AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 8, Number 8, 1992 Mary Ann Liebert, Inc., Publishers
Identification of Large Glycosylated Proteins Recognized by Monoclonal Antibodies Against HIV-1 gag Proteins
Downloaded by East Carolina University from www.liebertpub.com at 01/16/19. For personal use only.
A. PINTER, W.J. HONNEN, K. REVESZ, and R. HERZ
INTRODUCTION
RESULTS
gag GENE OF HIV encodes the major structural proteins of the viral core, and is expressed initially as a 55 kD polyprotcin which is myristoylated at its amino terminus.1'2 During the process of viral maturation, this precursor is proteolytically cleaved by the virus-encoded protease into four separate domains; the matrix protein pi7, which corresponds to the amino terminal domain of Pr55 and carries the myristic acid substitution, the capsid protein p24, and ribonucleoproteins p6 and p9. A frame-shifting mechanism leads to the production of small amounts of a larger product containing both gag and pol sequences that is the precursor of the mature pol products, protease, reverse transcriptase, and integrase.3 The myristic acid moieties of both Pr55 and p 17 are believed to be associated with the lipid bilayer, thereby targeting the gag proteins to the interior of the membrane. This is consistent with immunoelectron microscopy and molecular modeling studies, which support the association of p 17 with the inner surface of viral membranes.4"'5 We have isolated two monoclonal antibodies against pi7K"K which recognize viral-encoded antigens exposed on the surface of live HIV-infected cells. This reactivity was initially demonstrated by fluorescence microscopy, and confirmed by FACS analysis and by binding of a radioiodinated second antibody. Similar reactivities have been described by Ikuta et al.5" Radioimmunoprecipitation assays of infected cells labeled with ['1H]glucosamine demonstrated the presence of a complex of large glycoprotcins that are immunoprecipitated by these antibodies; these components were also recognized to a lesser extent by other antibodies against p 17 and p24. These results suggest that the HIV gag gene may encode one or more membrane-associated glycoproteins in addition to the normal precursor of the internal core proteins. These molecules may be analogous to the glycosylated cell surface gag-encoded proteins of the murine leukemia viruses, which are important targets for both ADCC and immunotoxin-based therapies. The HIV gag-related cell surface components may play a role in the generation of antibodies against p 17*"* that are characteristic of early stages of HIV infection, and they may also act as natural targets for the immune system and present potential targets for immuno-
We recently have isolated a number of rat monoclonal antibodies (MAbs) against HIV-I pnK'lf!, including two which recognize conserved cpitopes that are present on the surface of live HIV-infected cells.6 This reactivity can be demonstrated by fluorescence-activated cell sorting (Fig. 1). Live infected cells were incubated with monoclonal antibodies against pi7 and stained with an FITC-labeled second antibody. Analysis by flow cytometry demonstrated that MAbs G1 Ig 1 and G1 1 h3 bound to the surfaces of approximately 65% of the live cells, while MAb B4f8 stained only about 5% of the cells. All three MAbs reacted equally well with cells which were permeabilized with acetone priorto addition of the antibodies (not shown). Acontrol with no first antibody did not show appreciable staining to either live or permeabilized cells. The presence of cell surface gag proteins on live HIV-infected cells was further quantitated with a radiolabeled antibodybinding assay. Cells (at greater than 90% viability) were incubated with various antibodies at 37°C for 30 min, fixed with 0.5% para-formaldehyde, and then incubated with l25I-labeled sheep anti-rat Ig F(ab') fragments. The amount of specifically bound antibody was counted, and compared with several controls (Fig. 2). Significant levels of MAbs Gl Igl and Gl lh3 were bound to the intact cells; these levels were similar to that observed for a rat antiserum against gp 160. Much lower levels of anti-pl7 MAb B4f8 were bound, and there was essentially no binding of an anti-p24 MAb over that obtained with uninfected cells. This reaction of anti-pl7 antibodies with live cells was unexpected, since an analysis of the sequence of pi7 did not demonstrate any obvious signal peptides or transmembrane regions that could account for the presence of this protein on the cell surface. Molecules present on the surface of eucaryotic cells are usually glycosylated, and precedents exist for the cell surface expression of glycosylated forms of murine leukemia virus gag polyproteins.71" In order to determine whether any of the components recognized by the anti-p 17 antibodies corresponded to glycosylated products of HIV gag, immunoprecipitations were performed with lysates of HIV-infected cells that had been
The
therapy. Laboratory of Retroviral Biology,
Public Health Research Institute, 455 First Avenue, New York, NY 10016.
