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Structural constraints of HIV-1 Nef may curtail escape from HLA-B7-restricted CTL recognition
ELSEVIER
Immunology
Letters
55 (1997)
Rapid
Structural constraints of HIVHLA-B7-restricted Monika
Bauer ‘, Maria Klaus Eichmann
.’ Ahteilung
Virologie.
Institut
$2
Medizinische
d’ hnmunologie
Cellulaire ’ GBF,
Mikrohiologir ftir
HIV-I
Nef;
et Tissulaire.
Departtnent
CTL; SH3 domain;
URA
of Genetics.
author.
Tel.:
Nef may curtail escape from CTL recognition
CNRS
Unicersitiir
79 108 Freiburg, 625. Htjpital
38 I.?4 Brmmschweig.
Freihurg,
79 104 Freiburg.
‘,
Grrmutt~
Gertnatq
Pitir-SalpCrriPre.
75013
Prtri.r. Frcmw
Germane
1996
HLA-B7
+ 49 761 2036614:
0165-2478/97, $17.00 c‘ 1997 Elsevier Science PII S0165-2478(96)02679-X
und Hj,girne,
I I November
The Nef proteins of human and simian immunodeficiency viruses (HIV and SIV) are expressed early in the virus life cycle and are essential for efficient virus replication in primary cells in vitro and in vivo [1,2]. As a consequence, viruses lacking a functional nef gene have an attenuated phenotype [3-51. The mechanism of Nef function is not entirely defined. Nef expression has been shown to down regulate surface proteins, e.g. CD4 and by interacting with cellular signal-transducing proteins such as the src family tyrosine kinases and the serine/threonine mitogen-activated protein kinase (MAPK), may change the activation state of Tlymphocytes [6-81. From the recently determined crystal structure of an HIV-l Nef/Fyn(R961) complex, the amino acids engaged in folding and protein-protein interaction were determined and span Arg-71 to Val148 and Arg-178 to Phe-203 of Nef [9]. Besides being of important regulatory function, Nef is a potent inducer of antiviral cellular immune responses. In about 40-60% of HIV seropositives, Nefspecific HLA-restricted CD8-positive cytotoxic T-lymphocytes (CTL) have been demonstrated [ 10,111. HIV-specific CTL correlate with the reduction of primary viremia and are believed to control the level of * Corresponding 2036639.
I
Immunbiologir.
Accepted
Kqwwcls:
note
Lucchiari-Hartz b, Reinhard Maier ‘, Gaby Haas ‘, Brigitte Autran b, Ronald Frank d, Bernhard Maier b, Andreas Meyerhans ‘I.* b Max-Planek-lnslitut
’ Lohorctroire
119- 122
fax:
B.V. All rights
+ 49 761
reserved
virus replication in the asymptomatic phase of the infection [12-161. Consequently, Nef is an interesting antigen for HIV vaccination trials. This is even more so as the Nef-specific CTL precursor frequencies in healthy seronegative blood donors are unexpectedly high [17]. To identify Nef-specific CTL epitopes within the structured SH3 binding domain, the HLA-B7 binding and CTL recognition of several peptides were investigated. HLA-B7 is a common haplotype in the caucasian population. It uses proline as a dominant anchor amino acid at position 2 and a hydrophobic anchor at the C-terminus of CTL epitopes [18,19]. While proline at position 2 is essential for efficient binding, the C-terminal anchor amino acid can be leucine, valine, isoleucine, phenylalanine and methionine (data not shown). The prolines in the SH3 binding domain of HIV-l Nef are highly conserved, even between different clades. and contribute either to the tertiary protein structure or to a direct interaction with associated cellular proteins [7,8,20]. The HIV-l Nef protein contains three fragments (amino acids 68-76, 77-85, 1288137) consistent with the known peptide motifs for HLA-B7 (Fig. 1). The corresponding peptides were synthesized with an AMS 422 automated peptide synthesizer (Abimed AnalysenTechnik GmBH, Langenfeld, Germany) as previously
