JOURNAL OF VIROLOGY, Dec. 1990, p. 6252-6256
Vol. 64, No. 12
0022-538X/90/126252-05$02.00/0 Copyright C 1990, American Society for Microbiology
Soluble CD4 Enhances Simian Immunodeficiency Virus SIVagm Infection ALBRECHT WERNER, GUDRUN WINSKOWSKY, AND REINHARD KURTH* Paul-Ehrlich-Institute, Paul-Ehrlich-Strasse 51-59, D-6070 Langen 1, Federal Republic of Germany Received 30 April 1990/Accepted 27 July 1990
The CD4 molecule is expressed on T-helper cells and serves as the cellular receptor for the human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and for the simian immunodeficiency viruses SIVmac and SIVagm. HIV-1, HIV-2, and SIVmac infectivity can be blocked by monoconal antibodies (MAbs) directed against the CD4 molecule and by soluble CD4 proteins (sCD4). In the present study, we demonstrated not only lack of inhibition, but 10- to 100-fold sCD4-dependent enhancement of SIVagm infectivity of human T-cell lymphoma lines, although SIVagm infection was blocked by MAbs OKT4a and Leu3a. SIVagm enhancement with sCD4 was suppressed by MAbs OKT4a and Leu3a to levels observed without addition of sCD4. The infectivity of all four tested SIVagm variants was enhanced by sCD4 on all tested lymphoma cell lines. These results suggest a second step (second or secondary receptor) required for enhancing virus entry into the cell and may have serious implications for approaches to the treatment of acquired immunodeficiency syndrome on the basis of modified sCD4 molecules.
The 55-kDa CD4 glycoprotein is expressed on T-helper/ inductor lymphocytes (6, 14) and is thought to be involved in the interaction with major histocompatibility complex class II-bearing cells (9). In humans and higher primates, CD4 and its structurally related simian analogs also serve as the cellular receptors for human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and for simian immunodeficiency viruses SIVmaC and SIVagm (6, 14, 15, 21, 23). The highaffinity interaction between HIV-1 and HIV-2 envelope protein gpl20 and cellular CD4 can be inhibited in vitro by both low concentrations of soluble CD4 (sCD4) and by a number of CD4-specific monoclonal antibodies (MAbs) (7, 8, 12, 26, 27, 29), resulting in complete blocking of virus infectivity. In preliminary experiments in vivo, human sCD4 has been shown to possess some therapeutic effect in the treatment of SIVmac-infected rhesus monkeys (28). Consequently, clinical trials have been initiated in the United States to treat HIV-infected individuals. Infectivity of SIVagm can be blocked by the same MAbs directed against the CD4 molecule as HIV-1 and HIV-2 can (6, 15). These findings are not surprising, considering that the putative CD4-binding sites of SIVagm and HIV are relatively conserved (17, 21). However, in contrast to the findings with HIV, blocking of SIVagm infectivity with sCD4 is not possible. On the contrary, sCD4 enhances SIVagm infectivity (1). In the present study we also report that sCD4 can block infectivity of HIV-1 but enhances infectivity of SIVagm. In addition, the enhancement of SIVagm infectivity with sCD4 can be suppressed by MAbs OKT4a and Leu3a, but not by OKT4, to levels measured without sCD4 and OKT4a. Binding of recombinant sCD4 to SIVagm may activate viral binding sites or fusion domains, resulting in enhanced cell infection.
were kindly donated by R. C. Gallo, National Institutes of Health, Bethesda, Md., and we received the CEM-SS cell line, a subclone of CEM, from P. Nara, National Cancer Institute, Frederick, Md. All cell lines were maintained in RPMI 1640 containing 10% heat-inactivated fetal calf serum (FCS) and antibiotics. Virus stocks and assays. Assays were carried out with the HTLV-IIIB strain of HIV-1 and with several SIVagm isolates from African green monkeys, as described previously (15). SIVagm and HIV-1 (HTLV-IIIB) virus stocks were prepared and titrated as described previously (30). Virus stocks were diluted in RPMI 1640 containing 10% FCS and antibiotics to a concentration of 2 x 102 50% tissue culture infective doses (TCID50) per ml. Then 0.5 ml of the stocks was supplemented with an equal volume of full-length sCD4 (3.2 ,ug/ml or with concentrations of sCD4 in the range of 0.1 to 6.4 jig/ml). sCD4 polypeptide containing extracellular amino acid residues 1 to 374 of the human CD4 cell receptor prepared from supernatant cultures of Spodoptera frugiperda host cells infected with recombinant Autographa californica nuclear polyhedrosis virus containing the corresponding gene sequence for human sCD4 was kindly provided by T. B. Helting, Pharmacia Genetic Engineering Inc., La Jolla, Calif. sCD4, either full length or containing the first 153 amino-terminal amino acids (Vl and part of the V2 domain), was also purified from correspondingly transfected Escherichia coli (by Pharmacia Genetic Engineering Inc.) and showed a comparable SIVagm infection-enhancing activity. Because the yield of sCD4 from E. coli was much lower than that from the supernatant of S. frugiperda host cells, the experiments illustrated here were performed with sCD4 derived from these cells. All virus-sCD4 mixtures were incubated for 1 h at 37°C. Molt4 clone 8 cells (5 x 105), cultured in 24-well plates (Costar), were infected with the mixtures in duplicate. Cultures infected with the virus-sCD4 mixture received tissue culture medium containing 3.2 jig of sCD4 per ml. Reverse transcriptase (RT) activity was assayed three times a week (30). Virus titrations in the presence of sCD4 and MAbs to CD4. SIVagm and HIV-1 stocks were titrated in RPMI 1640 con-
MATERIALS AND METHODS Cell lines. The human T-cell lymphoma-derived cell line Molt4 clone 8 was obtained from M. Hayami (13), H9 cells *
Corresponding author. 6252
VOL. 64, 1990
CD4 ENHANCEMENT OF SIVagm INFECTION
6253
MAbs OKT4, OKT4a, and Leu3a, at a 1:10 dilution (5 ,ug of tissue culture medium per ml). The cells were seeded in 24-well plates (5 x 105 cells in 500 ,ul per well), and virus dilutions (with or without sCD4) were added. After 2 days, the cells were transferred to 50-ml tissue culture flasks and cultured for a further 30 days without addition of sCD4 or MAb, respectively. Endpoints of virus titrations were determined by measurement of RT activity in cell culture supernatants. RT assays. RT assays were performed following the procedure previously published (30). MAbs. MAbs OKT4a and OKT4 were purchased from Ortho Diagnostics Inc., Heidelberg, Federal Republic of Germany; Leu3a was obtained from Becton Dickinson Inc., Heidelberg, Federal Republic of Germany.
