the syncytia already formed remained unchanged (not shown). Table 2 Effect of CaM on fusion of TF228.1.16 cells with SupT1 cells Treatment
% fusion
CaM (mg/ml)
2.5 h
8h
32 h
0 1.6 5 15 45
65 99 63 9 11 20 9 8 0 0
859 12 849 9 33 9 8 B1 0
859 16 80 9 7 359 9 B1 0
TF228.1.16 cells were mixed with SupT1 cells (1:1 cell ratio) and transferred to a 96-well plate (105 cells per well in 200 ml of culture medium). After 2.5, 8 and 32 h of co-cultivation in the presence or absence of CaM (mg/ml), three random fields of cells were photographed, and the percentage fusion was evaluated as indicated in Table 1.
To determine if CaM can inhibit the fusion induced by recombinant vaccinia viruses (VV) expressing HIV-1 envelope, 293/CD4 + cells were infected with VVTG9.1 (HIV-1 LAI)(Kieny et al., 1988) or vPE17 (HIV-1 BH-8) and incubated in the presence of CaM. VV vPE17 expresses a truncated form of 747 aa (aa 748–851 deleted) of the env protein (Earl et al., 1991). In parallel, 293/CD4 + cells were infected with VVH/F, a recombinant vaccinia virus which expresses both glycoproteins of measles virus (MV), the haemagglutinin (HA) and fusion (F) proteins. Both glycoproteins are necessary to induce syncytia formation in susceptible cells (Wild et al., 1991). As shown in Fig. 2, CaM inhibited the fusion induced by the HIV-1 gp160 but not MV glycoproteins. Dextran sulfate, suramin and heparin are known to inhibit HIV-induced cell fusion by electrostatic interaction with the envelope protein (Callahan, 1994). We have examined the effect of these compounds on MV-induced cell fusion. Following treatment with the drug, Hela T4 cells were infected with VVH/F (5 pfu/cell) and 16 h later the effect on fusion was examined. Syncytium formation in HeLa T4 cells infected by the recombinant vaccinia virus expressing both MV glycoproteins (VVH/F) was almost completely (more than 90%) blocked by 9 mg/ml of DXSF. 10 000, 18 mg/ml of suramin and 30 mg/ml of heparin sodium salt (not shown). To extend our observation to other primate retroviruses, we used recombinant vaccinia viruses expressing SIVmac (VVSIV) and HIV-2 (VVHIV2) envelope glycoproteins (Otteken et al., 1993). SIV glycoproteins are similar in structure and function to the envelope protein of HIV-1 and HIV-2 and share approximately 75% amino acid sequence identity with the envelope glycoprotein of HIV-2 and approximately 40% with that of HIV-1. VVSIV and VVHIV-2 did not induce syncytia in 293/CD4 + cells (not shown) but did in SupT1 cells. Infection of SupT1 cells with the VV recombinants showed that syncytia appeared more rapidly with VVHIV-1 than the other two recombinants. In addition, the syncytia were larger and more widespread in VVHIV-1-infected SupT1 cells than those observed in VVHIV-2 and particularly VVSIV-infected SupT1 cells (Fig. 3).
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Fig. 2. Effect of CaM on syncytia formation in 293/CD4 + cells infected with recombinant vaccinia viruses expressing HIV-1 or measles virus envelope glycoproteins. The cells were infected with vaccinia recombinant viruses (5 pfu/cell) in the presence (right panels) or absence of CaM (10 mg/ml): VVH/F, expressing the measles virus haemagglutinin and fusion proteins (Wild et al., 1991), VVTG9.1 expressing the full-length HIV-1 LAI envelope protein (Kieny et al., 1988), vPE17 expressing an env protein aa 1 – 747 (HIV-1 IIIB, clone BH-8) (Earl et al., 1991). The cells were photographed 24 h after infection.
To examine the effect of CaM on fusion, SupT1 cells were infected with recombinant vaccinia viruses VVTG9.1, vPE17, VVHIV-2 or VVSIV in the presence or absence of CaM. As a vaccinia control we used VVMVHA which expresses the haemagglutinin of measles virus. VVMVHA is unable by itself to induce syncytia formation (Wild et al., 1991). As shown in Fig. 3, CaM inhibited the fusion induced by HIV-1 (panel B),
HIV-2 (panel A) and SIV (panel C) envelope glycoproteins. In addition we found that CaM was less efficiently bound to its target than Con A. When the TF228.1.16 cells were incubated in the presence of either Con A (10 mg/ml) or CaM (0.34 mg/ml) for 18 h and after washing co-cultured with untreated 293/CD4 + , the CaM-pretreated cells underwent fusion but not the Con A-pretreated cells (not
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Fig. 3. Effect of CaM on syncytia formation in SupT1 cells infected with recombinant vaccinia viruses expressing HIV-1, HIV-2 or SIV envelope glycoproteins. Cells were infected with vaccinia recombinant viruses (5 pfu/cell) in the presence or absence of CaM (10 mg/ml). VVMVHA expressing the measles virus haemagglutinin (Wild et al., 1991), vv9002 (ADP270.1) expressing SIVmac env gp160 (SIVmac32H[pJ5] clone)(termed VVSIV) (obtained through the MRC AIDS Directed Programme), VVenv863 expressing the HIV-2ben envelope protein (termed VVHIV-2) (Otteken et al., 1993). In panels A and C, the cells were photographed 24 h after infection at 200× magnification; in panel B, 10 h after infection at 100× magnification.
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shown). This may indicate that CaM bound to its target with a low affinity.
