JOURNAL OF VIROLOGY, Nov. 1991, p. 6325-6330
Vol. 65, No. 11
0022-538X/91/116325-06$02.00/0 Copyright C) 1991, American Society for Microbiology
Inhibition of Human Immunodeficiency Virus Type 1-Induced Cell Fusion by Recombinant Human Interferons DAVID E. WELLS,' SUBHENDRA CHATTERJEE,"2 MARK J. MULLIGAN,3 AND RICHARD W. COMPANSl* Departments of Microbiology,' Pediatrics,2 and Medicine,3 University of Alabama at Birmingham, Birmingham, Alabama 35294 Received 3 December 1990/Accepted 25 July 1991
Pretreatment of HeLa T4 cells with recombinant alpha, beta, or gamma interferon (IFN) was found to significantly inhibit syncytium formation induced by the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein. All three IFNs were found to be potent inhibitors of fusion in a system in which Spodoptera frugiperda cells, infected with a baculovirus recombinant expressing the HIV-1 envelope protein, were cocultivated with HeLa T4 cells. In addition, these IFNs were also found to block HeLa T4 cell fusion induced by the HIV-1 envelope proteins expressed from a vaccinia virus recombinant. Furthermore, the IFNs inhibited cell fusion between HIV-1 envelope glycoprotein-expressing cells and either immortalized or fresh CD4+ lymphocytes pretreated with the IFNs. These results suggest that further testing of human IFNs for therapy of HIV-1 infection will be of interest.
Infection with the human immunodeficiency virus (HIV) involves a series of steps initiated by binding of the viral glycoprotein, gpl20, to the host cell receptor, the CD4 molecule (1, 8, 15). Binding is thought to be followed by the insertion of the N terminus of the fusogenic glycoprotein gp4l (transmembrane glycoprotein) into the host cell membrane, triggering fusion of the viral and cellular membranes and concurrent entry of the viral genome into the cell (12, 24). Multinucleated giant cells (syncytia), which are frequently observed in vitro in HIV type 1 (HIV-1)-infected cultures, are probably formed by a similar mechanism. When expressed on the surfaces of cells, the gpl20/41 complex can induce cell-to-cell fusion with CD4+ cells (13, 22), and such cell fusion is considered a major mode of virus transmission within the host. In this investigation, we have used recombinant human alpha, beta, and gamma interferons (IFN-a, -13, and -y) to investigate their effects on cell fusion induced by the HIV-1 envelope glycoprotein. The results indicate that pretreatment of cells with any of these IFNs significantly inhibits the syncytium formation induced by HIV envelope glycoproteins. Recently, we have expressed biologically active envelope proteins of HIV-1 using a vaccinia virus recombinant (18) and using a baculovirus recombinant (25), and we have demonstrated their capacity to induce cell fusion with CD4+ cells. In the present study, both expression systems have been used to evaluate the potential of recombinant human IFNs to inhibit the cell fusion induced by the HIV-1 envelope protein. We previously reported that cocultivation of HeLa T4 cells with insect cells infected with a baculovirus recombinant expressing HIV gp160 resulted in extensive cell fusion (25). Using this assay system, we observed that pretreatment of the HeLa T4 cells for 18 h with 100 IU of IFN-a, -1B, or --y per ml completely inhibited syncytium formation induced by the HIV-1 envelope protein (Fig. 1). Figure 1A shows untreated HeLa T4 cells exhibiting extensive syncytium formation after cocultivation with Spodoptera frugiperda cells expressing the HIV-1 envelope *
Corresponding author.
