Identification and antigenicity of the major envelope glycoprotein of of lymphadenopathy-associated virus

VIROLOGY

144, 283-289

(1985)

Identification

and Antigenicity of the Major Envelope Glycoprotein of Lymphadenopathy-Associated Virus

L. MONTAGNIER,~ F. CLAVEL,B. KRUST,S. CHAMARET,F. REY, F. BARR~SINOUSSI, AND J. C. CHERMANN Vim1 Onw@

Unit, Institut Pasteur, 25 rue du Dr. Rwux, 7572.4Paris Cidex,

15,

France

Received January 14, 1985;accepted March 7, 1985 The major envelope glycoprotein of the causative agent of Acquired Immune Deficiency Syndrome (AIDS) lymphadenopathy-associated virus (LAV) has been identified and characterized. The glycoprotein has an apparent molecular weight of llO,OOO-120,000 under denaturing conditions in polyacrylamide gel electrophoresis. Upon deglycosylation by a specific endoglycosydase, its size is reduced to 80,000. Cellular precursors of this glycoprotein have been detected with apparent molecular weight of 150,000and 135,000. Nearly all AIDS and pre-AIDS patients have detectable antibodies against this viral glycoprotein. 0 1985 Academic Press, Inc.

Previous studies have shown that a novel type of human retrovirus is the causative agent of Acquired Immune Deficiency Syndrome (AIDS) and related diseases. We first isolated this virus from cultured T lymphocytes of a homosexual man with lymphadenopathy syndrome (LAS) (1) and thereafter named it lymphadenopathy-associated virus (LAV) (2). Viruses antigenically and morphologically related to LAV were subsequently isolated from patients with AIDS and LAS belonging to the main groups at risk for AIDS (2-4). The etiological role of the virus in AIDS is supported by its frequent isolation from AIDS and pre-AIDS patients, and its rare isolation from healthy individuals, its selective tropism for lymphocytes expressing the T4 molecule, the induction of cytopathic effect in such cells, and by serological data indicating prevalence of LAV infection in AIDS, pre-AIDS patients, and healthy individuals at risk for AIDS (5-9). Recently, retroviruses with similar antigenic, morphological properties have been isolated in several other laboratories (10, 12) and have also been shown to be strongly associated with AIDS (11, 1.3).

Various names (IDAV, HTLV-III, ARV) have been given to some of these isolates, but clearly they are antigenically and molecularly related to LAV (15, 34, 39). The relatedness of LAV or HTLV-III to the two other known human retroviruses, HTLV-I and -11 (16, 17), which are both C-type oncoviruses, is at present a matter of debate. According to Gallo and colleagues, the HTLV-III main proteins are distantly antigenically related to those of HTLV-I and -11 (14). We and the CDC group did not find significant homology by immune precipitation with patients’ sera (1, 18) or homologous competition radioimmune assay (9) of LAV ~2.5 with HTLV-I and HTLV-II ~24s. In contrast to Arya et al. (19), we have not found significant nucleic acid homology between LAV genomic DNA and HTLV-II and -1 DNAs, even under low-stringency hybridization conditions (20). Furthermore, recent sequence data (34,38-40) have shown a very close homology between LAV and HTLVIII but an absence of homology with HTLV-I and -11, suggesting that LAV may be the prototype of a new group of human retroviruses. We report here the identification of the major envelope protein of the virus, a ’ Author to whom requests for reprints should be high molecular weight glycoprotein which raises antibodies in nearly all tested AIDS addressed. 283

0042~6822/85 Copyright All rights

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0 1985 by Academic Press, Inc. of reproduction in any form reserved.

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llO-

18FIG. 1. SDS-PAGE analysis (12.5% acrylamide) of ?Xabeled LAV proteins, before and after immune precipitation with specific sera. LAVl-infected normal lymphocytes or LAVl-producing CEM cells (ATCC CCLl19) were incubated with 200 &i/ml of [?+zysteine (Amersham, 1400 Ci/m&f) for 16 br in cysteine-free RPM1 16-40 medium supplemented with 10% fetal calf serum and lo-’ M B-mercaptoethanol. The crude supernatant was centrifuged at 10,000 g for 10 min to remove cell debris and then pelleted at 60,000 g for 110 min. The pellet was resuspended in RIPA buffer (1) and aliquots were immunoprecipitated with sera (A) and then electrophoresed upon denaturation (1) or directly electrophoresed (B). (A) Lane 1: virus produced by T lymphocytes, precipitated with RUB serum (LAS patient from which LAVl was isolated). Lane 2: same virus preparation as 1, immunoprecipitated by serum of a healthy laboratory worker, B.S. Lane 3: virus produced by CEM, precipitated by RUB serum. Lane 4: virus produced by CEM, precipitated by normal serum (B.S.). Lane 5: Supernatant of control uninfected CEM, precipitated by serum RUB. Arrows indicate, from top to bottom: gpll0, ~25, ~18, ~13. (B) Lane 1: LAVl produced by CEM. Lane 2: HTLV-III produced by H9 cell line. Lane 3: Supernatant of uninfected H9 cells. M: Wlabeled molecular weight markers (phosphorylase B, 93,000; bovine serum albumin, 46,000; carbonic anhydrase, 30,000; lysosyme, 15,000).