1341
PINTER ET AL.
1342
INTENSITY
INTENSITY
INTENSITY
INTENSITY
Downloaded by East Carolina University from www.liebertpub.com at 01/16/19. For personal use only.
G11h3
FIG. 1. Detection of cell-surface staining of HIV-1 -infected cells by M Abs against pi 7""*. Live H9 cells infected with HTLV-IIIb were incubated with anti-pl7 MAbs Gl Igl, Gl lh3, or B4f8 diluted into complete medium. After washing, the cells were treated with FITC-labeled second antibody, washed, and analyzed using a Coulter fluorescent-activated cell sorter. The samples were calibrated with a standard preparation of fixed blood cells, and the percentage of live cells stained with each antibody was
quantitated.
(lanes A and B) and by a chimpanzee anti-gpl20 serum (lane C). Similar components were also immunoprecipitated although considerably less well, by two other monoclonal antibodies against p 17 (lanes G and H) and by hyperimmune rat sera prepared against recombinant p 17 (lane F) and p24 (lane E). None of these components were recognized by a hyperimmune
labeled for 24 hours with [3H]glucosamine (Fig. 3). Antibodies Gl lgl (lane I) and Gl lh3 (lane J) reacted with a complex of proteins, consisting predominantly of a closely-spaced doublet of approximately 150 kD along with another doublet of approximately 90-100 kD. These proteins appeared distinct from the env proteins gp!20 and gpl60 recognized by a human immune
serum
15000
¡I
10000
Infected Uninfected
o
5000 a.
E
a.
r~i G11h3
G11g1
B4f8
M—i
anti-gpl 60 Q10E3
Antibody
Quantitation of cell surface expression of pi7*"* epitopes by radioimmunoassay. Live H9 cells infected with the HTLV-IIIb strain of HIV-1 were treated with anti-pl7 MAbs Gl lh3, Gl lgl, and B4f8, with anti-p24 rat MAb QI0e3, and as a positive control, rat anti-gpl60 serum. Binding to uninfected H9 cells was also analyzed for each antibody. Binding was measured indirectly with [l25I]anti-rat F(ab')2 fragments. Nonspecific binding, measured in the absence of first antibody, was subtracted for each sample. The large differences in binding between infected and uninfected cells reflect the specific recognition of cell surface epitopes by the anti-pl7 MAbs and the anti-env serum. FIG. 2.
EXPRESSION OF
gag-ENCODED D
E
PROTEINS F
gp160
H
I
•
gpl20
Downloaded by East Carolina University from www.liebertpub.com at 01/16/19. For personal use only.
G
1343
FIG. 3. Radioimmunoprecipitation of [3H]glucosamine-labeled lysates of HIV-infected H9 cells. Cells infected with the HTLV-IIIb strain of HIV-1 were labeled with either 35S-labeled or with 3H-labeled glucosamine (lanes B-J) for 24 h, and cell lysates were immunoprecipitated with the following antibodies: human immune serum (lanes A and B);
cysteine (Lane A)
chimpanzee anti-gpl20 serum (lane C); rat anti-p66KI serum (laneD); rat anti-p24 serum (lane E); rat anti-pl7 serum (lane F); anti-pl7 MAb B4f8 (lane G); anti-pl7 MAb D3b3 (lane H); anti-pl7 MAb Gl lgl (lane I); and anti-pl7 MAb Gl lh3 (lane J). Lanes B and C were exposed for 1 day, lane J for 4 days, and lanes D-I for 21 days.
prepared against recombinant RT (lane D) or when the antibody was omitted (not shown).