protein. In contrast, when HeLa T4 cells pretreated with 100 IU of IFN-a, -,B, or -y per ml were cocultivated with S. frugiperda cells expressing the HIV-1 envelope protein, no cell fusion was detectable (Fig. 1B through D, respectively). Syncytium formation was not detected in controls consisting of pretreated or untreated HeLa cells (lacking the CD4+ receptor) cocultivated with S. frugiperda cells expressing the envelope protein (data not shown). These results indicate that pretreatment of the HeLa T4 cells with any of these human IFNs induces a state of complete resistance to cell fusion induced by the HIV-1 envelope glycoproteins. In order to evaluate the effect of IFNs on the cell fusion induced by the HIV-1 envelope protein with another assay system, HeLa T4 cells were cocultivated with HeLa cells infected with a vaccinia virus recombinant expressing gpl60 of HIV-1 (18). Extensive cell fusion developed when untreated HeLa T4 cells were cocultivated with HeLa cells expressing the vaccinia virus recombinant-derived envelope protein (Fig. 2A). Pretreatment of the HeLa T4 cells with 100 IU of IFN-oa, -13, or -y per ml significantly inhibited such syncytium formation (Fig. 2B through D, respectively), although IFN-a did not appear to block cell fusion completely at this concentration. In addition to these two systems, we also evaluated the effects of IFNs on the ability of a CD4+ leukemia cell line (Jurkat) to fuse with HIV-1 envelope glycoprotein-expressing cells. The HIV-1 env-J vaccinia virus recombinant was used to infect HSB cells (a CD4- lymphoid cell line); after 18 h, Jurkat cells that had been pretreated for 18 h with 0, 500, or 1,000 IU of the IFNs per ml were cocultivated with the envelope glycoprotein expressing HSB cells. Syncytium formation was evaluated following 8 h of cocultivation. Partial (500 IU/ml) and nearly complete (1,000 IU/ml) inhibition of cell fusion was observed (Fig. 3). Three independent experiments yielded similar results; no toxic effects of the IFNs on Jurkat cells were observed for up to 48 h. Cell fusion assays with fresh human peripheral blood mononuclear cells preincubated with the IFNs yielded similar inhibition results (data not shown). To determine whether the interaction between the HIV-1 envelope gene and the target cell is directly affected by cloned IFNs, monolayers of HeLa T4 cells were coculti6325
6326
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FIG. 1. Effect of recombinant human IFNs on syncytium formation induced by baculovirus-expressed HIV-1 envelope protein. (A) A confluent monolayer of HeLa T4 cells was cocultivated for 24 h with S. frugiperda cells expressing HIV-1 gpl60. S. frugiperda cells were infected at a multiplicity of infection of 1 with a recombinant baculovirus expressing gpl60, as described previously (25). A significant fractiori of the cocultivated cells formed multinucleated giant cells. (B through D) Confluent monolayers of HeLa T4 cells were pretreated for 18 h with 100 IU of recombinant human IFN-ax, -P, or -y per ml, respectively, and then cocultivated for 24 h with S. frugiperda cells expressing gpl60. Recombinant human IFN-a and --y (2 x 106 and 5.5 x 106 IU/ml, respectively) were provided by Schering-Plough Corp., Bloomfield, N.J., and recombinant human IFN-P (5.4 x 107 IU/ml) was obtained from Cetus Corp., Emeryville, Calif. Pretreatment of the cells with each IFN completely prevented syncytium formation.
VOL. 65, 1991
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FIG. 2. Effect of recombinant human IFNs on syncytium formation induced by vaccinia virus env-1-expressed HIV-1 gpl60. (A) Confluent monolayers of HeLa T4 cells were cocultivated for 24 h with HeLa cells expressing the HIV-1 envelope protein. HeLa cells were infected at a multiplicity of infection of 0.1 with vaccinia virus env-l, a vaccinia virus recombinant that expresses the HIV-1 envelope glycoprotein. Extensive cell fusion is evident. (B through D) Confluent monolayers of HeLa T4 cells were pretreated for 18 h with 100 IU of IFN-a, -1, or -^y per ml, respectively, and then cocultivated with HeLa cells expressing HIV-1 gpl6O. Pretreatment of cells with these IFNs significantly inhibited the multinucleate cell formation.