106
105 10 03
102
107 b
0
5
10
15
20
25
days post infection
FIG. 1. RT activity in supernatants of standardized Molt4 clone 8 cell cultures during the course of infection with HIV-1 (a) and SIVa,gm (b). O, Cells infected with untreated HIV-1 or SIVagm;E*, cells infected with HIV-1 or SIVagm preincubated with sCD4.
10[ -
tamning 10% FCS and antibiotics. Virus inocula (102 TCID50) were supplemented with equal volumes of sCD4 at a con10~~~~ centration of 8 ,ug/ml and incubated for 1 h at 37°C. Target cells were incubated for 1 h at 4°C with or without purified
RESULTS RT activity in cell culture supernatants of Molt4 clone 8 during the course of infection with HIV-1 and SIVgm in the presence of sCD4. During the characterization of novel SIVagm isolates (4, 15, 17), we observed that several human CD4-positive T-lymphoma lines could readily be infected. Some of these lines allowed SIVagm replication to high titers (15). Infection was inhibited by those MAbs which also prevented HIV-1 and HIV-2 infection, e.g., OKT4a and Leu3a. In an extension of these experiments (30), we also tried to block SIVagm infection by sCD4. In clear contrast to the situation confirmed in HIV-1 control experiments, sCD4 was observed to enhance SIVagm infection in vitro (Fig. 1). Infection of Molt4 clone 8 lymphoma cells with 102 TCID50 of HIV-1 or SIVagm per 0.5 ml pretreated (1 h at 37°C) with 1.6 ,ug of sCD4 per 0.5 ml resulted in inhibition of HIV-1 replication (Fig. la) but accelerated appearance of syncytium-forming infected cells and earlier detection of RT activity in SIVagm culture supernatants compared with cultures infected by SIVagm without preincubation with sCD4 (Fig. lb). Adding the virus strains and sCD4 concomitantly to the target cells led to both a reduced inhibition of HIV-1 replication and reduced enhancement of SIVagm replication (not shown), thus yielding intermediate results. RT activity of Molt4 clone 8 cell culture supernatants
800000s.1
700000-_
400000-
07
20000
0
0.1
0.2
0.4
0.8
1.6
3.2
6.4
sCD4 (jig/mnl) FIG. 2. RT activity of Molt4 clone 8 cell culture supernatants infected with 102 TCID50 of HIV-1 (-) or SIVagm (X), plotted as a function of sCD4 concentration. RT activity of HIV-1-infected cultures was assayed at day 16 after infection, and RT activity of SIVgm-infected cultures was assayed at day 9 after infection.
6254
J. VIROL.
WERNER ET AL.
106 a
b
c
d
a
b
c
d
a
b
c
d
A
103-7 1U
B
106
104
103 Molt4/8 H9 CEM-SS FIG. 3. Effects of sCD4 (m) and MAb OKT4a (m ) on HIV-1 (A) and SIVagm (B) replication in Molt4 clone 8, H9, and CEM-SS lymphoma cells. Addition of sCD4 to virus inocula blocked (HIV-1) or enhanced (SIVagm) virus growth (compare columns a and b). Preincubation of target cells with MAb OKT4a (or Leu3a; data not shown) abolished growth of both HIV-1 and SIVagm (compare columns a and c). Concomitant preincubation of virus inocula with sCD4 and of target cells with MAb OKT4a led to a differential result (columns a and d); although HIV infectivity was abolished, SIVagm titers remained as high as in the control cultures (column a). OKT4 MAb, unable to block SIVagm infection (30), had no effect on the enhanced infection of SIVagm-sCD4 complexes (virus growth as in column a).