rat serum
first
DISCUSSION These experiments identify large HIV-1-coded glycoproteins that are immunoprecipitated by certain anti-pl7 MAbs. One possibility is that these represent HIV env products that are associated with, and are therefore coprecipitated by anti-pl7 antibodies. Although the larger band precipitated by anti-pl7 antibodies is similar in size to gpl60, the number and overall pattern of bands seen in the anti-pl 7 lanes is distinct from those observed in the anti-gp70 immunoprecipitates. For several retroviruses an alternative pathway for the expression of gag is utilized that results in the formation of large, membraneassociated glycoproteins. For the murine leukemia viruses (MuLV), these molecules exist in the form of two cell-surface glycoproteins of approximately 80,000-90,000 daltons7-1" that are secreted from infected cells as proteolytic fragments of 55 and 40 kD."12 Such molecules synthesized by endogenous viruses related to the Gross strain of MuLV have been shown to correspond to the Gross Cell Surface Antigen (GCSA), which functions as an efficient target for antibody- and cell-mediated cytotoxicity reactions.I314 The mechanism of expression of the glycosylated gag proteins of MuLV has been shown to involve initiation of translation of gag at a CUG codon located 264 nucleotides upstream of the normal gag initiation site.15 This results in the introduction of a novel N-terminal domain that includes several hydrophobic regions that might function as
leaders sequences for membrane insertion. Analyses of deletion mutants of MuLV have shown that this molecule is not essential for replication of the virus in tissue culture. '6 but may facilitate infection.15 The most obvious interpretation of the results described in the present study is that the glycosylated components immunoprecipitated by anti-pl7 antibodies represent similar products of HI V-1. The structure and mechanism of expression of such molecules is unclear. Examination of HIV gag and upstream sequences does not identify an in-frame sequence 5' to the gag initiation codon that can serve an analogous function to the 5' leader sequence of the glycosylated gag product of MuLV. Potential signal peptide sequences in the 5' untranslated sequence of HIV-1 are not available even after consideration of possible splicing or frame-shifting mechanisms. Another feature that distinguishes the HIV molecules from their MuLV counterparts is their large size. This suggests that these molecules may contain significant regions of pol sequences in addition to the gag regions identified by immunological reactivites. or may be a reflection of extensive glycosylation. The lack of recognition of these proteins by a hyperimmune serum prepared against recombinant reverse transcriptasc may indicate that this region of the molecule is either absent or modified, perhaps by glycosylation. The elucidation of the structure and origin of these molecules requires further immunological and biochemical studies, including determination of the extent and nature of glycosylation and amino terminal and internal sequences. The presence of HIV gag products on the surface of infected cells may have significance toward both the natural development of antiviral antibodies and the generation of protective immune responses in infected patients. Antibodies against both gag and pol products are produced in high titers early in infection, and evidence has been presented for a correlation between the loss of antibodies against both pi7 and p24 and progression to AIDS.17 It is possible that the cell surface molecules described in this study play a role in the generation of such antibodies. Furthermore, these molecules may act as targets for such protective immune functions as antibodydependent cellular cytotoxicity (ADCC) and antibody-dependent complement-mediated cytotoxicity (ACC). One of the monoclonal antibodies that recognizes this complex on the cell surface (G1 lgl) reacts with a highly conserved epitope also found on both LAV-2 and SIV isolates.6 This antibody will be useful to determine the universality of expression of these molecules by different viral isolates and in different cell types, including peripheral blood lymphocytes and macrophages. If they are expressed in vivo, the cell surface gag molecules may provide additional targets for immunotoxin therapy, using the MAbs described in this paper or their humanized counter-
parts.1«19
ACKNOWLEDGMENTS We thank Dr. Fred Valentine for assistance in performing the cell sorting experiment, and Dr. Sam Kayman for valuable discussions concerning this study. This work was supported by Public Health Service Grants AI-72659 and AI-23884 and CFAR Grant AI-27742.
1344
PINTER ET AL.
REFERENCES
10.