patients. This finding may have important implications for the serological diagnosis of LAV infection and determination of its role in AIDS pathogeny. Previous studies of LAV proteins had revealed three major proteins, ~25, ~18, ~13, presumably components of the internal core of the virus

21). Upon metabolic labeling, p25 is the main protein labeled by r5S]methionine, which indicates a high methionine content, while labeling with a mixture of 14Camino acids or with [35S]cysteine also detects two smaller proteins, p18 and p13 [(81) and Fig. 11. These proteins are not present in a control preparation of uninfected cells and are antigenic in patients. Approximately 50% of two groups of Caucasian AIDS patients had antibodies against p25 (8, 9) while 94% of the group of Zairian AIDS patients were positive (22). Most of the p25 seropositive patients also have antibodies against ~18 and ~13, a few are p18 positive while being p25 negative. The envelope protein(s) should not be expected to be as abundant as the core proteins in viral preparation, but should be highly antigenic, unless genetic variation could make the host tolerant to it, as it is the case for equine infectious anemia virus (EIAV) glycoprotein (32). Upon [35S]methionine labeling, it was difficult to detect a specific protein band which was not present in the supernatant of uninfected control cells. However, a high molecular weight protein could be detected using heavy metabolic labeling with [35S]cysteine followed by immune precipitation with selected sera. T lymphocytes of a normal donor infected with LAW (I), or CEM cells infected with the same isolate were labeled with [35Slcysteine in cysteine-free medium. In order to avoid a possible alteration of the viral envelope and the subsequent loss of the glycoprotein upon purification, in the first set of experiments, the labeled virus was not further purified by zonal centrifugation. The clarified supernatant of cell culture was pelleted and the pellet lysed in RIPA buffer and incubated with various human sera. A preparation of supernatant of uninfected cells was treated similarly and run in parallel. As shown in Figs. 1 and 4, a protein of approximately llOK-120K could be specifically immunoprecipitated by sera of pre-AIDS or AIDS patients, in addition to the core proteins, and not by sera of normal healthy blood donors or of labo(18,

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1200 -93 -69 -46

FIG. 2. SDS-PAGE analysis of purified [35S]cysteine-labelled LAVl produced on CEM cells was labeled as described in Fig. 1. The crude supernatant was centrifuged at 10,666 g for 10 min and 2 ml were layered on top of a Nycodenz (Nyegaard, Oslo) density gradient (18) (5.35%). After centrifugation to equilibrium (SW27, Spinco rotor, 25,066 rpm for 16 hr), fractions were collected. Aliquots of each fraction were taken for reverse transcriptase assay (1) and for immune precipitation with RUB serum. The reverse transcriptase peak corresponding to the virus band was in lanes 5 and 6. Lane 1 corresponds to gradient’s bottom, lane 16 to its top. Arrows indicate the positions of p25 and gpll0. -

ratory workers. The protein was absent in supernatants of uninfected T lymphocytes, T- or B-cell lines. Its viral origin was further assessed by its association with the band of purified virus in Nycodenz gradients, coinciding with that of reverse transcriptase activity and the ~25 protein (Fig. 2). However, a certain amount of labeled 1lOK protein remained at the top of the gradient, suggesting it was released by the cells or by the virus during the centrifugation in a free form. The glycoprotein nature of the llOK120K protein, referred to below as gpll0, was assessed in three ways:

nose were then precipitated by sera of a LAS patient (RUB) shown in this laboratory to precipitate all four viral proteins (~13, ~18, ~25, and ~110) and a control serum. The immunoprecipitates were then electrophoresed under denaturing conditions. A single band of 110K could be detected only in the eluate from infected cells precipitated by the RUB serum. (2) Endo-p-N-methylglycosaminidae-H (endo-H) treatment. To further assess the glycosylation of gpll0, the [?S]cysteinelabeled viral lysate was immunoprecipitated by the RUB serum. The immune complexes were then bound to protein ASepharose 4B and, after elution, treated (1) Binding to lectins. Preliminary experiments had shown that LAV could be with endo-H (0.25 pg) (26) at 3’7” for precipitated by concanavalin A or bind to different periods of time, or with buffer a Con A-Sepharose column and be eluted only as a control. SDS gel analysis of the eluted antigens showed the disappearance by the leetin-specific sugar, a-methylmannose. This indicated that the envelope of the 110K band and the appearance of glycoprotein of the virus could be purified a band of approximately 80K after 3 hr treatment (Fig. 3). The other protein with the help of concanavalin A or lentil lectin. We therefore labeled the virus with bands were not changed by endo-H treatr5S]cysteine and the detergent-lysed virus ment indicating the absence of protease was adsorbed on a column of either Con in the endo-H preparation. Endo-H is A-Sepharose or lentil-Sepharose (23). The known to separate proximally oligosaccharides from polypeptides. glycoproteins eluted with a-methylman-