infected with HIV-1 and SIVag,m as a function of sCD4 concentration. sCD4-mediated enhancement of SIVagm infectivity was dependent on the concentration of sCD4. As shown in Fig. 2, HIV-1 infection was reduced by 1.6 jig of sCD4 per ml added to both virus inoculum and culture medium and totally prevented at sCD4 concentrations at and above 3.2 ,g/ml. In contrast, infection by SIVagm became demonstrable at day 9 postinfection only if sCD4 was added at 0.4-,g/ml or higher concentrations. Effects of sCD4 and CD4-specific MAbs on HIV-1 and SIVagm replication in Molt4 clone 8, H9, and CEM-SS lymphoma cells. HIV-1 and SIVagm blocking or enhancement of infectivity by sCD4 was not target cell specific (Fig. 3, compare columns a and b). Preincubation of target cells with MAb OKT4a (or Leu3a; data not shown) abolished infectivity of both HIV-1 and SIVagm via the CD4 receptor (Fig. 3, column c), whereas MAb OKT4, which is known not to
interfere with HIV-1 and SIVagm (16) infectivity, had no blocking effect. Preincubation of virus inocula with sCD4 and target cells with MAb OKT4a or Leu3a again prevented HIV-1 infection as expected (Fig. 3, column d), whereas SIVagm infectivity was restored to approximately normal levels. Thus, addition of sCD4 to SIVagm enabled the virus to overcome the block in infectivity caused by the binding of MAbs OKT4a and Leu3a to the CD4 cell receptor. This phenomenon has now been observed with all four distinguishable SIVagm variants isolated in our laboratory. DISCUSSION The mechanism of the 10- to 100-fold enhancement of SIVagm infection by sCD4 is difficult to explain at present. The CD4-binding domain of SIVagm gp140 could be localized by amino acid sequence comparison with the binding domain
VOL. 64, 1990
CD4 ENHANCEMENT OF SIVagm INFECTION
in the gp120 outer envelope glycoprotein of HIV-1 (3). The HIV domain (in the conserved region C3 of gp120) was defined by MAbs capable of blocking the gpl20-CD4 interaction and by in vitro mutagenesis of gp120 (22, 31). gp120 mutagenesis and peptide studies suggest a discontinuous nature of the viral region binding to the Vl surface loop of CD4 (2). The affinity constant for the CD4-gp120 interaction is on the order of 10-9 M (18). This high-affinity binding may well be diminished in the heterologous interaction between the primate SIVagm gpl40 and human CD4. It is conceivable that inadequate SIVagm gp140 binding to human CD4 may lead to conformational changes of SIVagm gp140 and/or gp45 envelope proteins, possibly resulting in the formation of a second virus envelope receptor or in the expression of efficient fusion domains. A fusion domain has already been described for the HIV gp4l transmembrane protein (10), allowing the virus to infect cells in a pH-independent manner (20, 24). A comparable fusion domain has also been recognized in the SIVagm gp45 transmembrane protein (3). Allan and coworkers, who were able to block the SIVagm enhancement of sCD4 with nonneutralizing sera from infected African green monkeys, discussed that antibodies directed against the conserved regions including the fusion domain of the SIVagm transmembrane protein may be responsible for the observed abrogation of sCD4-mediated enhancement (1). Additional experiments have now been initiated to explore a possible host range extension of SIVagm associated with sCD4. Expression of an effective fusion domain should allow the infection of a wider range of cells of higher primates and humans in a pH-independent manner, whereas expression of a putative second viral receptor may extend the tropism in a pH-dependent manner to only a limited variety of (e.g., CD4-negative) target cells. The recent descriptions of pHdependent infections of CD4-negative nonlymphoma cell lines, which cannot be blocked by sCD4 or anti-CD4 MAbs, provide suggestive evidence for the existence of a second, CD4-independent pathway of HIV and SIV infections (11, 16, 19, 25, 30). If enhancement of SIVagm infectivity is due to improper binding to human sCD4, caution and extensive in vitro pretesting is required for molecularly modified sCD4 (27) designed to act as therapeutic agents in HIV-infected patients, because modification may result in inadequate sCD4HIV gp120 interaction and enhancement of HIV infectivity.
Molecularly cloned SIVagm3 is highly divergent from other SIVagm isolates and is biologically active in vitro and in vivo. J. Virol. 63:5119-5123. Clapham, P. R., J. N. Weber, D. Whitby, K. McIntosh, A. G. Dalgleish, P. J. Maddon, K. C. Deen, R. W. Sweet, and R. A. Weiss. 1989. Soluble CD4 blocks the infectivity of diverse strains of HIV and SIV for T-cells and monocytes but not for brain and muscle cells. Nature (London) 337:368-373. Dalgleish, A. G., P. C. L. Beverly, P. R. Clapham, D. H. Crowford, M. F. Greaves, and R. A. Weiss. 1984. The CD 4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature (London) 312:763-767. Deen, K. C., J. S. McDougal, R. Inacker, G. Folena-Wasserman, J. Arthos, J. Rosenberg, P. J. Maddon, R. Axel, and R. W. Sweet. 1988. A soluble form of CD4 (T4) protein inhibits AIDS virus infection. Nature (London) 331:82-84. Fisher, R. A., J. M. Bertonis, W. Meier, V. A. Johnson, D. S. Costopoulos, T. Liu, R. Tizard, B. D. Walker, M. S. Hirsch, R. T. Schooley, and R. A. Flavell. 1988. HIV infection is blocked in vitro by recombinant soluble CD4. Nature (London) 331:7678. Gay, D., P. Maddon, R. Sekaoy, M. A. Talle, M. Godfrey, E. Long, G. Goldstein, L. Chess, R. Axel, J. Kappler, and P. Marrack. 