1. Pal R. Reitz MS Jr. Tschachler E, Gallo RC. Sarngadharan MG, and Veronese FM: Myristoylation OÍ gag proteins of HIV-1 plays an important role in virus assembly. AIDS Res Human Retroviruses 1990;6:721-730. 2. Veronese FM, Copeland TD, Oroszlan S. Gallo RC. and Sarngadharan MG: Biochemical and immunological analysis of human immunodeficiency virus gag gene products pi 7 and p24, J. Virol. 1988;62:795-801. 3. Jacks T, Power MD. Masiarz FR, Luciw PA. Barr PJ. and Varmus HE: Characterization ofribosomal frameshifting in HIV-1 gag-pol
Downloaded by East Carolina University from www.liebertpub.com at 01/16/19. For personal use only.
expression. Nature 1988;331:280-283. 4. Andreassen H, Bohr H. Bohr J. Brunak S, Bugge T. Cotterill RMJ, Jacobsen C, Kusk P. Lautrup B. Petersen SB. Saermark T, and Ulrich K: Analysis of the secondary structure of the human immunodeficiency virus (HIV) proteins pi7. gpl20, and gp4l by computer modeling based on neural network methods. J AIDS 1990;3:615-622. 5.
Niedrig M, Rabanus JP, L'Age Stehr J. Gelderblom HR. and Pauli G: Monoclonal antibodies directed against human immunodeficiency virus (HIV) gag proteins with specificity for conserved epitopes in HIV-I. HIV-2 and simian immunodeficiency virus. J
Gen. Virol. 1988;69:2109-2114. 5a. IkutaK.MoritaC. MiyakeS. ltoT. Okabayashi M. SanoK, Nakai M. Hirai K, and Kato S: Expression of human immunodeficiency virus type I (HIV-I) gag antigens on the surface of a cell line persistently infected with HIV-1 that highly expresses HIV-I
antigen. Virology 1989:170:408-417. Shang F. Huang H. Revesz K. Chen H,
Herz R, and Pinter A: Characterization of monoclonal antibodies against the HIV matrix protein, pH1-''"-': identification of an epitope exposed on the surface of infected cells. J Virol 1991;65:4798-4804. 7. Pillemer EA, Koistra DA. Witte ON, and Weissman IL: Monoclonal antibody to the amino-terminal L sequence of murine leukemia virus glycosylated gag polyproteins demonstrates their unusual orientation in the cell membrane. J Virol 1986:57:413421. 8. Saris CJM, Eenbergen JV, Liskam RMJ. and Bloemers HPJ: Structure of glycosylated and unglycosylated gag and gag-pol precursor proteins of Moloney murine leukemia virus. J Virol
6.
9.
1983:46:841-859. Yoshiki T, and Fleissner E: A
Tung J,
leukemia virus
on
1976;9:573-578.
the surface of
core
polyprotein of murine
mouse
leukemia cells. Cell
Tung J. Pinter A. and Fleissner E: Two species oftype-C viral core polyprotein on AKR mouse leukemia cells. J Virol 1977:23:430-
435. 11. Edwards SA. and Fan H: G«g-related polyproteins of Moloney murine leukemia virus: evidence for independent synthesis of glycosylated and unglycosylated forms. J Virol 1979:30:551-563. 12. Schultz AM, and Oroszlan S: Murine leukemia virus gag polyproteins: the peptide chain unique to Pr80 is located at the amino
Virology 1978;91:481-486. Snyder HW. Stocken E. and Fleissner E: Characterization of molecular species carrying Gross cell surface antigen. J Virol terminus.
13.
1977:23:302-314. 14. Ledbetter J, Nowinski RC. and Emery S: Viral proteins expressed on the surface of murine leukemia cells. J Virol 1977:22:65-73. 15. Prats AC. Billy GD, Wang P. and Darlix J: CUG initiation codon used for the synthesis of a cell surface antigen coded by the murine leukemia virus. J Mol Biol 1989:205:363-372. 16. Schwartzberg P, Colicelli J, and Goff SP: Deletion mutants of Moloney murine leukemia virus which lack glycosylated gag protein are replication competent. J Virol 1983;46:538-546. 17. Mehta. SU. Rupprecht KR, Hunt JC, Kramer DE, Mcrae BJ, Allen RG, Dawson GJ, and Devare SG: Prevalence of antibodies to the core protein pi7, a serological marker during HIV-1 infection, AIDS Res Human Retroviruses 1990;6:443-454. 18. Vitetta ES, Fulton RJ, May RD, Till M, and Uhr JW: Redesigning nature's poisons to create anti-tumor reagents. Science
1987;238:1098-1104. 19.
