10
Biochimica et Biophvswa Acta. 1096 (1991) 10 i3
Elscvim BBADIS 61007
Review
Molecular interactions between HIV and the T lymphocyte R.E. Phillips Institute of Molecular Medicine, University of Oxford ( U.K.)
(Received 21 June 1990)
Key words: HIV; T lymphocyte; Lymphocyte-virus interaction; Immune recognition; Receptor binding
Contents I.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10
II. Molecular basis of HIV binding to and fusion with the lymphocyte membrane . . . . . . . . . . . . . . . . A. CD4 binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. The CD4 binding site on gpl20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. HIV fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. T cell immunity to HIV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Cytoxic T lymphocytes (CTL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10 10 11
III. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12
I. Introduction The techniques of molecular biology have clarified several critical interactions between the h u m a n immunodeficiency virus (HIV) and the l y m p h o i d cell population. Firstly, the H I V receptor and some detail of how the virus enters lymphocytes have been defined. Secondly, the m e c h a n i s m b y which cytotoxic T lymphocytes recognise and kill target cells infected by H I V has been elucidated. Other studies have begun to show how the H I V virus persists in the i m m u n e system and how the virus might evade the i m m u n e response and so ultimately overwhelm the host. This review will concentrate on the receptor and i m m u n e recognition.
Abbreviations: HIV, human immunodeficiency virus; CTL, cytotoxic T lymphocytes; MHC, major histocompatibility complex; NP, nucleoprotein. Correspondence: R.E. Phillips, Institute of Molecular Medicine, John Radcliffe Hospital, U.K.
11 11 11
II. Molecular basis of H1V binding to and fusion with the lymphocyte surface membrane H-A. CD4 binding
In 1981, lymphocytes with the cell-surface marker C D 4 were f o u n d to be selectively depleted in patients with A I D S . [1]. W h e n H I V was isolated, in vitro studies showed that T cells beating the C D 4 marker were favoured targets for H I V [2]. Monoclonal antibodies which b o u n d C D 4 were added to lymphocyte cultures: fusion between HIV-infected and CD4-uninfected cells was inhibited and pseudotype vesicular stomatitis viruses carrying H I V envelope glycoprotein had much diminished infectivity [3]. Other studies showed that a n t i - C D 4 m o n o c l o n a l antibodies inhibited infection of peripheral blood lymphocytes [2] and the direct binding of H I V to CD4-positive cells [4]. Later work showed that, in infected cells, C D 4 molecules were complexed with the external glycoprotein of the H I V envelope (gpl20). C D 4 alone appeared to be sufficient for binding since insertion of C D 4 c D N A into C D 4 negative h u m a n cells conferred susceptibility to
0925-4439/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
11 HIV infection [5]. However, when mouse L-cells were made to express human CD4, HIV bound but did not infect these hybrid cells, implying that other molecules on the cell membrane or within the cytoplasm were required. Both CD4 and gpl20 can be produced in pure form by recombinant techniques. When tested in vitro, binding affinity between these molecules was high [6], so permitting a series of studies aimed at mapping the precise site on CD4 where gpl20 binds. Mutational analysis indicated that the V1 region of CD4 was critical for binding [9-11], but fine mapping in V1 has produced conflicting results [9,12]. Most authors agree that amino acids somewhere in the region spanned by positions 25 to 58 are involved. This part of the molecule contains a complementarity determining region, CDR2, which is thought to assume a loop structure [12]. This loop may bind clefts in the HIV envelope formed by conserved amino acid motifs, which are physically inaccessible to antibodies.
H-B. The CD4 binding site on gp120 The HIV envelope has a glycoprotein precursor (gp160) which is cleaved to form the outer virion envelope protein and the transmembrane glycoprotein gp41. Deletion of the carbox3~ terminus of gpl20 prevents binding to CD4 but mutations elsewhere in the molecule also reduce binding [13]. However, since the three-dimensional conformation of gpl20 is likely to be a critical determinant of CD4 binding, mutational analysis may not be the best way to define the precise details of the g p l 2 0 - C D 4 interaction. Single amino acid substitutions in gpl20 can alter receptor binding of HIV as well as alter tissue tropism
H-D. T cell immunity to H I V Work done over the last 15 years or so has revolutionised understanding of the way T cells interact with viruses and these advances have turned out to have had great relevance to HIV infection.