vated with S. frugiperda cells infected 24 h previously with a baculovirus recombinant expressing the HIV-1 envelope protein. IFN-a, -13, or --y was added at a concentration of 100 IU/ml at the beginning of the cocultivation period. Formation of syncytia could be detected at low levels as early as 6 h after cocultivation of HeLa T4 cells with insect cells expressing HIV-1 envelope proteins. No significant inhibitory effect of the IFNs was observed under these conditions (data not shown). These results indicate that the IFNs used in these studies do not directly affect the biological activities of the HIV-1 envelope protein and that pretreatment of the target cells with IFN is required for induction of a fusionresistant state, although under other conditions (such as higher concentrations of IFN or longer treatment), a direct effect of IFN cannot be excluded, as it has been reported that IFN induces changes in cell membrane fluidity (2, 19). A quantitative analysis of the effects of IFNs on HIV-1induced syncytium formation in the S. frugiperda-HeLa T4 cell system revealed that IFN--y apparently is most effective, with a concentration of 10 IU/ml being sufficient to almost completely prevent syncytium formation (Fig. 4). For both
of the other IFNs, partial inhibition was observed at concentrations as low as 1 IU/ml. Thus, each of these IFNs is capable of affecting syncytium formation at low concentrations. To determine the effects of these IFNs on the cell surface expression of CD4 molecules, Jurkat cells were treated with 1,000 IU of IFN-a, -1, or --y per ml for 18 h. The expression of CD4 molecules on the cell surface was determined by immunofluorescence staining with OKT4 or OKT4A (Ortho, Inc.), monoclonal antibodies that recognize CD4 epitopes. Specific staining was quantitated by flow cytometry (FACStar; Becton Dickinson and Co.). The results (Fig. 5) indicate that the IFNs have no significant effects on the levels of CD4 molecules present on the cell surfaces. Previously it has been reported that human IFNs inhibited viral replication and syncytium formation caused by D-type retroviruses and certain other enveloped viruses. These investigations indicated that syncytium formation and replication by the D-type retrovirus Mason-Pfizer monkey virus could be inhibited by human IFNs (3, 4). These results are
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FIG. 3. Inhibitory effect of recombinant human IFNs on Jurkat cell fusion to HIV-1 envelope glycoprotein expressing cells. At 18 h before cocultivation, CD4-HSB cells were infected with vaccinia virus env-1, and Jurkat cells were incubated in the presence of 0, 500, or 1,000 IU of IFN per ml. At 8 h following cocultivation of the cell lines, cell fusion was evaluated. (A) Control (no IFN pretreatment of Jurkat cells); (B through D) nearly complete inhibition of cell fusion after pretreatment of Jurkat cells with 1,000 IU of IFN-a, -,3, or --y per ml respectively.
relevant because of reports associating D-type retroviruses with an AIDS of macaque monkeys (9, 14, 23). In other studies it was shown that recombinant human IFNs significantly reduce herpes simplex virus type 1 replication and block herpes simplex virus type 1-induced cell fusion in homologous and heterologous cells (5-7). Furthermore, it was demonstrated that "fusion from without" induced by Sendai virus could be specifically inhibited by homologous IFNs (2), suggesting that the human IFNs may have a significant effect on the interactions of viral envelope glycoproteins with host cells. This latter effect may prevent virus budding and release from IFN-treated cells, as suggested for some retroviruses (4, 21). At present, none of the antiviral drugs tested is completely satisfactory for treating AIDS and AIDS-related diseases. Because of the propensity of HIV-1 for genetic and structural variation, combinations of antiretroviral agents will likely be necessary to combat development of resistance. It has been reported that human IFN-a has a suppressive effect on HIV replication in vitro (11, 20). Recently, the effectiveness and antiretroviral activities of recombinant human IFN-a in AIDS-related Kaposi's sarcoma in a clinical trial
reported (10). In addition, human IFN-P and -_y have been shown to block the replication of HIV in vitro (16, 17). The present results suggest a novel mechanism by which HIV-1 replication may also be prevented in vivo. Inhibition of HIV-1-induced membrane fusion is likely to prevent the spread of HIV infection in vivo, since both entry of released viruses into susceptible cells and cell-to-cell spread (via fusion) would be prevented. The concentrations of IFNs found to be capable of preventing syncytium formation in the present study are readily attainable in vivo. The present results, therefore, suggest that further investigation of the effects of IFNs for therapy of human HIV-1 infection is warranted. were
We thank Cyndi Tanner and Lawrence Melsen for excellent technical assistance, Randall J. Owens for providing the vaccinia virus env-1 recombinant, and Peter Cresswell for the HSB cell line. This research was supported by grants Al 25784, AI 27290, Al 28147, Al 00912, and Al 25120 from the National Institute of Allergy and Infectious Diseases. David E. Wells was supported in part by Institutional Research Service Award Al 07150 from the National Institute of Allergy and Infectious Diseases.