M. Baurr et al. : Imnw~olog~~ Letters 55 (1997) I19- I.?.?
120
N40P?:
m
MAPK E
d
10
20
high affinity binding to SH3 domain
PPT
30
150
160
170
180
190
200
Fig. 1. HIV-I Nef protein: schematic structure and location of three HLA-B7 restricted CTL epitopes. The number of the amino acid positions in HIV-I Nef is shown. Secondary structural elements of the Nef,,,, are indicated by boxes (p-strands. z-helices, polyproline type II helix (PPII)). Residues involved in the direct interaction with SH3 of Fyn[R961] are labeled with asterisks, residues involved in the tertiary interaction between the Nef,.,,, and SH3 of Fyn[R961] are labeled with dots. Residues essential for maximal binding of Nef to Lck are indicated with squares. The location of the myristylation signal (m). variable duplication (d). putative phosphorylation site of protein kinase C (PKC). binding site of serineithreonine mitogen-activated protein kinase (MAPK), predicted beta-turn and the polypurine tract are indicated by lines. Amino acid sequences of major intrapatient variants for the three HLA-B7-restricted CTL epitopes are given [23]. The amino acid one letter code is: A, Ala: C, Cys; D, Asp; E, Glu; F, Phe; G. Gly; H, His; I, Be; K, Lys; L. Leu; M, Met; N, Asn; P. Pro; Q, Gin; R. Arg; S. Ser; T. Thr; V. Val; W, Trp; and Y. Tyr.
described [21]. Their binding capacity to HLA-B7 was measured by HLA-B7 stabilization on the transportdeficient hybrid cell line T2/B7 (provided by P. Cresswell). Briefly, cells were incubated with 50, 75 or 100 ,uM of peptide in serum free medium for 15 h at 37°C. Staining for indirect immunofluorescence was made with ME1 (anti-HLA-B27, -B7, -Bw22) as first antibody and FITC-labeled anti-mouse IgG (Sigma) as a second antibody. Fluorescence intensities were measured on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). The fluorescence ratio (FR) is defined as mean fluorescence experimental sample to mean fluorescence background. Fluorescence values are the mean of triplicate determinations and vary less than 10% for each peptide. HLA-B7-restricted cellular cytotoxicity against the various peptides was determined in a standard chromium release assay with peptide-pulsed HLA-B7-matched Epstein-Barr virus transformed Bcells (EBV B-cells) as target cells and peptide-pulsed mismatched targets as control. Effector cells were derived from peripheral blood mononuclear cells (PBMC) of four HIV-infected individuals with the HLA-B7 haplotype and stimulated with irradiated autologous PHA-activated PBMC for lo- 15 days. Specific lysis for a peptide concentration of 10 ,LLM at different effector to target cell ratios is shown in Fig. 2B. All Nef peptides tested bound HLA-B7 in a comparable manner to a known HLA-B7-restricted CTL epitope of EBV (Fig. 2A) [22]. Substitution of the C-terminal anchor amino acids (Nef85L, Nef851 or Nef85V) as frequently observed in Nef variants of HIV seropositives did not alter binding efficiency. This is in agreement with the previously observed flexibility of the HLA-B7 F-pocket to accommodate different hydro-
phobic side chains [19]. Except for proline at position 2, the level of binding was relatively insensitive towards amino acid substitutions and even length variation (nine or ten amino acids). The respective prolines in Nef are P69, P78 and P129 and are well conserved in HIV-l. They are involved either in direct contact with cellular proteins, in the formation of the polyproline type II x-helix, the structural element common to all. SH3 binding domains, or the arrangement of the antiparallel /J’ strands and helices, respectively. Thus the structural requirements for Nef folding seem to prevent sequence variation that would result in loss of HLA-B7 binding of the corresponding peptides. All the Nef peptides that bound to HLA-B7 represent HLA-B7-restricted CTL epitopes (Fig. 2B). CD8positive effector cells from four different HIV infected individuals have been tested and at least one of the epitopes was recognized after polyclonal stimulation of the PBMC with PHA. Thus the respective peptides not only bound HLA-B7 in vitro but seem to be correctly processed and presented in vivo. Despite the relative conservation of the epitopes Nef 68876, 77-85 and 1288137 in HIV-l patients, a number of mutations has been demonstrated [23]. However, as the corresponding variant peptides sensitized target cells for lysis and the CTL response in HIV-infected patients is able to adapt to antigenic variation [l I], Nef gene variation in vivo seems not to intervene with recognition of the whole protein. In summary, three HLA-B7-restricted CTL epitopes in HIV-l Nef have been identified. They carry the known peptide motif, bind efficiently to HLA-B7 and are specifically recognized by CTL from HIV-l infected individuals. Due to the structural properties of proline in general and the importance of the three prolines in
122
M. Bauer et al. /hnmunolog_v Letters 55 (1997) 119-122
Nef protein folding, the possibility of escape from HLA-B7-restricted CTL recognition seems curtailed.