II-E. Cytotoxic T lymphocytes (CTL) In 1974 Blanden and colleagues identified lymphocytes which specifically recognised viral antigen [19]. Shortly afterwards, Zinkernagel and Doherty made the crucial observation that some T lymphocytes were capable of recognising lymphocytic choriomeningitis virus only when they expressed Class 1 molecules of the major histocompatibility complex (MHC) which were identical with the host's Class 1 antigens [20,21] (Fig. 1). Subsequently, this subpopulation of T lymphocytes (called cytotoxic because they lyse infected target cells) was found to bear a surface glycoprotein CD8 [22]. On the other hand lymphocytes bearing CD4 were found to express Class 11 M H C molecules. Class 1 MHC allelles had a critical influence on recognition of targets: minor
T-Cell
_~
.=ptor
[14].
H-C. H I V fusion Recent studies have defined some of the molecular prerequisites necessary for the fusion of HIV-infected ceils to form syncytia. This work is important, since the cytopathic effect of the virus appears to involve cell fusion. Surface expression of gp120/41 alone was sufficient to permit fusion of CD4 cells; productive viral infection or other viral components were not required. [15,16]. G p l 2 0 is highly glycosylated and removal of carbohydrate interfered with fusion [17], but this may have been because conformational changes altered CD4 binding. Viruses containing a mutation which blocked gpl60 cleavage could replicate but syncitia did not form [18], evidence consistent with findings in influenza and Sendai virus infections that cleavage of the spike protein precursor is a necessary step for cell membrane fusion.
cular }X
Antigen-presenting cell (APC)
"
Fig. 1. A model of the molecular components thought to be involved in the interaction between T lymphocytes and antigen-presenting cells. The antigen binds to a groove on the MHC molecule which consists of a complex of three a-chains and /32 microglobulin.The T cell receptor, which is a complex of a and /3 constant and variable regions, is thought to recognise residues of both the antigen and the MHC molecule. Subsidiary molecules are also necessary for this interaction.
12 differences in the Class 1 gene product could dramatically alter CTL recognition [23,24]. These principles influenced work on the T-cell response to influenza. Initially it was believed that CTL were directed mainly towards the surface-coat glycoproteins of influenza, since this material was always present on the surface of infected cells in large amounts and was thus the most likely target [25]. However, influenza virus CTL lysed infected target cells with comparable efficiency, although there was substantial variation in the surface glycoprotein [26,27]. This suggested that other, possibly more conserved, viral antigens might be the CTL target. This proved to be the case. When CTL were cloned the antigens recognised were internal proteins which were much less polymorphic than the surface haemagglutinin [28]. The specificity of one murine CTL clone for the viral nucleoprotein (NP) was confirmed by experiments in which the cDNA encoding this protein was transfected into mouse L cells [29]. These studies immediately raised the question of how viral components that are not transmembrane proteins and do not have recognisable amino-terminal leader sequences are transported to the plasma membrane of the target cell where CTL recognition is likely to occur. Full-length nucleoprotein was not essential, since L cells which expressed large fragments of NP were recognised [30]. Fragments which lacked sequences with any leader-like homology were recognised just as efficiently as full-length NP. Thus, a further prediction of these studies was that if short peptides suffice as CTL target epitopes then these peptides might arise under physiological circumstances and be transported to the cell surface by a novel mechanism. Over the last 5 years in an elegant series of experiments, Townsend and his colleagues have confirmed most of these predictions. [31-33]. Short synthetic peptides proved sufficient to act as CTL epitopes when added in vitro to cells expressing self M H C Class 1 molecules [31]. Later work has shown that peptides restored a defect in the association of /~2 microglobulin and murine Class 1 heavy chains, thus providing more evidence that peptides formed in vitro associate with Class 1 molecules at some undetermined intracellular site [32]. In human populations, dominant influenza epitopes were generally found to be specific for H L A Class 1 molecules [34]. The simplest explanation for these observations is that each HLA Class 1 allelle will only bind a particular epitope. Studies of mutant epitope peptides has shown that certain sequence alterations were critical [35] for CTL recognition, implying that some peptide sequence changes could either prevent binding by the Class 1 molecule or interfere with T cell receptor recognition. A few years ago several groups began to apply the lessons learnt from study of CTL responses to influenza viruses to HIV infection [36-38].