VOL. 65,
1991
NOTES
REFERENCES
100
c
1. Bedinger, P., A. Moriarty, R. C. von Borstel II, N. J. Donovan, K. S. Steimer, and D. R. Littman. 1988. Internalization of the human immunodeficiency virus does not require the cytoplasmic domain of CD4. Nature (London) 334:162-165. 2. Chatterjee, S., H. C. Cheung, and E. Hunter. 1982. Interferon inhibits Sendai virus-induced cell fusion: an effect on cell
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Natl. Acad. Sci. USA 79:835-839. 1980. Inhibition of Mason-Pfizer monkey virus-induced syncytium formation in normal human cells by homologous interferon. Virology 104:487-490. 4. Chatterjee, S., and E. Hunter. 1987. Recombinant human interferons inhibit replication of Mason-Pfizer monkey virus in primate cells. Virology 157:548-551. 5. Chatterjee, S., E. Hunter, and R. Whitley. 1985. Effect of cloned human interferons on protein synthesis and morphogenesis of herpes simplex virus. J. Virol. 56:419-425. 6. Chatterjee, S., A. D. Lakeman, R. J. Whitley, and E. Hunter. 1984. Effect of cloned human interferons on the replication of and cell fusion induced by herpes simplex virus. Virus Res. membrane fluidity. Proc.
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3. Chatterjee, S., and E. Hunter.
c
tn c
40
r-
De
20
IFNs
1:81-87. 7. Chatterjee, S., and R. J.
U/mn
FIG. 4. Dose dependence of the inhibition by recombinant human IFNs of HIV-induced syncytium formation. HeLa T4 cells IFNs were pretreated with IFN-(x (0), IFN-P (0), or IFN--y cocultivated with S. frugiperda cells expressing the HIV-1 envelope protein, and the extent of syncytium formation was quantitated. HeLa T4 cells were pretreated with IFNs and cocultivated with S. frugiperda cells expressing HIV-1 gpl60 as described in the legend to Fig. 1. Five random fields of cells were photographed at low magnification, and the numbers of syncytia present were counted.
([1).
400
1217. 11. Ho, D. D., K. L. Hartshorn, T. R. Rota, C. A. Andrews, J. C. Kaplan, R. T. Schooley, and M. S. Hirsch. 1985. Recombinant human interferon alpha-A suppresses HTLV-III replication in vitro. Lancet i:602-604. 12. Kowalski, M., J. Patz, L. Basinipour, T. Dorfiman, W. C. Goh, E. Terwilliger, A. Dayton, C. Rose, W. Haseltine, and J. Sodroski. 1987. Functional regions of the envelope glycoprotein of human immunodeficiency virus type 1. Science 237:1351-1355. 13. Maddon, P. J., A. G. Dalgleish, J. S. McDougal, P. R. Clapham, R. A. Weiss, and R. Axel. 1986. The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the
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Whitley. 1989. Effect of recombinant hybrid human interferon on replication and morphogenesis of HSV-1 in monkey cells. Virus Res. 12:33-42. 8. Dalgeleish, A. G., P. C. L. Beverley, P. R. Clapham, D. H. Crawford, M. F. Greaves, and R. A. Weiss. 1984. The CD4(T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature (London) 312:763-767. 9. Daniel, M. D., N. W. King, N. L. Letvin, R. D. Hunt, P. K. Sehgal, and R. C. Desrosiers. 1984. A new type D retrovirus isolated from macaques with an immunodeficiency syndrome. Science 223:602-605. 10. De Wit, R., J. K. M. E. Schattenkerk, C. A. B. Boucher, P. J. M. Bakker, K. H. N. Veenhof, and S. A. Danner. 1988. Clinical and virological effects of high-dose recombinant interferon-a, in disseminated AIDS-related Kaposi's sarcoma. Lancet i:1214-
brain. Cell 47:333-348.