1987. Functional interaction between human T-cell protein CD4 and the major histocombatibility complex HLA-DR antigen. Nature (London) 328:626-629. Gonzalez-Scarano, F., M. N. Waxham, A. Ross, and J. A. Hoxie. 1987. Sequence similarities between human immunodeficiency virus gp4l and paramyxovirus fusion proteins. AIDS Res. 3:245-25. Harouse, J. M., C. Kunsch, H. T. Hartle, M. A. Laughlin, J. A. Hoxie, B. Wigdal, and F. Gonzalez-Scarano. 1989. CD4-independent infection of human neuronal cells by human immunodeficiency virus type 1. J. Virol. 63:2527-2533. Hussey, R. E., N. E. Richardson, M. Kowalski, N. R. Brown, H.-C. Chang, R. F. Siliciano, T. Dorfman, B. Walker, J. Sodroski, and E. L. Reinherz. 1988. A soluble CD4 protein selectively inhibits HIV replication and syncytium formation. Nature (London) 331:78-81. Kikukawa, R., Y. Koyanagi, S. Harada, N. Kobayashi, M. Hatanaka, and N. Yamamoto. 1986. Differential susceptibility to the acquired immunodeficiency syndrome retrovirus in cloned cells of human leukemic T-cell line Molt-4. J. Virol. 57:11591162. Klatzmann, D., E. Champagne, and S. Chamaret. 1984. T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV. Nature (London) 312:767-768. Kraus, G., A. Werner, M. Baier, D. Binninger, F. J. Ferdinand, S. Norley, and R. Kurth. 1989. Isolation of human immunodeficiency virus-related simian immunodeficiency viruses from African green monkeys. Proc. Natl. Acad. Sci. USA 86:28922896. Kunsch, C., H. T. Hartle, and B. Wigdahl. 1989. Infection of human fetal dorsal root ganglion glial cells with human immunodeficiency virus type 1 involves an entry mechanism independent of the CD4 T4a epitope. J. Virol. 63:5054-5061. Kurth, R., G. Kraus, A. Werner, S. Hartung, P. Centner, M. Baier, S. Norley, and J. Lower. 1988. Animal retrovirus models and vaccines. J. Acquired Immune Defic. Syndr. 1:284-294. Laski, L. A., G. Nakamura, D. H. Smith, C. Fennie, C. Shimasaki, E. Patzner, P. Berman, T. Gregory, and D. J. Capon. 1987. Delineation of a region of the human immunodeficiency virus type 1 gpl20 glycoprotein critical for interaction with the CD4 receptor. Cell 50:975-985. Li, X. L., T. Moudgill, H. V. Vinters, and D. D. Ho. 1990. CD4-independent, productive infection of a neuronal cell line by human immunodeficiency virus type 1. J. Virol. 64:1383-1387. McClure, M. O., M. Marsh, and R. A. Weiss. 1988. HIV-1 enters CD4 positive cells by a pH independent mechanism. EMBO J. 7:513-518. McClure, M. O., Q. J. Sattenau, P. C. L. Beverley, J. P. Hearn, A. K. Fitzgerald, A. J. Zuckerman, and R. A Weiss. 1987. HIV infection of primate lymphocytes and conservation of the CD4
ACKNOWLEDGMENTS We thank S. Norley for discussion and critical reading of the manuscript and B. Brandi for editorial assistance. This work was partly supported by a grant to R.K. from the German Ministry of Research and Technology. LITERATURE CITED 1. Allan, J. S., J. Strauss, and D. W. Buck. 1990. Enhancement of SIV infection with soluble receptor molecules. Science 247:
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17. 18.
1084-1088. 2. Arthos, J., K. C. Deen, M. A. Chaikin, J. A. Fornwald, G. Sathe, Q. J. Sattenau, P. R. Clapham, R. A. Weiss, J. S. McDougal, C. Pietropaolo, R. Axel, A. Truneh, P. J. Maddon, and R. W. Sweet. 1989. Identification of the residues in human CD4 critical for the binding of HIV. Cell 57:469-481. 3. Baier, M., C. Garber, C. Muller, K. Cichutek, and R. Kurth. 1990. Complete nucleotide sequence of a simian immunodeficiency virus from African green monkeys: a novel type of
intragroup divergence. Virology 176:216-221. 4. Baier, M., A. Werner, K. Cichutek, C. Garber, G. Kraus, F. J. Ferdinand, S. Hartung, T. S. Papas, and R. Kurth. 1989.
19. 20. 21.
6255
6256
WERNER ET AL.
receptor. Nature (London) 330:487-489. 22. McKeating, J. A., and R. L. Willey. 1989. Structure and function of the HIV envelope. AIDS 3:35-41. 23. Sattenau, Q. J., P. R. Clapham, R. A. Weiss, P. C. L. Beverley, L. Montagnier, M. F. Alhalabi, J.-C. Gluckmann, and D. Klatzmann. 1988. The human and simian immunodeficiency viruses HIV-1, HIV-2 and SIV interact with similar epitopes on their cellular receptor. AIDS 2:101-105. 24. Stein, B. S., S. D. Gowda, J. D. Lifson, R. C. Penhallon, K. G. Bensch, and E. G. Engelman. 1987. pH-independent HIV entry into CD4+ T cells via virus envelope fusion to the plasma membrane. Cell 49:659-668. 25. Tateno, M., F. Gonzalez-Scarano, and J. A. Levy. 1989. Human immunodeficiency virus can infect CD4-negative human fibroblastoid cells. Proc. Natl. Acad. Sci. USA 86:4287-4290. 26. Traunecker, A., W. Luke, and K. Karjalainen. 1988. Soluble CD4 molecules neutralize human immunodeficiency virus type
J. VIROL. 1. Nature (London) 331:84-86. 27. Traunecker, A., J. Schneider, H. Kiefer, and K. Karjalainen. 1989. Highly efficient neutralisation of HIV with recombinant CD4-immunoglobulin molecules. Nature (London) 339:68-70. 28. Watanabe, M., K. A. Reimann, P. A. DeLong, T. Liu, R. A. Fisher, and N. L. Letvin. 1989. Effect of recombinant soluble CD4 in rhesus monkeys infected with simian immunodeficiency virus of macaques. Nature (London) 337:267-270. 29. Weiss, R. A. 1988. Receptor molecule blocks HIV. Nature (London) 331:15. 30. Werner, A., G. Winskowsky, S. G. Norley, and R. Kurth. 1990. Productive infection of both CD4+ and CD4- human cell lines with HIV-1, HIV-2 and SIVagm. AIDS 4:537-544. 31. Willey, R. L., E. K. Ross, A. J. Buckler-White, T. S. Theodore, and M. A. Martin. 1989. Functional interaction of constant and variable domains of human immunodeficiency virus type 1 gpl20. J. Virol. 63:3595-3600.