It is now clear that the majority of infected individuals do have circulating HIV-specific CTL specificities that can be mapped to several gene loci, including internal proteins not expressed on the cell surface. Although CTL directed against the surface coat protein env have been detected [37], other investigators have found CTL specific for the pol [36] and gag genes [38]. These responses were HLA Class 1 restricted and using synthetic peptides Nixon et al. were able to carry out fine mapping of an HLA-B27 restricted epitope. More recently epitopes restricted by other Class I molecules have been defined and in longitudinal studies, individuals of defined HLA status have been found to shift from one dominant epitope specificity to another over several months (Phillips, Nixon, Gotch and McMichael, personal communication). Others have shown that single amino acid substitutions altered CTL specificities for gp120 [39]. This could be one way that HIV evades immune recognition. IlL Conclusion
Molecular analysis has defined how HIV binds to the CD4 receptor and how cytotoxic T-cells recognise antigenic targets of the virus. When attempts are made to analyse these interactions in detail by mutating DNA sequences thought to be critical, ambiguities appear. This is because amino acid substitutions can exert effects in different ways: for example, directly at a binding site or at a distance by altering the conformation of a molecule. We need better ways of analysing the structure of peptide chain interactions. Although X-ray crystallography can provide excellent and precise information about molecular structure, promising new techniques including N M R should provide a more rapid means of analysing multiple peptide chain interactions. Whether this sort of structural analysis allows the synthesis of therapeutic reagents remains to be seen. References 1 Gottlieb, M.S., Schroff, R., Schanker, H.M., Weisman, J.D., Fan, P.T., Wolf, R.A. and Saxon, A. (1981) N. Engl. J. Med. 305, 1425-1431. 2 Klatzmann, D., Champagne, E., Chamaret, S., Gruest, J., Guetard, D., Hencend, T., Gluckman, J.C. and Montagnier, L. (1984) Science 225, 59-63. 3 Dalgleish, A.G., Beverley, P.C., Clapman, P.R., Crawford, D.H., Greaves, M.F. and Weiss, R.A. (1984) Nature 312, 763-767. 4 McDougal, J.S., Mawle, A., Cort, S.P., Nicholson, J.K., Cross, G.D., Scheppler-Campbell, J.A., Hicks, D. and Sligh, J. (1985) J. Immunol. 135, 3151-3161. 5 Maddon, P.J., Dalgleish, A.G., McDougal, J.S., Clapman, P.R., Weiss, R.A. and Axel, R. (1986) Cell 47, 333-348. 6 Lasky, L.A., Nakamura, G., Smith, D.H., Fennie, C., Shimasaki, C., Patzer, E., Berman, P., Gregory, T. and Capon, D.J. (1987) Cell 50, 975-985. 7 Ref. deleted. 8 Ref. deleted.