Berger EA. Clouse KA, Chaudhary VK, Chakrabarti S, FitzGerald DJ. Pastan I. and Moss B: CD4-Pseudomonas exotoxin hybrid protein blocks the spread of human immunodeficiency virus infection in vitro and is active against cells expressing the envelope glycoproteins from diverse primate immunodeficiency retroviruses.
Proc Nati Acad Sei (USA) 1989;86:9539-9543.
Address
reprint requests
to:
A. Pinter Laboratory of Retroviral Biology Public Health Research Institute 455 First Avenue New York, NY 10016
Identification of Large Glycosylated Proteins Recognized by Monoclonal Antibodies Against HIV-1 gag Proteins
Downloaded by East Carolina University from www.liebertpub.com at 01/16/19. For personal use only.
A. PINTER, W.J. HONNEN, K. REVESZ, and R. HERZ
INTRODUCTION
RESULTS
gag GENE OF HIV encodes the major structural proteins of the viral core, and is expressed initially as a 55 kD polyprotcin which is myristoylated at its amino terminus.1'2 During the process of viral maturation, this precursor is proteolytically cleaved by the virus-encoded protease into four separate domains; the matrix protein pi7, which corresponds to the amino terminal domain of Pr55 and carries the myristic acid substitution, the capsid protein p24, and ribonucleoproteins p6 and p9. A frame-shifting mechanism leads to the production of small amounts of a larger product containing both gag and pol sequences that is the precursor of the mature pol products, protease, reverse transcriptase, and integrase.3 The myristic acid moieties of both Pr55 and p 17 are believed to be associated with the lipid bilayer, thereby targeting the gag proteins to the interior of the membrane. This is consistent with immunoelectron microscopy and molecular modeling studies, which support the association of p 17 with the inner surface of viral membranes.4"'5 We have isolated two monoclonal antibodies against pi7K"K which recognize viral-encoded antigens exposed on the surface of live HIV-infected cells. This reactivity was initially demonstrated by fluorescence microscopy, and confirmed by FACS analysis and by binding of a radioiodinated second antibody. Similar reactivities have been described by Ikuta et al.5" Radioimmunoprecipitation assays of infected cells labeled with ['1H]glucosamine demonstrated the presence of a complex of large glycoprotcins that are immunoprecipitated by these antibodies; these components were also recognized to a lesser extent by other antibodies against p 17 and p24. These results suggest that the HIV gag gene may encode one or more membrane-associated glycoproteins in addition to the normal precursor of the internal core proteins. These molecules may be analogous to the glycosylated cell surface gag-encoded proteins of the murine leukemia viruses, which are important targets for both ADCC and immunotoxin-based therapies. The HIV gag-related cell surface components may play a role in the generation of antibodies against p 17*"* that are characteristic of early stages of HIV infection, and they may also act as natural targets for the immune system and present potential targets for immuno-
We recently have isolated a number of rat monoclonal antibodies (MAbs) against HIV-I pnK'lf!, including two which recognize conserved cpitopes that are present on the surface of live HIV-infected cells.6 This reactivity can be demonstrated by fluorescence-activated cell sorting (Fig. 1). Live infected cells were incubated with monoclonal antibodies against pi7 and stained with an FITC-labeled second antibody. Analysis by flow cytometry demonstrated that MAbs G1 Ig 1 and G1 1 h3 bound to the surfaces of approximately 65% of the live cells, while MAb B4f8 stained only about 5% of the cells. All three MAbs reacted equally well with cells which were permeabilized with acetone priorto addition of the antibodies (not shown). Acontrol with no first antibody did not show appreciable staining to either live or permeabilized cells. The presence of cell surface gag proteins on live HIV-infected cells was further quantitated with a radiolabeled antibodybinding assay. Cells (at greater than 90% viability) were incubated with various antibodies at 37°C for 30 min, fixed with 0.5% para-formaldehyde, and then incubated with l25I-labeled sheep anti-rat Ig F(ab') fragments. The amount of specifically bound antibody was counted, and compared with several controls (Fig. 2). Significant levels of MAbs Gl Igl and Gl lh3 were bound to the intact cells; these levels were similar to that observed for a rat antiserum against gp 160. Much lower levels of anti-pl7 MAb B4f8 were bound, and there was essentially no binding of an anti-p24 MAb over that obtained with uninfected cells. This reaction of anti-pl7 antibodies with live cells was unexpected, since an analysis of the sequence of pi7 did not demonstrate any obvious signal peptides or transmembrane regions that could account for the presence of this protein on the cell surface. Molecules present on the surface of eucaryotic cells are usually glycosylated, and precedents exist for the cell surface expression of glycosylated forms of murine leukemia virus gag polyproteins.71" In order to determine whether any of the components recognized by the anti-p 17 antibodies corresponded to glycosylated products of HIV gag, immunoprecipitations were performed with lysates of HIV-infected cells that had been
The
therapy. Laboratory of Retroviral Biology,
Public Health Research Institute, 455 First Avenue, New York, NY 10016.