3. Discussion In the present study, we have shown that CaM inhibits retroviral glycoprotein-induced fusion and not that of measles virus. BJAB and 293 cells stably transfected with human CD4 are highly susceptible to HIV-1 infection (Reil et al., 1994; Kru¨ger et al., 1996). CaM inhibited cell-cell and recombinant-induced fusion mediated by HIV env protein using different cell types as targets. The fusion between TF228.1.16 cells and HeLaT4 cells (HeLa cells stably expressing CD4) or H4/CD4 (CD4 positive glial cell line obtained by transfection of human H4 neuroglioma cells) (Volsky et al., 1992) was also inhibited by CaM (not shown). If the binding of CaM to its target protein requires the presence of Ca2 + , the fusion-inhibitory effect of CaM does not seem to be due to a deprivation of Ca2 + in the medium. Troponin C and parvalbumin also bind Ca2 + with high affinity (Scha¨fer and Heizmann, 1996). Nevertherless, it is possible that the MV-mediated cell fusion may be less Ca2 + dependent than the HIV-mediated cell fusion. The calcium-binding subunit of the troponin complex (troponin C) is highly similar in both amino acid sequence and three dimensional structure to CaM (Babu et al., 1985; Strynadka and James, 1989). Like CaM, the C-domain of troponin C has two high-affinity sites for Ca2 + that also bind Mg2 + , whereas the N-domain has two low-affinity, Ca2 + -specific sites (Grabarek et al., 1992). This suggests that the effect of CaM may be specific. In addition, despite sequence similarities between HIV gp41 and paramyxovirus fusion proteins (GonzalezScarano et al., 1987), CaM inhibited HIV-mediated cell fusion but not MV-mediated cell fusion, whereas DXSF.10 000, suramin and heparin inhibited both. Using [125I]CaM overlays of SDS-PAGE gels or NMR and CD spectroscopy, previous studies showed that the cytoplasmic domain of the HIV-1 or SIV glycoproteins but not the gp120 interacted with CaM (Srinivas et al., 1993; Yuan et al.,
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1995). Using synthetic peptide analogs, Srinivas et al., 1993 demonstrated the presence of two CaMbinding domains (768–788 and 826–854) in the intracellular carboxy-terminal segment of gp41. The truncated form expressed by vPE17 lacks these two CaM-binding domains. Miller et al., 1993 showed lack of CaM binding by a construct containing the first 185 residues of gp41. This construct was purified and then subjected to SDSPAGE followed by overlay in situ with [125I]CaM. However, during the purification procedures and SDS-PAGE the proteins may be denatured, undergo conformational changes, lose their affinity for CaM and, consequently, low-affinity CaMbinding sites may not be detected. Recently several groups have identified proteins that work in conjunction with cell-surface CD4 enabling HIV-1 to infect cells (Alkhatib et al., 1996; Deng et al., 1996; Drajic et al., 1996; Feng et al., 1996). These cell-surface receptors belong to the family of seven-transmembrane-spanning (7 tm), G-proteincoupled receptors. Several regions located in the extracellular domain of gp41 are conserved among HIV-1, HIV-2 and SIV isolates and possibly interacts with gp120 (Stoiber et al., 1994) or with the second-receptor molecule (Ebenbichler et al., 1996). It is tempting to suggest that CaM interferes with the binding of gp41 to gp120 or to the co-receptor. CaM may mask a site on a protein necessary for fusion and prevent it from interacting with another. However, CaM may also induce a structural change in the protein target or react specifically with regions involved in the fusion process, i.e. the fusion domain or the leucine zipper-like motif (Dubay et al., 1992; Wild et al., 1992). The leucine zipper-like motif has been demonstrated to bind to the host cell membrane bringing the viral and cellular membrane closer and facilitating fusion (Rabenstein and Shin, 1995). CaM is a small ubiquitous eukaryotic protein expressed extra- and intracellularly, which binds and activates many classes of proteins, including protein kinases, calcium pump, cytoskeletal proteins. An extracellular role for CaM has been demonstrated as it can act as a growth factor for a variety of cells in culture, including human leukemia lymphocytes (Gorbachevskaya et al.,
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1983; Crocker et al., 1988; Dalley et al., 1996). To our knowledge no CaM receptor has been identified. However, it cannot be excluded that the extracellular portion of a molecule necessary for fusion has a recognition site for CaM. It would be an attractive hypothesis to postulate that extracellular CaM may mediate Ca2 + entry into the cell; Ca2 + , as second messenger may control the biological process involved in HIV-mediated cell fusion. Similarly, SDF-1, a chemokine and a biological ligand for the HIV-1 entry cofactor LESTR (CXCR-4) has been demonstrated to induce an increase in intracellular free Ca2 + in CXCR-4-transfected cells (Bleul et al., 1996). Recently, molecules which inhibit the Na + /K + / 2Cl − cotransporter function by direct binding, have been shown to block the cytopathic effect of HIV-1 (Voss et al., 1996). CaM may regulate a Ca2 + activated ion channel via direct binding to the channel. However, the importance of our observation has to be determined. Further studies are necessary to determine if extracellular CaM can prevent cells from HIV infection or modulate the life-cycle of the virus in infected individuals.
Acknowledgements We thank Drs M.P. Kie´ny, B. Moss and A. Otteken for generously providing recombinant vaccinia viruses, Drs Z.L. Jonak, H. Hauser, D.J. Volsky for cell lines. We would like also to thank Dr H.C. Holmes of the ADP reagent programme for reagents and Bernadette Maret for editorial assistance. E.M. held a SIDACTION fellowship.
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