13 9 Peterson, A. and Seed, B. (1988) Cell 54, 65-72. 10 Landau, N.R., Warton, M. and Littman, D.R. (1988) Nature 334, 159-162. 11 Clayton, L.K., Hussey, R.E., Steinbrich, R., Ramachandran, H., Husain, Y. and Rinherz, E.L. (1988) Nature 335, 363-366. 12 Arthos, J., Deen, K.C., Chaikin, M.A., Fornwald, J.A., Sathe, G., Sattentau, Q.J., Clapham, P.R., Weiss, R.A., McDougall, J.S., Pietropaolo, C., Axel, R., Trunek, A., Maddon, P.J. and Sweet, R.W. (1989) Cell 57, 469-481. 13 Kowalski, M., Potz, J., Basiripour, L., Dorfman, T., Goh, W.H., Terwilliger, E., Drayton, A., Rosen, C., Haseltine, W. and Sodroski, J. (1987) Science 237, 1351-1355. 14 Cordonnier, A., Montagnier, L. and Emerman, M. (1989) Nature 340, 571-574. 15 Lifson, J.D., Feinberg, M.B., Reyes, G.R., Rabin, L., Banapour, B., Chakrabarti, S., Moss, B., Wang-Staal, F., Steimer, K.S. and Engleman, E.G. (1986) Nature 323, 725-728. 16 Sodroski, J., Gok, W.C., Rosen, C. Campbell, R. and Haseltine, W.A. (1986) Nature 322, 470-474. 17 Matthews, T.J., Weinhold, K.J., Lyerly, H.K., Langlois, A.J., Wigzell, H. and Bolognesi, D.P. (1987) Proc. Natl. Acad. Sci. USA 84, 5424-5428. 18 McCune, J.M., Rabin, L.B., Feinberg, M.B., Lieberman, M., Kosek, J.C., Reyes, G.R. and Weissman, I.L. (1988) Cell 53, 55-67. 19 Blanden, R.V. (1974) Transplant Rev. 19, 56-88. 20 Zinkernagel, R.M. and Doherty, P.C. (1974) Nature 248, 701-702. 21 Zinkernagel, R.M. and Doherty, P.C. (1974) Nature 251, 547-548. 22 Biddison, W.E., Sheaner, R.M. and Chang, T.W. (1981) J. Immunol. 127, 487-491. 23 Nathenson, S.G., Geliebter, J., Pfattenbach, G.M. and Zeff, R.A. (1986) Annu. Rev. Immunol. 4, 471-502. 24 Parham, P., Lomen, C.E., Lawler, D.A., Ways, J.P., Holmes, N., Coppin, H.L., Salter, R.D., Wan, A.M. and Ennis, P.D. (1988) Proc. Natl. Acad. Sci. USA 85, 4005-4009.
25 McMichael, A.V. and Askonas, B.A. (1987) Eur. J. Immunol. 8, 705-711. 26 Zweerink, H.T., Coumeidge, S.A., Skehel, J.J., Crumpton, M.J. and Askonas, B.A. (1977) Nature 267, 354-356. 27 McMichael, A.J., Ting, A., Zweerink, H.T. and Askonas, B.A. (1977) Nature 270, 524. 28 Townsend, A.R. and Skehel, J.J. (1982) Nature 300, 655. 29 Townsend, A.R., McMichael, A.J., Carter, N.P., Huddleston, J.A. and Browrdee, G.G. (1984) Cell 39, 13-25. 30 Townsend, A.R., Gotch, F.M. and Davey, J. (1985) Cell 42, 457. 31 Townsend, A.R., Rothbard, J., Gotch, F.M., Bahadur, G., Wraith, D. and McMichael, A.J. (1986) Cell 44, 959. 32 Townsend, A.R., Ohlen, C., Bastin, J., Ljunggren, H.-G., Foster, L. and Karre, K. (1989) Nature 340, 443-448. 33 Cerundolo, V., Alexander, J., Anderson, K., Lamb, C., Cresswell, P., McMichael, A., Gotch, F. and Townsend, A. (1990) Nature 345, 449-452. 34 McMichael, A.J., Gotch, F.M. and Rothbard, J. (1986) J. Exp. Med. 164, 1397-1406. 35 Gotch, F.M., McMichael, A.J. and Rothbard, J. (1988) J. Exp. Med. 168, 2045-2057. 36 Walker, B.D., Chakrabati, S., Moss, B., Paradis, T.J., Flyrm, T., Dumo, A.G., Blumberg, R.S., Kaplan, J.C., Hirsch, M.S. and Schooley, R.T. (1987) Nature 328, 345-348. 37 Plata, F., Autran, B., Martins, L.P., Wain-Hobson, S., Raphael, M., Mayaud, C., Dennis, M., Guillon, J.M. and Debre, P. (1987) Nature 328, 348-351. 38 Nixon, D.F., Townsend, A.R., Elfin, J.G., Rizza, C.R., GaUway, J. and McMichael, A.J. (1988) Nature 336, 484-487. 39 Takahashi, H., Merli, S., Putney, S.D., Houghten, R., Moss, B., Germain, R.N. and Berzofsky, J.A. (1989) Science 246, 118-121.