1341
PINTER ET AL.
1342
INTENSITY
INTENSITY
INTENSITY
INTENSITY
Downloaded by East Carolina University from www.liebertpub.com at 01/16/19. For personal use only.
G11h3
FIG. 1. Detection of cell-surface staining of HIV-1 -infected cells by M Abs against pi 7""*. Live H9 cells infected with HTLV-IIIb were incubated with anti-pl7 MAbs Gl Igl, Gl lh3, or B4f8 diluted into complete medium. After washing, the cells were treated with FITC-labeled second antibody, washed, and analyzed using a Coulter fluorescent-activated cell sorter. The samples were calibrated with a standard preparation of fixed blood cells, and the percentage of live cells stained with each antibody was
quantitated.
(lanes A and B) and by a chimpanzee anti-gpl20 serum (lane C). Similar components were also immunoprecipitated although considerably less well, by two other monoclonal antibodies against p 17 (lanes G and H) and by hyperimmune rat sera prepared against recombinant p 17 (lane F) and p24 (lane E). None of these components were recognized by a hyperimmune
labeled for 24 hours with [3H]glucosamine (Fig. 3). Antibodies Gl lgl (lane I) and Gl lh3 (lane J) reacted with a complex of proteins, consisting predominantly of a closely-spaced doublet of approximately 150 kD along with another doublet of approximately 90-100 kD. These proteins appeared distinct from the env proteins gp!20 and gpl60 recognized by a human immune
serum
15000
¡I
10000
Infected Uninfected
o
5000 a.
E
a.
r~i G11h3
G11g1
B4f8
M—i
anti-gpl 60 Q10E3
Antibody
Quantitation of cell surface expression of pi7*"* epitopes by radioimmunoassay. Live H9 cells infected with the HTLV-IIIb strain of HIV-1 were treated with anti-pl7 MAbs Gl lh3, Gl lgl, and B4f8, with anti-p24 rat MAb QI0e3, and as a positive control, rat anti-gpl60 serum. Binding to uninfected H9 cells was also analyzed for each antibody. Binding was measured indirectly with [l25I]anti-rat F(ab')2 fragments. Nonspecific binding, measured in the absence of first antibody, was subtracted for each sample. The large differences in binding between infected and uninfected cells reflect the specific recognition of cell surface epitopes by the anti-pl7 MAbs and the anti-env serum. FIG. 2.
EXPRESSION OF
gag-ENCODED D
E
PROTEINS F
gp160
H
I
•
gpl20
Downloaded by East Carolina University from www.liebertpub.com at 01/16/19. For personal use only.