Vol. 64, No. 12
0022-538X/90/126252-05$02.00/0 Copyright C 1990, American Society for Microbiology
Soluble CD4 Enhances Simian Immunodeficiency Virus SIVagm Infection ALBRECHT WERNER, GUDRUN WINSKOWSKY, AND REINHARD KURTH* Paul-Ehrlich-Institute, Paul-Ehrlich-Strasse 51-59, D-6070 Langen 1, Federal Republic of Germany Received 30 April 1990/Accepted 27 July 1990
The CD4 molecule is expressed on T-helper cells and serves as the cellular receptor for the human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and for the simian immunodeficiency viruses SIVmac and SIVagm. HIV-1, HIV-2, and SIVmac infectivity can be blocked by monoconal antibodies (MAbs) directed against the CD4 molecule and by soluble CD4 proteins (sCD4). In the present study, we demonstrated not only lack of inhibition, but 10- to 100-fold sCD4-dependent enhancement of SIVagm infectivity of human T-cell lymphoma lines, although SIVagm infection was blocked by MAbs OKT4a and Leu3a. SIVagm enhancement with sCD4 was suppressed by MAbs OKT4a and Leu3a to levels observed without addition of sCD4. The infectivity of all four tested SIVagm variants was enhanced by sCD4 on all tested lymphoma cell lines. These results suggest a second step (second or secondary receptor) required for enhancing virus entry into the cell and may have serious implications for approaches to the treatment of acquired immunodeficiency syndrome on the basis of modified sCD4 molecules.
The 55-kDa CD4 glycoprotein is expressed on T-helper/ inductor lymphocytes (6, 14) and is thought to be involved in the interaction with major histocompatibility complex class II-bearing cells (9). In humans and higher primates, CD4 and its structurally related simian analogs also serve as the cellular receptors for human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and for simian immunodeficiency viruses SIVmaC and SIVagm (6, 14, 15, 21, 23). The highaffinity interaction between HIV-1 and HIV-2 envelope protein gpl20 and cellular CD4 can be inhibited in vitro by both low concentrations of soluble CD4 (sCD4) and by a number of CD4-specific monoclonal antibodies (MAbs) (7, 8, 12, 26, 27, 29), resulting in complete blocking of virus infectivity. In preliminary experiments in vivo, human sCD4 has been shown to possess some therapeutic effect in the treatment of SIVmac-infected rhesus monkeys (28). Consequently, clinical trials have been initiated in the United States to treat HIV-infected individuals. Infectivity of SIVagm can be blocked by the same MAbs directed against the CD4 molecule as HIV-1 and HIV-2 can (6, 15). These findings are not surprising, considering that the putative CD4-binding sites of SIVagm and HIV are relatively conserved (17, 21). However, in contrast to the findings with HIV, blocking of SIVagm infectivity with sCD4 is not possible. On the contrary, sCD4 enhances SIVagm infectivity (1). In the present study we also report that sCD4 can block infectivity of HIV-1 but enhances infectivity of SIVagm. In addition, the enhancement of SIVagm infectivity with sCD4 can be suppressed by MAbs OKT4a and Leu3a, but not by OKT4, to levels measured without sCD4 and OKT4a. Binding of recombinant sCD4 to SIVagm may activate viral binding sites or fusion domains, resulting in enhanced cell infection.
were kindly donated by R. C. Gallo, National Institutes of Health, Bethesda, Md., and we received the CEM-SS cell line, a subclone of CEM, from P. Nara, National Cancer Institute, Frederick, Md. All cell lines were maintained in RPMI 1640 containing 10% heat-inactivated fetal calf serum (FCS) and antibiotics. Virus stocks and assays. Assays were carried out with the HTLV-IIIB strain of HIV-1 and with several SIVagm isolates from African green monkeys, as described previously (15). SIVagm and HIV-1 (HTLV-IIIB) virus stocks were prepared and titrated as described previously (30). Virus stocks were diluted in RPMI 1640 containing 10% FCS and antibiotics to a concentration of 2 x 102 50% tissue culture infective doses (TCID50) per ml. Then 0.5 ml of the stocks was supplemented with an equal volume of full-length sCD4 (3.2 ,ug/ml or with concentrations of sCD4 in the range of 0.1 to 6.4 jig/ml). sCD4 polypeptide containing extracellular amino acid residues 1 to 374 of the human CD4 cell receptor prepared from supernatant cultures of Spodoptera frugiperda host cells infected with recombinant Autographa californica nuclear polyhedrosis virus containing the corresponding gene sequence for human sCD4 was kindly provided by T. B. Helting, Pharmacia Genetic Engineering Inc., La Jolla, Calif. sCD4, either full length or containing the first 153 amino-terminal amino acids (Vl and part of the V2 domain), was also purified from correspondingly transfected Escherichia coli (by Pharmacia Genetic Engineering Inc.) and showed a comparable SIVagm infection-enhancing activity. Because the yield of sCD4 from E. coli was much lower than that from the supernatant of S. frugiperda host cells, the experiments illustrated here were performed with sCD4 derived from these cells. All virus-sCD4 mixtures were incubated for 1 h at 37°C. Molt4 clone 8 cells (5 x 105), cultured in 24-well plates (Costar), were infected with the mixtures in duplicate. Cultures infected with the virus-sCD4 mixture received tissue culture medium containing 3.2 jig of sCD4 per ml. Reverse transcriptase (RT) activity was assayed three times a week (30). Virus titrations in the presence of sCD4 and MAbs to CD4. SIVagm and HIV-1 stocks were titrated in RPMI 1640 con-
MATERIALS AND METHODS Cell lines. The human T-cell lymphoma-derived cell line Molt4 clone 8 was obtained from M. Hayami (13), H9 cells *
Corresponding author. 6252
VOL. 64, 1990
CD4 ENHANCEMENT OF SIVagm INFECTION
6253
MAbs OKT4, OKT4a, and Leu3a, at a 1:10 dilution (5 ,ug of tissue culture medium per ml). The cells were seeded in 24-well plates (5 x 105 cells in 500 ,ul per well), and virus dilutions (with or without sCD4) were added. After 2 days, the cells were transferred to 50-ml tissue culture flasks and cultured for a further 30 days without addition of sCD4 or MAb, respectively. Endpoints of virus titrations were determined by measurement of RT activity in cell culture supernatants. RT assays. RT assays were performed following the procedure previously published (30). MAbs. MAbs OKT4a and OKT4 were purchased from Ortho Diagnostics Inc., Heidelberg, Federal Republic of Germany; Leu3a was obtained from Becton Dickinson Inc., Heidelberg, Federal Republic of Germany.