Acknowledgements This work was supported by grants of the ‘Deutsche Forschungsgemeinschaft’ (M.B., R.M., A.M.), the French Agence Nationale de Recherches sur le SIDA (G.H., B.A.) and by Biotechnology program EC Biomed (M.L., K.E.). We thank Otto Haller for continuous support and Hanspeter Pircher and Simon WainHobson for critical comments on the manuscript.
References Ill Cullen. B.R. (1994) Virology 205, I. VI Ratner, L.. and Niederman, T.M.J. (1995) Curr. Top. Microbial. Immunol.
193, 169. H.W., Ringler, D.J., Mori, K., Panicali, D.L., Sehgal. P.K., Daniel, M.D. and Desrosiers, R.C. (1991) Cell 65, 651. F., Greenough, T.C.. Brettler, D.B.. Sullivan, J.L., 141 Kirchhoff, and Desrosiers, R.C. (1995) New Engl. J. Med. 332, 228. PI Deacon, N.J., Tsykin, A., Solomon, A., Smith, K., LudfordMenting, M., Hooker, D.J., McPhee, D.A., Greenwdy, A.L., Ellett. A., Chatfield, C., Lawson, V.A., Crowe. S.. Maerz, A.. Sonza. S., Learmont, J., Sullivan, J.S., Cunningham, A.. Dwyer, D., Dowton, D. and Mills. J. (1995) Science 270, 988. PI Trono. D. (1995) Cell 82, 189. 171 Saksela, K., Cheng. G. and Baltimore, D. (1995) EMBO J. 14. 484. A., Azad. A., Mils, J. and McPhee. D. (1996) J. PI Greenway, Viral. 70, 6701. [91 Lee. C.H., Saksela, K., Mirza, U.A., Chait. B.T. and Kuriyan. J. (1996) Cell 85, 931.
[31 Kestler,
[lo] Lamhamedi-Cherradi, S., Culmann-Penciolelh, B., Guy, B., Kieny, M.P., Dreyfus, F., Saimot, A.G.. Sereni, D.. Sicard, D.. Levy. J.P. and Gomard. E. (1992) AIDS 6, 1249. [I l] Haas. G., Plikat, U., Debre. P., Lucchiari, M.. Katlama, C.. Dudoit. Y.. Bonduelle, O., Bauer, M., Ihlenfeldt, H-G., Jung, G.. Maier, B., Meyerhans, A. and Autrdn, B. (1996) J. Immunol. (in press). [12] Safrit, J.T.. Andrews, C.A., Zhu, T., Ho, D.D. and Koup. R.A. (1994) J. Exp. Med. 179, 463. [13] Koup, R.A., Safrit, J.T., Cao, Y., Andrews. C.A.. McLeod, G.. Borkowsky, W.F. and Ho, D.D. (1994) J. Viral. 68, 4650. [14] Borrow, P., Lewicki, H., Hahn, B.H., Shaw. G.M. and Oldstone, M.B. (1994) J. Viral. 68, 6103. [I51 Rinaldo, C., Huang, X.L., Fan, Z.F., Ding, M., Beltz, L., Logar, A., Panicali, D.M.. Liebmann, J., Cottrill, M. and Gupta, P. (1995) J. Viral. 69, 5838. [16] Pantaleo, G., Menzo, S., Vaccarezza, M., Graziosi. C.. Cohen, O.J., Demarest. J.F., Montefiori, D., Orenstein, J.M., Fox. C.. Schrager, L.K., Margolick, J.B., Buchbinder, S., Giorgi. J.V. and Fauci, A.S. (1995) New Engl. J. Med. 332, 209. [I71 Lucchiari, M., Niedermann, G.. Leipner, C.. Meyerhans. A., Eichmann. K. and Maier, B. (1994) Int. Immunol. 6. 1739. [I81 Maier. R.. Falk, K.. Roetzschke. O., Maier, B.. Gnau, V., Stefanovic, S., Jung, G., Rammensee, H-G. and Meyerhans, A. ( 1994) Immunogenetics 40, 306. [I91 Huczko. E.L., Bodnar. W.M.. Benjamin, D., Sakaguchi, K.. Zhu, N.Z.. Shabanowitz, J.. Henderson, R.A., Appella, E.. Hunt, D.F. and Engelhard, V.H. (1993) J. Immunol. 