G
1343
FIG. 3. Radioimmunoprecipitation of [3H]glucosamine-labeled lysates of HIV-infected H9 cells. Cells infected with the HTLV-IIIb strain of HIV-1 were labeled with either 35S-labeled or with 3H-labeled glucosamine (lanes B-J) for 24 h, and cell lysates were immunoprecipitated with the following antibodies: human immune serum (lanes A and B);
cysteine (Lane A)
chimpanzee anti-gpl20 serum (lane C); rat anti-p66KI serum (laneD); rat anti-p24 serum (lane E); rat anti-pl7 serum (lane F); anti-pl7 MAb B4f8 (lane G); anti-pl7 MAb D3b3 (lane H); anti-pl7 MAb Gl lgl (lane I); and anti-pl7 MAb Gl lh3 (lane J). Lanes B and C were exposed for 1 day, lane J for 4 days, and lanes D-I for 21 days.
prepared against recombinant RT (lane D) or when the antibody was omitted (not shown).
rat serum
first
DISCUSSION These experiments identify large HIV-1-coded glycoproteins that are immunoprecipitated by certain anti-pl7 MAbs. One possibility is that these represent HIV env products that are associated with, and are therefore coprecipitated by anti-pl7 antibodies. Although the larger band precipitated by anti-pl7 antibodies is similar in size to gpl60, the number and overall pattern of bands seen in the anti-pl 7 lanes is distinct from those observed in the anti-gp70 immunoprecipitates. For several retroviruses an alternative pathway for the expression of gag is utilized that results in the formation of large, membraneassociated glycoproteins. For the murine leukemia viruses (MuLV), these molecules exist in the form of two cell-surface glycoproteins of approximately 80,000-90,000 daltons7-1" that are secreted from infected cells as proteolytic fragments of 55 and 40 kD."12 Such molecules synthesized by endogenous viruses related to the Gross strain of MuLV have been shown to correspond to the Gross Cell Surface Antigen (GCSA), which functions as an efficient target for antibody- and cell-mediated cytotoxicity reactions.I314 The mechanism of expression of the glycosylated gag proteins of MuLV has been shown to involve initiation of translation of gag at a CUG codon located 264 nucleotides upstream of the normal gag initiation site.15 This results in the introduction of a novel N-terminal domain that includes several hydrophobic regions that might function as
leaders sequences for membrane insertion. Analyses of deletion mutants of MuLV have shown that this molecule is not essential for replication of the virus in tissue culture. '6 but may facilitate infection.15 The most obvious interpretation of the results described in the present study is that the glycosylated components immunoprecipitated by anti-pl7 antibodies represent similar products of HI V-1. The structure and mechanism of expression of such molecules is unclear. Examination of HIV gag and upstream sequences does not identify an in-frame sequence 5' to the gag initiation codon that can serve an analogous function to the 5' leader sequence of the glycosylated gag product of MuLV. Potential signal peptide sequences in the 5' untranslated sequence of HIV-1 are not available even after consideration of possible splicing or frame-shifting mechanisms. Another feature that distinguishes the HIV molecules from their MuLV counterparts is their large size. This suggests that these molecules may contain significant regions of pol sequences in addition to the gag regions identified by immunological reactivites. or may be a reflection of extensive glycosylation. The lack of recognition of these proteins by a hyperimmune serum prepared against recombinant reverse transcriptasc may indicate that this region of the molecule is either absent or modified, perhaps by glycosylation. The elucidation of the structure and origin of these molecules requires further immunological and biochemical studies, including determination of the extent and nature of glycosylation and amino terminal and internal sequences. The presence of HIV gag products on the surface of infected cells may have significance toward both the natural development of antiviral antibodies and the generation of protective immune responses in infected patients. Antibodies against both gag and pol products are produced in high titers early in infection, and evidence has been presented for a correlation between the loss of antibodies against both pi7 and p24 and progression to AIDS.17 It is possible that the cell surface molecules described in this study play a role in the generation of such antibodies. Furthermore, these molecules may act as targets for such protective immune functions as antibodydependent cellular cytotoxicity (ADCC) and antibody-dependent complement-mediated cytotoxicity (ACC). One of the monoclonal antibodies that recognizes this complex on the cell surface (G1 lgl) reacts with a highly conserved epitope also found on both LAV-2 and SIV isolates.6 This antibody will be useful to determine the universality of expression of these molecules by different viral isolates and in different cell types, including peripheral blood lymphocytes and macrophages. If they are expressed in vivo, the cell surface gag molecules may provide additional targets for immunotoxin therapy, using the MAbs described in this paper or their humanized counter-
parts.1«19
ACKNOWLEDGMENTS We thank Dr. Fred Valentine for assistance in performing the cell sorting experiment, and Dr. Sam Kayman for valuable discussions concerning this study. This work was supported by Public Health Service Grants AI-72659 and AI-23884 and CFAR Grant AI-27742.