106
105 10 03
102
107 b
0
5
10
15
20
25
days post infection
FIG. 1. RT activity in supernatants of standardized Molt4 clone 8 cell cultures during the course of infection with HIV-1 (a) and SIVa,gm (b). O, Cells infected with untreated HIV-1 or SIVagm;E*, cells infected with HIV-1 or SIVagm preincubated with sCD4.
10[ -
tamning 10% FCS and antibiotics. Virus inocula (102 TCID50) were supplemented with equal volumes of sCD4 at a con10~~~~ centration of 8 ,ug/ml and incubated for 1 h at 37°C. Target cells were incubated for 1 h at 4°C with or without purified
RESULTS RT activity in cell culture supernatants of Molt4 clone 8 during the course of infection with HIV-1 and SIVgm in the presence of sCD4. During the characterization of novel SIVagm isolates (4, 15, 17), we observed that several human CD4-positive T-lymphoma lines could readily be infected. Some of these lines allowed SIVagm replication to high titers (15). Infection was inhibited by those MAbs which also prevented HIV-1 and HIV-2 infection, e.g., OKT4a and Leu3a. In an extension of these experiments (30), we also tried to block SIVagm infection by sCD4. In clear contrast to the situation confirmed in HIV-1 control experiments, sCD4 was observed to enhance SIVagm infection in vitro (Fig. 1). Infection of Molt4 clone 8 lymphoma cells with 102 TCID50 of HIV-1 or SIVagm per 0.5 ml pretreated (1 h at 37°C) with 1.6 ,ug of sCD4 per 0.5 ml resulted in inhibition of HIV-1 replication (Fig. la) but accelerated appearance of syncytium-forming infected cells and earlier detection of RT activity in SIVagm culture supernatants compared with cultures infected by SIVagm without preincubation with sCD4 (Fig. lb). Adding the virus strains and sCD4 concomitantly to the target cells led to both a reduced inhibition of HIV-1 replication and reduced enhancement of SIVagm replication (not shown), thus yielding intermediate results. RT activity of Molt4 clone 8 cell culture supernatants
800000s.1
700000-_
400000-
07
20000
0
0.1
0.2
0.4
0.8
1.6
3.2
6.4
sCD4 (jig/mnl) FIG. 2. RT activity of Molt4 clone 8 cell culture supernatants infected with 102 TCID50 of HIV-1 (-) or SIVagm (X), plotted as a function of sCD4 concentration. RT activity of HIV-1-infected cultures was assayed at day 16 after infection, and RT activity of SIVgm-infected cultures was assayed at day 9 after infection.
6254
J. VIROL.
WERNER ET AL.
106 a
b
c
d
a
b
c
d
a
b
c
d
A
103-7 1U
B
106
104
103 Molt4/8 H9 CEM-SS FIG. 3. Effects of sCD4 (m) and MAb OKT4a (m ) on HIV-1 (A) and SIVagm (B) replication in Molt4 clone 8, H9, and CEM-SS lymphoma cells. Addition of sCD4 to virus inocula blocked (HIV-1) or enhanced (SIVagm) virus growth (compare columns a and b). Preincubation of target cells with MAb OKT4a (or Leu3a; data not shown) abolished growth of both HIV-1 and SIVagm (compare columns a and c). Concomitant preincubation of virus inocula with sCD4 and of target cells with MAb OKT4a led to a differential result (columns a and d); although HIV infectivity was abolished, SIVagm titers remained as high as in the control cultures (column a). OKT4 MAb, unable to block SIVagm infection (30), had no effect on the enhanced infection of SIVagm-sCD4 complexes (virus growth as in column a).