151, 2572. [20] Myers. G., Korber, B., Hahn. B.H., Jeang, K.T.. Mellors, J.W., McCutchen. F.E., Henderson, L.E. and Pavlakis, G.N. (1995) Human Retroviruses and AIDS. Los Alamos National Laboratory, Los Alamos, New Mexico. [21] Nietfeld. W.. Bauer. M.. Fevrier. M., Maier, R., Holzwarth, B., Frank, R., Maier, B., Riviere,Y. and Meyerhans. A. (1995) J. Immunol. 154, 2188. [22] Hill, A., Worth, A.. Elliot, T., Rowland-Jones, S., Brooks, J.. Rickinson, A. and McMichael, A. (1995) Eur. J. Immunol. 25. 18. [23] Shugars, D.C.. Smith, M.S., Glueck, D.H.. Nantermet, P.V., Seillier-Moiseiwitsch, F. and Swanstrom, R. (1993) J. Viral. 67, 4639.
Immunology
Letters
55 (1997)
Rapid
Structural constraints of HIVHLA-B7-restricted Monika
Bauer ‘, Maria Klaus Eichmann
.’ Ahteilung
Virologie.
Institut
$2
Medizinische
d’ hnmunologie
Cellulaire ’ GBF,
Mikrohiologir ftir
HIV-I
Nef;
et Tissulaire.
Departtnent
CTL; SH3 domain;
URA
of Genetics.
author.
Tel.:
Nef may curtail escape from CTL recognition
CNRS
Unicersitiir
79 108 Freiburg, 625. Htjpital
38 I.?4 Brmmschweig.
Freihurg,
79 104 Freiburg.
‘,
Grrmutt~
Gertnatq
Pitir-SalpCrriPre.
75013
Prtri.r. Frcmw
Germane
1996
HLA-B7
+ 49 761 2036614:
0165-2478/97, $17.00 c‘ 1997 Elsevier Science PII S0165-2478(96)02679-X
und Hj,girne,
I I November
The Nef proteins of human and simian immunodeficiency viruses (HIV and SIV) are expressed early in the virus life cycle and are essential for efficient virus replication in primary cells in vitro and in vivo [1,2]. As a consequence, viruses lacking a functional nef gene have an attenuated phenotype [3-51. The mechanism of Nef function is not entirely defined. Nef expression has been shown to down regulate surface proteins, e.g. CD4 and by interacting with cellular signal-transducing proteins such as the src family tyrosine kinases and the serine/threonine mitogen-activated protein kinase (MAPK), may change the activation state of Tlymphocytes [6-81. From the recently determined crystal structure of an HIV-l Nef/Fyn(R961) complex, the amino acids engaged in folding and protein-protein interaction were determined and span Arg-71 to Val148 and Arg-178 to Phe-203 of Nef [9]. Besides being of important regulatory function, Nef is a potent inducer of antiviral cellular immune responses. In about 40-60% of HIV seropositives, Nefspecific HLA-restricted CD8-positive cytotoxic T-lymphocytes (CTL) have been demonstrated [ 10,111. HIV-specific CTL correlate with the reduction of primary viremia and are believed to control the level of * Corresponding 2036639.