1344
PINTER ET AL.
REFERENCES
10.
1. Pal R. Reitz MS Jr. Tschachler E, Gallo RC. Sarngadharan MG, and Veronese FM: Myristoylation OÍ gag proteins of HIV-1 plays an important role in virus assembly. AIDS Res Human Retroviruses 1990;6:721-730. 2. Veronese FM, Copeland TD, Oroszlan S. Gallo RC. and Sarngadharan MG: Biochemical and immunological analysis of human immunodeficiency virus gag gene products pi 7 and p24, J. Virol. 1988;62:795-801. 3. Jacks T, Power MD. Masiarz FR, Luciw PA. Barr PJ. and Varmus HE: Characterization ofribosomal frameshifting in HIV-1 gag-pol
Downloaded by East Carolina University from www.liebertpub.com at 01/16/19. For personal use only.
expression. Nature 1988;331:280-283. 4. Andreassen H, Bohr H. Bohr J. Brunak S, Bugge T. Cotterill RMJ, Jacobsen C, Kusk P. Lautrup B. Petersen SB. Saermark T, and Ulrich K: Analysis of the secondary structure of the human immunodeficiency virus (HIV) proteins pi7. gpl20, and gp4l by computer modeling based on neural network methods. J AIDS 1990;3:615-622. 5.
Niedrig M, Rabanus JP, L'Age Stehr J. Gelderblom HR. and Pauli G: Monoclonal antibodies directed against human immunodeficiency virus (HIV) gag proteins with specificity for conserved epitopes in HIV-I. HIV-2 and simian immunodeficiency virus. J
Gen. Virol. 1988;69:2109-2114. 5a. IkutaK.MoritaC. MiyakeS. ltoT. Okabayashi M. SanoK, Nakai M. Hirai K, and Kato S: Expression of human immunodeficiency virus type I (HIV-I) gag antigens on the surface of a cell line persistently infected with HIV-1 that highly expresses HIV-I
antigen. Virology 1989:170:408-417. Shang F. Huang H. Revesz K. Chen H,
Herz R, and Pinter A: Characterization of monoclonal antibodies against the HIV matrix protein, pH1-''"-': identification of an epitope exposed on the surface of infected cells. J Virol 1991;65:4798-4804. 7. Pillemer EA, Koistra DA. Witte ON, and Weissman IL: Monoclonal antibody to the amino-terminal L sequence of murine leukemia virus glycosylated gag polyproteins demonstrates their unusual orientation in the cell membrane. J Virol 1986:57:413421. 8. Saris CJM, Eenbergen JV, Liskam RMJ. and Bloemers HPJ: Structure of glycosylated and unglycosylated gag and gag-pol precursor proteins of Moloney murine leukemia virus. J Virol
6.
9.
1983:46:841-859. Yoshiki T, and Fleissner E: A
Tung J,
leukemia virus
on
1976;9:573-578.
the surface of
core
polyprotein of murine
mouse
leukemia cells. Cell
Tung J. Pinter A. and Fleissner E: Two species oftype-C viral core polyprotein on AKR mouse leukemia cells. J Virol 1977:23:430-
435. 11. Edwards SA. and Fan H: G«g-related polyproteins of Moloney murine leukemia virus: evidence for independent synthesis of glycosylated and unglycosylated forms. J Virol 1979:30:551-563. 12. Schultz AM, and Oroszlan S: Murine leukemia virus gag polyproteins: the peptide chain unique to Pr80 is located at the amino
Virology 1978;91:481-486. Snyder HW. Stocken E. and Fleissner E: Characterization of molecular species carrying Gross cell surface antigen. J Virol terminus.
13.
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