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K. G. Osborn, N. W. Lerche, P. V. Henrickson, L. 0. Arthur, R. V. Gilden, M. Gravell, W. T. London, J. L. Sever, J. A. Levy, R. J. Munn, and M. B. Gardner. 1984. Simian AIDS: isolation of a type D retrovirus and transmission of the disease. Science
14. Marx, P. A., D. H. Maul, Moody, L. J. Lowenstine, R.
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223:1083-1086. 15. McDougal, J. S., M. S. Kennedy, J. M. Sligh, S. P. Cort, A. Mawle, and J. K. A. Nicholson. 1986. Binding of HTLV-III/LAV to T4+ T cells by a complex of the 110K viral protein and the T4
molecule. Science 231:382-385. Michaelis, B., and J. A. Levy. 1989. HIV replication can be blocked by recombinant human interferon beta. AIDS 3:27-31. 17. Nakashima, H., T. Yoshida, S. Harada, and N. Yamamoto. 1986. Recombinant human interferon gamma suppresses HIV replication in vitro. Int. J. Cancer 38:433-436. 18. Owens, R. J., and R. W. Compans. 1989. Expression of the
16. II
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I
,
e1
la, FITC
lo
I I
1 lo
I
I
I
IIII .11101 2 I 1a
l
I-
FITC FLUORESCENCE FLUORESCENCE FIG. 5. Lack of effect of recombinant human IFNs on Jurkat cell
surface expression of CD4. Immunofluorescence staining with OKT4A monoclonal antibody (fluorescein isothiocyanate [FITC] conjugated) was analyzed by flow cytometry. (A) Control (no IFN treatment of the cells prior to staining). (B through D) Before immunofluorescence staining, cells were pretreated (18 h) with 1,000 or --y per ml, respectively. x axis, intensity of IU IFN-a, fluorescence; y axis, number of cells. Similar results were obtained with monoclonal antibody OKT4 (data not shown). -p,
human
immunodeficiency virus envelope glycoprotein
is re-
stricted to basolateral surfaces of polarized epithelial cells. J. Virol. 63:978-982. 19. Pfeffer, L. M., F. R. Landsberger, and I. Tamm. 1981. Betainterferon-induced time-dependent changes in the plasma membrane lipid bilayer of cultured cells. J. Interferon Res. 1:613620. 20. Poli, G., J. M. Orenstein, A. Kinter, T. M. Folks, and A. S.
6330
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Fauci. 1989. Interferon-ox but not AZT suppresses HIV expression in chronically infected cell lines. Science 244:575-577. 21. Sen, G. C., and N. H. Sarkar. 1980. Effects of interferon on the production of murine mammary tumor virus by mammary tumor cells in culture. Virology 102:431-443. 22. Sodroski, J., W. C. Goh, C. Rosen, K. Campbell, and W. A. Haseltine. 1986. Role of the HTLV-III/LAV envelope in syncytium formation and cytopathicity. Nature (London) 322:470474. 23. Stromberg, K., R. E. Benveniste, L. 0. Arthur, H. Rabin, W. E. Giddnes, Jr., H. D. Ochs, W. R. Morton, and C.-C. Tsai. 1984.
J. VIROL. Characterization of exogenous type D retrovirus from a fibroma of a macaque with simian AIDS and fibromatosis. Science 224:289-292. 24. Veronese, F., A. L. De Vico, T. D. Copeland, S. Oroszlan, R. C. Gallo, and M. G. Sarngadharan. 1985. Characterization of gp-41 as the transmembrane protein coded by the HTLV-III/LAV envelope gene. Science 229:1402-1405. 25. Wells, D. E., and R. W. Compans. 1990. Expression and characterization of a functional human immunodeficiency virus envelope glycoprotein in insect cells. Virology 176:575-586.
Vol. 65, No. 11
0022-538X/91/116325-06$02.00/0 Copyright C) 1991, American Society for Microbiology
Inhibition of Human Immunodeficiency Virus Type 1-Induced Cell Fusion by Recombinant Human Interferons DAVID E. WELLS,' SUBHENDRA CHATTERJEE,"2 MARK J. MULLIGAN,3 AND RICHARD W. COMPANSl* Departments of Microbiology,' Pediatrics,2 and Medicine,3 University of Alabama at Birmingham, Birmingham, Alabama 35294 Received 3 December 1990/Accepted 25 July 1991
Pretreatment of HeLa T4 cells with recombinant alpha, beta, or gamma interferon (IFN) was found to significantly inhibit syncytium formation induced by the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein. All three IFNs were found to be potent inhibitors of fusion in a system in which Spodoptera frugiperda cells, infected with a baculovirus recombinant expressing the HIV-1 envelope protein, were cocultivated with HeLa T4 cells. In addition, these IFNs were also found to block HeLa T4 cell fusion induced by the HIV-1 envelope proteins expressed from a vaccinia virus recombinant. Furthermore, the IFNs inhibited cell fusion between HIV-1 envelope glycoprotein-expressing cells and either immortalized or fresh CD4+ lymphocytes pretreated with the IFNs. These results suggest that further testing of human IFNs for therapy of HIV-1 infection will be of interest.