I
Immunbiologir.
Accepted
Kqwwcls:
note
Lucchiari-Hartz b, Reinhard Maier ‘, Gaby Haas ‘, Brigitte Autran b, Ronald Frank d, Bernhard Maier b, Andreas Meyerhans ‘I.* b Max-Planek-lnslitut
’ Lohorctroire
119- 122
fax:
B.V. All rights
+ 49 761
reserved
virus replication in the asymptomatic phase of the infection [12-161. Consequently, Nef is an interesting antigen for HIV vaccination trials. This is even more so as the Nef-specific CTL precursor frequencies in healthy seronegative blood donors are unexpectedly high [17]. To identify Nef-specific CTL epitopes within the structured SH3 binding domain, the HLA-B7 binding and CTL recognition of several peptides were investigated. HLA-B7 is a common haplotype in the caucasian population. It uses proline as a dominant anchor amino acid at position 2 and a hydrophobic anchor at the C-terminus of CTL epitopes [18,19]. While proline at position 2 is essential for efficient binding, the C-terminal anchor amino acid can be leucine, valine, isoleucine, phenylalanine and methionine (data not shown). The prolines in the SH3 binding domain of HIV-l Nef are highly conserved, even between different clades. and contribute either to the tertiary protein structure or to a direct interaction with associated cellular proteins [7,8,20]. The HIV-l Nef protein contains three fragments (amino acids 68-76, 77-85, 1288137) consistent with the known peptide motifs for HLA-B7 (Fig. 1). The corresponding peptides were synthesized with an AMS 422 automated peptide synthesizer (Abimed AnalysenTechnik GmBH, Langenfeld, Germany) as previously
M. Baurr et al. : Imnw~olog~~ Letters 55 (1997) I19- I.?.?
120
N40P?:
m
MAPK E
d
10
20
high affinity binding to SH3 domain
PPT
30
150
160
170
180
190
200
Fig. 1. HIV-I Nef protein: schematic structure and location of three HLA-B7 restricted CTL epitopes. The number of the amino acid positions in HIV-I Nef is shown. Secondary structural elements of the Nef,,,, are indicated by boxes (p-strands. z-helices, polyproline type II helix (PPII)). Residues involved in the direct interaction with SH3 of Fyn[R961] are labeled with asterisks, residues involved in the tertiary interaction between the Nef,.,,, and SH3 of Fyn[R961] are labeled with dots. Residues essential for maximal binding of Nef to Lck are indicated with squares. The location of the myristylation signal (m). variable duplication (d). putative phosphorylation site of protein kinase C (PKC). binding site of serineithreonine mitogen-activated protein kinase (MAPK), predicted beta-turn and the polypurine tract are indicated by lines. Amino acid sequences of major intrapatient variants for the three HLA-B7-restricted CTL epitopes are given [23]. The amino acid one letter code is: A, Ala: C, Cys; D, Asp; E, Glu; F, Phe; G. Gly; H, His; I, Be; K, Lys; L. Leu; M, Met; N, Asn; P. Pro; Q, Gin; R. Arg; S. Ser; T. Thr; V. Val; W, Trp; and Y. Tyr.