Biochimica et Biophvswa Acta. 1096 (1991) 10 i3
Elscvim BBADIS 61007
Review
Molecular interactions between HIV and the T lymphocyte R.E. Phillips Institute of Molecular Medicine, University of Oxford ( U.K.)
(Received 21 June 1990)
Key words: HIV; T lymphocyte; Lymphocyte-virus interaction; Immune recognition; Receptor binding
Contents I.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10
II. Molecular basis of HIV binding to and fusion with the lymphocyte membrane . . . . . . . . . . . . . . . . A. CD4 binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. The CD4 binding site on gpl20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. HIV fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. T cell immunity to HIV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Cytoxic T lymphocytes (CTL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10 10 11
III. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12
I. Introduction The techniques of molecular biology have clarified several critical interactions between the h u m a n immunodeficiency virus (HIV) and the l y m p h o i d cell population. Firstly, the H I V receptor and some detail of how the virus enters lymphocytes have been defined. Secondly, the m e c h a n i s m b y which cytotoxic T lymphocytes recognise and kill target cells infected by H I V has been elucidated. Other studies have begun to show how the H I V virus persists in the i m m u n e system and how the virus might evade the i m m u n e response and so ultimately overwhelm the host. This review will concentrate on the receptor and i m m u n e recognition.
Abbreviations: HIV, human immunodeficiency virus; CTL, cytotoxic T lymphocytes; MHC, major histocompatibility complex; NP, nucleoprotein. Correspondence: R.E. Phillips, Institute of Molecular Medicine, John Radcliffe Hospital, U.K.
11 11 11
II. Molecular basis of H1V binding to and fusion with the lymphocyte surface membrane H-A. CD4 binding
In 1981, lymphocytes with the cell-surface marker C D 4 were f o u n d to be selectively depleted in patients with A I D S . [1]. W h e n H I V was isolated, in vitro studies showed that T cells beating the C D 4 marker were favoured targets for H I V [2]. Monoclonal antibodies which b o u n d C D 4 were added to lymphocyte cultures: fusion between HIV-infected and CD4-uninfected cells was inhibited and pseudotype vesicular stomatitis viruses carrying H I V envelope glycoprotein had much diminished infectivity [3]. Other studies showed that a n t i - C D 4 m o n o c l o n a l antibodies inhibited infection of peripheral blood lymphocytes [2] and the direct binding of H I V to CD4-positive cells [4]. Later work showed that, in infected cells, C D 4 molecules were complexed with the external glycoprotein of the H I V envelope (gpl20). C D 4 alone appeared to be sufficient for binding since insertion of C D 4 c D N A into C D 4 negative h u m a n cells conferred susceptibility to
0925-4439/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
11 HIV infection [5]. However, when mouse L-cells were made to express human CD4, HIV bound but did not infect these hybrid cells, implying that other molecules on the cell membrane or within the cytoplasm were required. Both CD4 and gpl20 can be produced in pure form by recombinant techniques. When tested in vitro, binding affinity between these molecules was high [6], so permitting a series of studies aimed at mapping the precise site on CD4 where gpl20 binds. Mutational analysis indicated that the V1 region of CD4 was critical for binding [9-11], but fine mapping in V1 has produced conflicting results [9,12]. Most authors agree that amino acids somewhere in the region spanned by positions 25 to 58 are involved. This part of the molecule contains a complementarity determining region, CDR2, which is thought to assume a loop structure [12]. This loop may bind clefts in the HIV envelope formed by conserved amino acid motifs, which are physically inaccessible to antibodies.