Infection with the human immunodeficiency virus (HIV) involves a series of steps initiated by binding of the viral glycoprotein, gpl20, to the host cell receptor, the CD4 molecule (1, 8, 15). Binding is thought to be followed by the insertion of the N terminus of the fusogenic glycoprotein gp4l (transmembrane glycoprotein) into the host cell membrane, triggering fusion of the viral and cellular membranes and concurrent entry of the viral genome into the cell (12, 24). Multinucleated giant cells (syncytia), which are frequently observed in vitro in HIV type 1 (HIV-1)-infected cultures, are probably formed by a similar mechanism. When expressed on the surfaces of cells, the gpl20/41 complex can induce cell-to-cell fusion with CD4+ cells (13, 22), and such cell fusion is considered a major mode of virus transmission within the host. In this investigation, we have used recombinant human alpha, beta, and gamma interferons (IFN-a, -13, and -y) to investigate their effects on cell fusion induced by the HIV-1 envelope glycoprotein. The results indicate that pretreatment of cells with any of these IFNs significantly inhibits the syncytium formation induced by HIV envelope glycoproteins. Recently, we have expressed biologically active envelope proteins of HIV-1 using a vaccinia virus recombinant (18) and using a baculovirus recombinant (25), and we have demonstrated their capacity to induce cell fusion with CD4+ cells. In the present study, both expression systems have been used to evaluate the potential of recombinant human IFNs to inhibit the cell fusion induced by the HIV-1 envelope protein. We previously reported that cocultivation of HeLa T4 cells with insect cells infected with a baculovirus recombinant expressing HIV gp160 resulted in extensive cell fusion (25). Using this assay system, we observed that pretreatment of the HeLa T4 cells for 18 h with 100 IU of IFN-a, -1B, or --y per ml completely inhibited syncytium formation induced by the HIV-1 envelope protein (Fig. 1). Figure 1A shows untreated HeLa T4 cells exhibiting extensive syncytium formation after cocultivation with Spodoptera frugiperda cells expressing the HIV-1 envelope *
Corresponding author.
protein. In contrast, when HeLa T4 cells pretreated with 100 IU of IFN-a, -,B, or -y per ml were cocultivated with S. frugiperda cells expressing the HIV-1 envelope protein, no cell fusion was detectable (Fig. 1B through D, respectively). Syncytium formation was not detected in controls consisting of pretreated or untreated HeLa cells (lacking the CD4+ receptor) cocultivated with S. frugiperda cells expressing the envelope protein (data not shown). These results indicate that pretreatment of the HeLa T4 cells with any of these human IFNs induces a state of complete resistance to cell fusion induced by the HIV-1 envelope glycoproteins. In order to evaluate the effect of IFNs on the cell fusion induced by the HIV-1 envelope protein with another assay system, HeLa T4 cells were cocultivated with HeLa cells infected with a vaccinia virus recombinant expressing gpl60 of HIV-1 (18). Extensive cell fusion developed when untreated HeLa T4 cells were cocultivated with HeLa cells expressing the vaccinia virus recombinant-derived envelope protein (Fig. 2A). Pretreatment of the HeLa T4 cells with 100 IU of IFN-oa, -13, or -y per ml significantly inhibited such syncytium formation (Fig. 2B through D, respectively), although IFN-a did not appear to block cell fusion completely at this concentration. In addition to these two systems, we also evaluated the effects of IFNs on the ability of a CD4+ leukemia cell line (Jurkat) to fuse with HIV-1 envelope glycoprotein-expressing cells. The HIV-1 env-J vaccinia virus recombinant was used to infect HSB cells (a CD4- lymphoid cell line); after 18 h, Jurkat cells that had been pretreated for 18 h with 0, 500, or 1,000 IU of the IFNs per ml were cocultivated with the envelope glycoprotein expressing HSB cells. Syncytium formation was evaluated following 8 h of cocultivation. Partial (500 IU/ml) and nearly complete (1,000 IU/ml) inhibition of cell fusion was observed (Fig. 3). Three independent experiments yielded similar results; no toxic effects of the IFNs on Jurkat cells were observed for up to 48 h. Cell fusion assays with fresh human peripheral blood mononuclear cells preincubated with the IFNs yielded similar inhibition results (data not shown). To determine whether the interaction between the HIV-1 envelope gene and the target cell is directly affected by cloned IFNs, monolayers of HeLa T4 cells were coculti6325
6326