described [21]. Their binding capacity to HLA-B7 was measured by HLA-B7 stabilization on the transportdeficient hybrid cell line T2/B7 (provided by P. Cresswell). Briefly, cells were incubated with 50, 75 or 100 ,uM of peptide in serum free medium for 15 h at 37°C. Staining for indirect immunofluorescence was made with ME1 (anti-HLA-B27, -B7, -Bw22) as first antibody and FITC-labeled anti-mouse IgG (Sigma) as a second antibody. Fluorescence intensities were measured on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). The fluorescence ratio (FR) is defined as mean fluorescence experimental sample to mean fluorescence background. Fluorescence values are the mean of triplicate determinations and vary less than 10% for each peptide. HLA-B7-restricted cellular cytotoxicity against the various peptides was determined in a standard chromium release assay with peptide-pulsed HLA-B7-matched Epstein-Barr virus transformed Bcells (EBV B-cells) as target cells and peptide-pulsed mismatched targets as control. Effector cells were derived from peripheral blood mononuclear cells (PBMC) of four HIV-infected individuals with the HLA-B7 haplotype and stimulated with irradiated autologous PHA-activated PBMC for lo- 15 days. Specific lysis for a peptide concentration of 10 ,LLM at different effector to target cell ratios is shown in Fig. 2B. All Nef peptides tested bound HLA-B7 in a comparable manner to a known HLA-B7-restricted CTL epitope of EBV (Fig. 2A) [22]. Substitution of the C-terminal anchor amino acids (Nef85L, Nef851 or Nef85V) as frequently observed in Nef variants of HIV seropositives did not alter binding efficiency. This is in agreement with the previously observed flexibility of the HLA-B7 F-pocket to accommodate different hydro-
phobic side chains [19]. Except for proline at position 2, the level of binding was relatively insensitive towards amino acid substitutions and even length variation (nine or ten amino acids). The respective prolines in Nef are P69, P78 and P129 and are well conserved in HIV-l. They are involved either in direct contact with cellular proteins, in the formation of the polyproline type II x-helix, the structural element common to all. SH3 binding domains, or the arrangement of the antiparallel /J’ strands and helices, respectively. Thus the structural requirements for Nef folding seem to prevent sequence variation that would result in loss of HLA-B7 binding of the corresponding peptides. All the Nef peptides that bound to HLA-B7 represent HLA-B7-restricted CTL epitopes (Fig. 2B). CD8positive effector cells from four different HIV infected individuals have been tested and at least one of the epitopes was recognized after polyclonal stimulation of the PBMC with PHA. Thus the respective peptides not only bound HLA-B7 in vitro but seem to be correctly processed and presented in vivo. Despite the relative conservation of the epitopes Nef 68876, 77-85 and 1288137 in HIV-l patients, a number of mutations has been demonstrated [23]. However, as the corresponding variant peptides sensitized target cells for lysis and the CTL response in HIV-infected patients is able to adapt to antigenic variation [l I], Nef gene variation in vivo seems not to intervene with recognition of the whole protein. In summary, three HLA-B7-restricted CTL epitopes in HIV-l Nef have been identified. They carry the known peptide motif, bind efficiently to HLA-B7 and are specifically recognized by CTL from HIV-l infected individuals. Due to the structural properties of proline in general and the importance of the three prolines in
122
M. Bauer et al. /hnmunolog_v Letters 55 (1997) 119-122
Nef protein folding, the possibility of escape from HLA-B7-restricted CTL recognition seems curtailed.
Acknowledgements This work was supported by grants of the ‘Deutsche Forschungsgemeinschaft’ (M.B., R.M., A.M.), the French Agence Nationale de Recherches sur le SIDA (G.H., B.A.) and by Biotechnology program EC Biomed (M.L., K.E.). We thank Otto Haller for continuous support and Hanspeter Pircher and Simon WainHobson for critical comments on the manuscript.
References Ill Cullen. B.R. (1994) Virology 205, I. VI Ratner, L.. and Niederman, T.M.J. (1995) Curr. Top. Microbial. Immunol.
193, 169. H.W., Ringler, D.J., Mori, K., Panicali, D.L., Sehgal. P.K., Daniel, M.D. and Desrosiers, R.C. (1991) Cell 65, 651. F., Greenough, T.C.. Brettler, D.B.. Sullivan, J.L., 141 Kirchhoff, and Desrosiers, R.C. (1995) New Engl. J. Med. 332, 228. PI Deacon, N.J., Tsykin, A., Solomon, A., Smith, K., LudfordMenting, M., Hooker, D.J., McPhee, D.A., Greenwdy, A.L., Ellett. A., Chatfield, C., Lawson, V.A., Crowe. S.. Maerz, A.. Sonza. S., Learmont, J., Sullivan, J.S., Cunningham, A.. Dwyer, D., Dowton, D. and Mills. J. (1995) Science 270, 988. PI Trono. D. (1995) Cell 82, 189. 171 Saksela, K., Cheng. G. and Baltimore, D. (1995) EMBO J. 14. 484. A., Azad. A., Mils, J. and McPhee. D. (1996) J. PI Greenway, Viral. 70, 6701. [91 Lee. C.H., Saksela, K., Mirza, U.A., Chait. B.T. and Kuriyan. J. (1996) Cell 85, 931.