H-B. The CD4 binding site on gp120 The HIV envelope has a glycoprotein precursor (gp160) which is cleaved to form the outer virion envelope protein and the transmembrane glycoprotein gp41. Deletion of the carbox3~ terminus of gpl20 prevents binding to CD4 but mutations elsewhere in the molecule also reduce binding [13]. However, since the three-dimensional conformation of gpl20 is likely to be a critical determinant of CD4 binding, mutational analysis may not be the best way to define the precise details of the g p l 2 0 - C D 4 interaction. Single amino acid substitutions in gpl20 can alter receptor binding of HIV as well as alter tissue tropism
H-D. T cell immunity to H I V Work done over the last 15 years or so has revolutionised understanding of the way T cells interact with viruses and these advances have turned out to have had great relevance to HIV infection.
II-E. Cytotoxic T lymphocytes (CTL) In 1974 Blanden and colleagues identified lymphocytes which specifically recognised viral antigen [19]. Shortly afterwards, Zinkernagel and Doherty made the crucial observation that some T lymphocytes were capable of recognising lymphocytic choriomeningitis virus only when they expressed Class 1 molecules of the major histocompatibility complex (MHC) which were identical with the host's Class 1 antigens [20,21] (Fig. 1). Subsequently, this subpopulation of T lymphocytes (called cytotoxic because they lyse infected target cells) was found to bear a surface glycoprotein CD8 [22]. On the other hand lymphocytes bearing CD4 were found to express Class 11 M H C molecules. Class 1 MHC allelles had a critical influence on recognition of targets: minor
T-Cell
_~
.=ptor
[14].
H-C. H I V fusion Recent studies have defined some of the molecular prerequisites necessary for the fusion of HIV-infected ceils to form syncytia. This work is important, since the cytopathic effect of the virus appears to involve cell fusion. Surface expression of gp120/41 alone was sufficient to permit fusion of CD4 cells; productive viral infection or other viral components were not required. [15,16]. G p l 2 0 is highly glycosylated and removal of carbohydrate interfered with fusion [17], but this may have been because conformational changes altered CD4 binding. Viruses containing a mutation which blocked gpl60 cleavage could replicate but syncitia did not form [18], evidence consistent with findings in influenza and Sendai virus infections that cleavage of the spike protein precursor is a necessary step for cell membrane fusion.
cular }X
Antigen-presenting cell (APC)
"
Fig. 1. A model of the molecular components thought to be involved in the interaction between T lymphocytes and antigen-presenting cells. The antigen binds to a groove on the MHC molecule which consists of a complex of three a-chains and /32 microglobulin.The T cell receptor, which is a complex of a and /3 constant and variable regions, is thought to recognise residues of both the antigen and the MHC molecule. Subsidiary molecules are also necessary for this interaction.