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FIG. 1. Effect of recombinant human IFNs on syncytium formation induced by baculovirus-expressed HIV-1 envelope protein. (A) A confluent monolayer of HeLa T4 cells was cocultivated for 24 h with S. frugiperda cells expressing HIV-1 gpl60. S. frugiperda cells were infected at a multiplicity of infection of 1 with a recombinant baculovirus expressing gpl60, as described previously (25). A significant fractiori of the cocultivated cells formed multinucleated giant cells. (B through D) Confluent monolayers of HeLa T4 cells were pretreated for 18 h with 100 IU of recombinant human IFN-ax, -P, or -y per ml, respectively, and then cocultivated for 24 h with S. frugiperda cells expressing gpl60. Recombinant human IFN-a and --y (2 x 106 and 5.5 x 106 IU/ml, respectively) were provided by Schering-Plough Corp., Bloomfield, N.J., and recombinant human IFN-P (5.4 x 107 IU/ml) was obtained from Cetus Corp., Emeryville, Calif. Pretreatment of the cells with each IFN completely prevented syncytium formation.
VOL. 65, 1991
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FIG. 2. Effect of recombinant human IFNs on syncytium formation induced by vaccinia virus env-1-expressed HIV-1 gpl60. (A) Confluent monolayers of HeLa T4 cells were cocultivated for 24 h with HeLa cells expressing the HIV-1 envelope protein. HeLa cells were infected at a multiplicity of infection of 0.1 with vaccinia virus env-l, a vaccinia virus recombinant that expresses the HIV-1 envelope glycoprotein. Extensive cell fusion is evident. (B through D) Confluent monolayers of HeLa T4 cells were pretreated for 18 h with 100 IU of IFN-a, -1, or -^y per ml, respectively, and then cocultivated with HeLa cells expressing HIV-1 gpl6O. Pretreatment of cells with these IFNs significantly inhibited the multinucleate cell formation.
vated with S. frugiperda cells infected 24 h previously with a baculovirus recombinant expressing the HIV-1 envelope protein. IFN-a, -13, or --y was added at a concentration of 100 IU/ml at the beginning of the cocultivation period. Formation of syncytia could be detected at low levels as early as 6 h after cocultivation of HeLa T4 cells with insect cells expressing HIV-1 envelope proteins. No significant inhibitory effect of the IFNs was observed under these conditions (data not shown). These results indicate that the IFNs used in these studies do not directly affect the biological activities of the HIV-1 envelope protein and that pretreatment of the target cells with IFN is required for induction of a fusionresistant state, although under other conditions (such as higher concentrations of IFN or longer treatment), a direct effect of IFN cannot be excluded, as it has been reported that IFN induces changes in cell membrane fluidity (2, 19). A quantitative analysis of the effects of IFNs on HIV-1induced syncytium formation in the S. frugiperda-HeLa T4 cell system revealed that IFN--y apparently is most effective, with a concentration of 10 IU/ml being sufficient to almost completely prevent syncytium formation (Fig. 4). For both
of the other IFNs, partial inhibition was observed at concentrations as low as 1 IU/ml. Thus, each of these IFNs is capable of affecting syncytium formation at low concentrations. To determine the effects of these IFNs on the cell surface expression of CD4 molecules, Jurkat cells were treated with 1,000 IU of IFN-a, -1, or --y per ml for 18 h. The expression of CD4 molecules on the cell surface was determined by immunofluorescence staining with OKT4 or OKT4A (Ortho, Inc.), monoclonal antibodies that recognize CD4 epitopes. Specific staining was quantitated by flow cytometry (FACStar; Becton Dickinson and Co.). The results (Fig. 5) indicate that the IFNs have no significant effects on the levels of CD4 molecules present on the cell surfaces. Previously it has been reported that human IFNs inhibited viral replication and syncytium formation caused by D-type retroviruses and certain other enveloped viruses. These investigations indicated that syncytium formation and replication by the D-type retrovirus Mason-Pfizer monkey virus could be inhibited by human IFNs (3, 4). These results are
6328
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FIG. 3. Inhibitory effect of recombinant human IFNs on Jurkat cell fusion to HIV-1 envelope glycoprotein expressing cells. At 18 h before cocultivation, CD4-HSB cells were infected with vaccinia virus env-1, and Jurkat cells were incubated in the presence of 0, 500, or 1,000 IU of IFN per ml. At 8 h following cocultivation of the cell lines, cell fusion was evaluated. (A) Control (no IFN pretreatment of Jurkat cells); (B through D) nearly complete inhibition of cell fusion after pretreatment of Jurkat cells with 1,000 IU of IFN-a, -,3, or --y per ml respectively.