[31 Kestler,
[lo] Lamhamedi-Cherradi, S., Culmann-Penciolelh, B., Guy, B., Kieny, M.P., Dreyfus, F., Saimot, A.G.. Sereni, D.. Sicard, D.. Levy. J.P. and Gomard. E. (1992) AIDS 6, 1249. [I l] Haas. G., Plikat, U., Debre. P., Lucchiari, M.. Katlama, C.. Dudoit. Y.. Bonduelle, O., Bauer, M., Ihlenfeldt, H-G., Jung, G.. Maier, B., Meyerhans, A. and Autrdn, B. (1996) J. Immunol. (in press). [12] Safrit, J.T.. Andrews, C.A., Zhu, T., Ho, D.D. and Koup. R.A. (1994) J. Exp. Med. 179, 463. [13] Koup, R.A., Safrit, J.T., Cao, Y., Andrews. C.A.. McLeod, G.. Borkowsky, W.F. and Ho, D.D. (1994) J. Viral. 68, 4650. [14] Borrow, P., Lewicki, H., Hahn, B.H., Shaw. G.M. and Oldstone, M.B. (1994) J. Viral. 68, 6103. [I51 Rinaldo, C., Huang, X.L., Fan, Z.F., Ding, M., Beltz, L., Logar, A., Panicali, D.M.. Liebmann, J., Cottrill, M. and Gupta, P. (1995) J. Viral. 69, 5838. [16] Pantaleo, G., Menzo, S., Vaccarezza, M., Graziosi. C.. Cohen, O.J., Demarest. J.F., Montefiori, D., Orenstein, J.M., Fox. C.. Schrager, L.K., Margolick, J.B., Buchbinder, S., Giorgi. J.V. and Fauci, A.S. (1995) New Engl. J. Med. 332, 209. [I71 Lucchiari, M., Niedermann, G.. Leipner, C.. Meyerhans. A., Eichmann. K. and Maier, B. (1994) Int. Immunol. 6. 1739. [I81 Maier. R.. Falk, K.. Roetzschke. O., Maier, B.. Gnau, V., Stefanovic, S., Jung, G., Rammensee, H-G. and Meyerhans, A. ( 1994) Immunogenetics 40, 306. [I91 Huczko. E.L., Bodnar. W.M.. Benjamin, D., Sakaguchi, K.. Zhu, N.Z.. Shabanowitz, J.. Henderson, R.A., Appella, E.. Hunt, D.F. and Engelhard, V.H. (1993) J. Immunol. 151, 2572. [20] Myers. G., Korber, B., Hahn. B.H., Jeang, K.T.. Mellors, J.W., McCutchen. F.E., Henderson, L.E. and Pavlakis, G.N. (1995) Human Retroviruses and AIDS. Los Alamos National Laboratory, Los Alamos, New Mexico. [21] Nietfeld. W.. Bauer. M.. Fevrier. M., Maier, R., Holzwarth, B., Frank, R., Maier, B., Riviere,Y. and Meyerhans. A. (1995) J. Immunol. 154, 2188. [22] Hill, A., Worth, A.. Elliot, T., Rowland-Jones, S., Brooks, J.. Rickinson, A. and McMichael, A. (1995) Eur. J. Immunol. 25. 18. [23] Shugars, D.C.. Smith, M.S., Glueck, D.H.. Nantermet, P.V., Seillier-Moiseiwitsch, F. and Swanstrom, R. (1993) J. Viral. 67, 4639.