12 differences in the Class 1 gene product could dramatically alter CTL recognition [23,24]. These principles influenced work on the T-cell response to influenza. Initially it was believed that CTL were directed mainly towards the surface-coat glycoproteins of influenza, since this material was always present on the surface of infected cells in large amounts and was thus the most likely target [25]. However, influenza virus CTL lysed infected target cells with comparable efficiency, although there was substantial variation in the surface glycoprotein [26,27]. This suggested that other, possibly more conserved, viral antigens might be the CTL target. This proved to be the case. When CTL were cloned the antigens recognised were internal proteins which were much less polymorphic than the surface haemagglutinin [28]. The specificity of one murine CTL clone for the viral nucleoprotein (NP) was confirmed by experiments in which the cDNA encoding this protein was transfected into mouse L cells [29]. These studies immediately raised the question of how viral components that are not transmembrane proteins and do not have recognisable amino-terminal leader sequences are transported to the plasma membrane of the target cell where CTL recognition is likely to occur. Full-length nucleoprotein was not essential, since L cells which expressed large fragments of NP were recognised [30]. Fragments which lacked sequences with any leader-like homology were recognised just as efficiently as full-length NP. Thus, a further prediction of these studies was that if short peptides suffice as CTL target epitopes then these peptides might arise under physiological circumstances and be transported to the cell surface by a novel mechanism. Over the last 5 years in an elegant series of experiments, Townsend and his colleagues have confirmed most of these predictions. [31-33]. Short synthetic peptides proved sufficient to act as CTL epitopes when added in vitro to cells expressing self M H C Class 1 molecules [31]. Later work has shown that peptides restored a defect in the association of /~2 microglobulin and murine Class 1 heavy chains, thus providing more evidence that peptides formed in vitro associate with Class 1 molecules at some undetermined intracellular site [32]. In human populations, dominant influenza epitopes were generally found to be specific for H L A Class 1 molecules [34]. The simplest explanation for these observations is that each HLA Class 1 allelle will only bind a particular epitope. Studies of mutant epitope peptides has shown that certain sequence alterations were critical [35] for CTL recognition, implying that some peptide sequence changes could either prevent binding by the Class 1 molecule or interfere with T cell receptor recognition. A few years ago several groups began to apply the lessons learnt from study of CTL responses to influenza viruses to HIV infection [36-38].
It is now clear that the majority of infected individuals do have circulating HIV-specific CTL specificities that can be mapped to several gene loci, including internal proteins not expressed on the cell surface. Although CTL directed against the surface coat protein env have been detected [37], other investigators have found CTL specific for the pol [36] and gag genes [38]. These responses were HLA Class 1 restricted and using synthetic peptides Nixon et al. were able to carry out fine mapping of an HLA-B27 restricted epitope. More recently epitopes restricted by other Class I molecules have been defined and in longitudinal studies, individuals of defined HLA status have been found to shift from one dominant epitope specificity to another over several months (Phillips, Nixon, Gotch and McMichael, personal communication). Others have shown that single amino acid substitutions altered CTL specificities for gp120 [39]. This could be one way that HIV evades immune recognition. IlL Conclusion
Molecular analysis has defined how HIV binds to the CD4 receptor and how cytotoxic T-cells recognise antigenic targets of the virus. When attempts are made to analyse these interactions in detail by mutating DNA sequences thought to be critical, ambiguities appear. This is because amino acid substitutions can exert effects in different ways: for example, directly at a binding site or at a distance by altering the conformation of a molecule. We need better ways of analysing the structure of peptide chain interactions. Although X-ray crystallography can provide excellent and precise information about molecular structure, promising new techniques including N M R should provide a more rapid means of analysing multiple peptide chain interactions. Whether this sort of structural analysis allows the synthesis of therapeutic reagents remains to be seen. References 1 Gottlieb, M.S., Schroff, R., Schanker, H.M., Weisman, J.D., Fan, P.T., Wolf, R.A. and Saxon, A. (1981) N. Engl. J. Med. 305, 1425-1431. 2 Klatzmann, D., Champagne, E., Chamaret, S., Gruest, J., Guetard, D., Hencend, T., Gluckman, J.C. and Montagnier, L. (1984) Science 225, 59-63. 3 Dalgleish, A.G., Beverley, P.C., Clapman, P.R., Crawford, D.H., Greaves, M.F. and Weiss, R.A. (1984) Nature 312, 763-767. 4 McDougal, J.S., Mawle, A., Cort, S.P., Nicholson, J.K., Cross, G.D., Scheppler-Campbell, J.A., Hicks, D. and Sligh, J. (1985) J. Immunol. 135, 3151-3161. 5 Maddon, P.J., Dalgleish, A.G., McDougal, J.S., Clapman, P.R., Weiss, R.A. and Axel, R. (1986) Cell 47, 333-348. 6 Lasky, L.A., Nakamura, G., Smith, D.H., Fennie, C., Shimasaki, C., Patzer, E., Berman, P., Gregory, T. and Capon, D.J. (1987) Cell 50, 975-985. 7 Ref. deleted. 8 Ref. deleted.
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