relevant because of reports associating D-type retroviruses with an AIDS of macaque monkeys (9, 14, 23). In other studies it was shown that recombinant human IFNs significantly reduce herpes simplex virus type 1 replication and block herpes simplex virus type 1-induced cell fusion in homologous and heterologous cells (5-7). Furthermore, it was demonstrated that "fusion from without" induced by Sendai virus could be specifically inhibited by homologous IFNs (2), suggesting that the human IFNs may have a significant effect on the interactions of viral envelope glycoproteins with host cells. This latter effect may prevent virus budding and release from IFN-treated cells, as suggested for some retroviruses (4, 21). At present, none of the antiviral drugs tested is completely satisfactory for treating AIDS and AIDS-related diseases. Because of the propensity of HIV-1 for genetic and structural variation, combinations of antiretroviral agents will likely be necessary to combat development of resistance. It has been reported that human IFN-a has a suppressive effect on HIV replication in vitro (11, 20). Recently, the effectiveness and antiretroviral activities of recombinant human IFN-a in AIDS-related Kaposi's sarcoma in a clinical trial
reported (10). In addition, human IFN-P and -_y have been shown to block the replication of HIV in vitro (16, 17). The present results suggest a novel mechanism by which HIV-1 replication may also be prevented in vivo. Inhibition of HIV-1-induced membrane fusion is likely to prevent the spread of HIV infection in vivo, since both entry of released viruses into susceptible cells and cell-to-cell spread (via fusion) would be prevented. The concentrations of IFNs found to be capable of preventing syncytium formation in the present study are readily attainable in vivo. The present results, therefore, suggest that further investigation of the effects of IFNs for therapy of human HIV-1 infection is warranted. were
We thank Cyndi Tanner and Lawrence Melsen for excellent technical assistance, Randall J. Owens for providing the vaccinia virus env-1 recombinant, and Peter Cresswell for the HSB cell line. This research was supported by grants Al 25784, AI 27290, Al 28147, Al 00912, and Al 25120 from the National Institute of Allergy and Infectious Diseases. David E. Wells was supported in part by Institutional Research Service Award Al 07150 from the National Institute of Allergy and Infectious Diseases.
VOL. 65,
1991
NOTES
REFERENCES
100
c
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3. Chatterjee, S., and E. Hunter.
c
tn c
40
r-
De
20
IFNs
1:81-87. 7. Chatterjee, S., and R. J.
U/mn
FIG. 4. Dose dependence of the inhibition by recombinant human IFNs of HIV-induced syncytium formation. HeLa T4 cells IFNs were pretreated with IFN-(x (0), IFN-P (0), or IFN--y cocultivated with S. frugiperda cells expressing the HIV-1 envelope protein, and the extent of syncytium formation was quantitated. HeLa T4 cells were pretreated with IFNs and cocultivated with S. frugiperda cells expressing HIV-1 gpl60 as described in the legend to Fig. 1. Five random fields of cells were photographed at low magnification, and the numbers of syncytia present were counted.
([1).
400
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16. II
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,
e1
la, FITC
lo
I I
1 lo
I
I
I
IIII .11101 2 I 1a
l
I-
FITC FLUORESCENCE FLUORESCENCE FIG. 5. Lack of effect of recombinant human IFNs on Jurkat cell
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human
immunodeficiency virus envelope glycoprotein
is re-
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