infected with HIV-1 and SIVag,m as a function of sCD4 concentration. sCD4-mediated enhancement of SIVagm infectivity was dependent on the concentration of sCD4. As shown in Fig. 2, HIV-1 infection was reduced by 1.6 jig of sCD4 per ml added to both virus inoculum and culture medium and totally prevented at sCD4 concentrations at and above 3.2 ,g/ml. In contrast, infection by SIVagm became demonstrable at day 9 postinfection only if sCD4 was added at 0.4-,g/ml or higher concentrations. Effects of sCD4 and CD4-specific MAbs on HIV-1 and SIVagm replication in Molt4 clone 8, H9, and CEM-SS lymphoma cells. HIV-1 and SIVagm blocking or enhancement of infectivity by sCD4 was not target cell specific (Fig. 3, compare columns a and b). Preincubation of target cells with MAb OKT4a (or Leu3a; data not shown) abolished infectivity of both HIV-1 and SIVagm via the CD4 receptor (Fig. 3, column c), whereas MAb OKT4, which is known not to
interfere with HIV-1 and SIVagm (16) infectivity, had no blocking effect. Preincubation of virus inocula with sCD4 and target cells with MAb OKT4a or Leu3a again prevented HIV-1 infection as expected (Fig. 3, column d), whereas SIVagm infectivity was restored to approximately normal levels. Thus, addition of sCD4 to SIVagm enabled the virus to overcome the block in infectivity caused by the binding of MAbs OKT4a and Leu3a to the CD4 cell receptor. This phenomenon has now been observed with all four distinguishable SIVagm variants isolated in our laboratory. DISCUSSION The mechanism of the 10- to 100-fold enhancement of SIVagm infection by sCD4 is difficult to explain at present. The CD4-binding domain of SIVagm gp140 could be localized by amino acid sequence comparison with the binding domain
VOL. 64, 1990
CD4 ENHANCEMENT OF SIVagm INFECTION
in the gp120 outer envelope glycoprotein of HIV-1 (3). The HIV domain (in the conserved region C3 of gp120) was defined by MAbs capable of blocking the gpl20-CD4 interaction and by in vitro mutagenesis of gp120 (22, 31). gp120 mutagenesis and peptide studies suggest a discontinuous nature of the viral region binding to the Vl surface loop of CD4 (2). The affinity constant for the CD4-gp120 interaction is on the order of 10-9 M (18). This high-affinity binding may well be diminished in the heterologous interaction between the primate SIVagm gpl40 and human CD4. It is conceivable that inadequate SIVagm gp140 binding to human CD4 may lead to conformational changes of SIVagm gp140 and/or gp45 envelope proteins, possibly resulting in the formation of a second virus envelope receptor or in the expression of efficient fusion domains. A fusion domain has already been described for the HIV gp4l transmembrane protein (10), allowing the virus to infect cells in a pH-independent manner (20, 24). A comparable fusion domain has also been recognized in the SIVagm gp45 transmembrane protein (3). Allan and coworkers, who were able to block the SIVagm enhancement of sCD4 with nonneutralizing sera from infected African green monkeys, discussed that antibodies directed against the conserved regions including the fusion domain of the SIVagm transmembrane protein may be responsible for the observed abrogation of sCD4-mediated enhancement (1). Additional experiments have now been initiated to explore a possible host range extension of SIVagm associated with sCD4. Expression of an effective fusion domain should allow the infection of a wider range of cells of higher primates and humans in a pH-independent manner, whereas expression of a putative second viral receptor may extend the tropism in a pH-dependent manner to only a limited variety of (e.g., CD4-negative) target cells. The recent descriptions of pHdependent infections of CD4-negative nonlymphoma cell lines, which cannot be blocked by sCD4 or anti-CD4 MAbs, provide suggestive evidence for the existence of a second, CD4-independent pathway of HIV and SIV infections (11, 16, 19, 25, 30). If enhancement of SIVagm infectivity is due to improper binding to human sCD4, caution and extensive in vitro pretesting is required for molecularly modified sCD4 (27) designed to act as therapeutic agents in HIV-infected patients, because modification may result in inadequate sCD4HIV gp120 interaction and enhancement of HIV infectivity.
Molecularly cloned SIVagm3 is highly divergent from other SIVagm isolates and is biologically active in vitro and in vivo. J. Virol. 63:5119-5123. Clapham, P. R., J. N. Weber, D. Whitby, K. McIntosh, A. G. Dalgleish, P. J. Maddon, K. C. Deen, R. W. Sweet, and R. A. Weiss. 1989. Soluble CD4 blocks the infectivity of diverse strains of HIV and SIV for T-cells and monocytes but not for brain and muscle cells. Nature (London) 337:368-373. Dalgleish, A. G., P. C. L. Beverly, P. R. Clapham, D. H. Crowford, M. F. Greaves, and R. A. Weiss. 1984. The CD 4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature (London) 312:763-767. Deen, K. C., J. S. McDougal, R. Inacker, G. Folena-Wasserman, J. Arthos, J. Rosenberg, P. J. Maddon, R. Axel, and R. W. Sweet. 1988. A soluble form of CD4 (T4) protein inhibits AIDS virus infection. Nature (London) 331:82-84. Fisher, R. A., J. M. Bertonis, W. Meier, V. A. Johnson, D. S. Costopoulos, T. Liu, R. Tizard, B. D. Walker, M. S. Hirsch, R. T. Schooley, and R. A. Flavell. 1988. HIV infection is blocked in vitro by recombinant soluble CD4. Nature (London) 331:7678. Gay, D., P. Maddon, R. Sekaoy, M. A. Talle, M. Godfrey, E. Long, G. Goldstein, L. Chess, R. Axel, J. Kappler, and P. Marrack. 1987. Functional interaction between human T-cell protein CD4 and the major histocombatibility complex HLA-DR antigen. Nature (London) 328:626-629. Gonzalez-Scarano, F., M. N. Waxham, A. Ross, and J. A. Hoxie. 1987. Sequence similarities between human immunodeficiency virus gp4l and paramyxovirus fusion proteins. AIDS Res. 3:245-25. Harouse, J. M., C. Kunsch, H. T. Hartle, M. A. Laughlin, J. A. Hoxie, B. Wigdal, and F. Gonzalez-Scarano. 1989. CD4-independent infection of human neuronal cells by human immunodeficiency virus type 1. J. Virol. 63:2527-2533. Hussey, R. E., N. E. Richardson, M. Kowalski, N. R. Brown, H.-C. Chang, R. F. Siliciano, T. Dorfman, B. Walker, J. Sodroski, and E. L. Reinherz. 1988. A soluble CD4 protein selectively inhibits HIV replication and syncytium formation. Nature (London) 331:78-81. Kikukawa, R., Y. Koyanagi, S. Harada, N. Kobayashi, M. Hatanaka, and N. Yamamoto. 1986. Differential susceptibility to the acquired immunodeficiency syndrome retrovirus in cloned cells of human leukemic T-cell line Molt-4. J. Virol. 57:11591162. Klatzmann, D., E. Champagne, and S. Chamaret. 1984. T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV. Nature (London) 312:767-768. Kraus, G., A. Werner, M. Baier, D. Binninger, F. J. Ferdinand, S. Norley, and R. Kurth. 1989. Isolation of human immunodeficiency virus-related simian immunodeficiency viruses from African green monkeys. Proc. Natl. Acad. Sci. USA 86:28922896. Kunsch, C., H. T. Hartle, and B. Wigdahl. 1989. Infection of human fetal dorsal root ganglion glial cells with human immunodeficiency virus type 1 involves an entry mechanism independent of the CD4 T4a epitope. J. Virol. 63:5054-5061. Kurth, R., G. Kraus, A. Werner, S. Hartung, P. Centner, M. Baier, S. Norley, and J. Lower. 1988. Animal retrovirus models and vaccines. J. Acquired Immune Defic. Syndr. 1:284-294. Laski, L. A., G. Nakamura, D. H. Smith, C. Fennie, C. Shimasaki, E. Patzner, P. Berman, T. Gregory, and D. J. Capon. 1987. Delineation of a region of the human immunodeficiency virus type 1 gpl20 glycoprotein critical for interaction with the CD4 receptor. Cell 50:975-985. Li, X. L., T. Moudgill, H. V. Vinters, and D. D. Ho. 1990. CD4-independent, productive infection of a neuronal cell line by human immunodeficiency virus type 1. J. Virol. 64:1383-1387. McClure, M. O., M. Marsh, and R. A. Weiss. 1988. HIV-1 enters CD4 positive cells by a pH independent mechanism. EMBO J. 7:513-518. McClure, M. O., Q. J. Sattenau, P. C. L. Beverley, J. P. Hearn, A. K. Fitzgerald, A. J. Zuckerman, and R. A Weiss. 1987. HIV infection of primate lymphocytes and conservation of the CD4
ACKNOWLEDGMENTS We thank S. Norley for discussion and critical reading of the manuscript and B. Brandi for editorial assistance. This work was partly supported by a grant to R.K. from the German Ministry of Research and Technology. LITERATURE CITED 1. Allan, J. S., J. Strauss, and D. W. Buck. 1990. Enhancement of SIV infection with soluble receptor molecules. Science 247:
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17. 18.
1084-1088. 2. Arthos, J., K. C. Deen, M. A. Chaikin, J. A. Fornwald, G. Sathe, Q. J. Sattenau, P. R. Clapham, R. A. Weiss, J. S. McDougal, C. Pietropaolo, R. Axel, A. Truneh, P. J. Maddon, and R. W. Sweet. 1989. Identification of the residues in human CD4 critical for the binding of HIV. Cell 57:469-481. 3. Baier, M., C. Garber, C. Muller, K. Cichutek, and R. Kurth. 1990. Complete nucleotide sequence of a simian immunodeficiency virus from African green monkeys: a novel type of
intragroup divergence. Virology 176:216-221. 4. Baier, M., A. Werner, K. Cichutek, C. Garber, G. Kraus, F. J. Ferdinand, S. Hartung, T. S. Papas, and R. Kurth. 1989.
19. 20. 21.
6255
6256
WERNER ET AL.
receptor. Nature (London) 330:487-489. 22. McKeating, J. A., and R. L. Willey. 1989. Structure and function of the HIV envelope. AIDS 3:35-41. 23. Sattenau, Q. J., P. R. Clapham, R. A. Weiss, P. C. L. Beverley, L. Montagnier, M. F. Alhalabi, J.-C. Gluckmann, and D. Klatzmann. 1988. The human and simian immunodeficiency viruses HIV-1, HIV-2 and SIV interact with similar epitopes on their cellular receptor. AIDS 2:101-105. 24. Stein, B. S., S. D. Gowda, J. D. Lifson, R. C. Penhallon, K. G. Bensch, and E. G. Engelman. 1987. pH-independent HIV entry into CD4+ T cells via virus envelope fusion to the plasma membrane. Cell 49:659-668. 25. Tateno, M., F. Gonzalez-Scarano, and J. A. Levy. 1989. Human immunodeficiency virus can infect CD4-negative human fibroblastoid cells. Proc. Natl. Acad. Sci. USA 86:4287-4290. 26. Traunecker, A., W. Luke, and K. Karjalainen. 1988. Soluble CD4 molecules neutralize human immunodeficiency virus type
J. VIROL. 1. Nature (London) 331:84-86. 27. Traunecker, A., J. Schneider, H. Kiefer, and K. Karjalainen. 1989. Highly efficient neutralisation of HIV with recombinant CD4-immunoglobulin molecules. Nature (London) 339:68-70. 28. Watanabe, M., K. A. Reimann, P. A. DeLong, T. Liu, R. A. Fisher, and N. L. Letvin. 1989. Effect of recombinant soluble CD4 in rhesus monkeys infected with simian immunodeficiency virus of macaques. Nature (London) 337:267-270. 29. Weiss, R. A. 1988. Receptor molecule blocks HIV. Nature (London) 331:15. 30. Werner, A., G. Winskowsky, S. G. Norley, and R. Kurth. 1990. Productive infection of both CD4+ and CD4- human cell lines with HIV-1, HIV-2 and SIVagm. AIDS 4:537-544. 31. Willey, R. L., E. K. Ross, A. J. Buckler-White, T. S. Theodore, and M. A. Martin. 1989. Functional interaction of constant and variable domains of human immunodeficiency virus type 1 gpl20. J. Virol. 63:3595-3600.