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-200 150 135= 110' 95, 80,

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FIG. 3. Effect of endoglycosidase H on LAV envelope glycoprotein and its cellular precursors. Lysates of LAV-infected MOLT4-T4 hybridoma syncitia, labeled for 12 hr with [36S]cysteine (lanes a to d) and of [?S]cysteine-labeled pelleted LAV virions (lanes e to h) were immunoprecipitated with the serum of a patient with persistent lymphadenopathy (B.R.U.). The immune complexes were bound to protein A-Sepharose beads, washed extensively, and eluted by heating at 95“ for 2 min in citrate buffer, pH 5.5, with 0.1% SDS and 5% aprotinin (Zymofren, Specia). The soluhilized antigens were then treated with 0.25 gg of endo-N-methylglycosaminidase-H (endo-H, Boehringer-Mannheim, FRG) in a final volume of 22 ~1, for different periods of time, at 37”C, and analyzed by SDS-polyacrylamide gel electrophoresis (7.5% acrylamide, 0.17% bisacrylamide). Lanes a and e: no treatment. Lanes b and f: 1 hr treatment. Lanes c and g: 2 hr treatment. Lanes d and h: 3 hr treatment. m = molecular weight markers.

(3) Labeling with [14C]slumsamine. After metabolic labeling of infected T lymphocytes with [‘4Clglucosamine for 48 hr, the pelleted virus was immunoprecipitated with a LAV-positive serum and analyzed by SDS-PAGE. A llOK-labeled band was specifically immunoprecipitated together with 70K and 42K bands.

In order to study the cellular precursors of the gpll0, we labeled LAV-producing cells with [35S]cysteine. A cellular hybrid, between normal T4 lymphocytes and the MOLT-4 cell line (41), proved to be particularly suitable for this study. Upon infection with LAVl, this line displays numerous giant polycaryons, arising by fusion between infected cells or by fusion between uninfected and infected cells. Giant syncitia were strongly fluorescent with patients’ sera in an indirect immunofluorescence assay, upon fixation with acetone, and could be easily separated by gravity deposition. After labeling for 3 or 12 hr, syncitia were lysed in detergent and the clarified lysate was immunoprecipitated

with LAV-positive serum, denatured, and analyzed by SDS-gel electrophoresis. After 3 hr labeling, a band of 150K was detected. Upon longer labeling (12 hr), another band of 135K appeared, suggesting that it was derived from the 150K precursor. No llOK-120K band was detected, confirming its association with free viral particles and not with budding virus. Upon endo-H treatment, the 150K-135K bands were reduced into two bands of 95K and 80K, respectively (Fig. 3). Although more work is required to establish the relationship of these various components, the present results could be best explained in the following way: the gp150 precursor corresponds to the whole coding capacity of the env gene as deduced from sequence (34), namely 95K for the protein moiety after removal of the carbohydrates. Upon the first proteolytic cleavage, which could take place either in the cytoplasm or at the cell membrane, the gp150 is converted into the gp135 form @OKafter carbohydrate removal). During virus morphogenesis, the gp135 is con-

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verted into gpllO-120, by partial enzymatic removal of carbohydrates, without proteolytic cleavage. The virus-associated gpll0 may itself be further processed during virus aging: besides the main llOK-120K band seen after % labeling of the virus, three other thin bands of ‘IOK, 40K, and 34K, respectively, could be specifically immunoprecipitated by patients’ sera. Since some of these sera did not precipitate any gag protein, it may be assumed that these proteins are antigenically related to gpll0, and are cleavage products of the latter. The same bands were more apparent after immunoblotting (Fig. 4). 1234 110 d

25-

18-

FIG. 4. Western blot analysis. Supernatants from noninfected H9 cells or from LAV and HTLV-III producing cell lines were concentrated by ultracentrifugation. The viral and control pellets (5-log total protein) were fractionated in an SDS containing 12% polyacrylamide gel, electrophoretically transferred onto nitrocellulose sheet, and immunoblotted as described ($6). The blot was incubated with a serum from a patient with lymphadenopathy syndrome (diluted l/200), washed, incubated with goat anti-human IgG coupled with peroxydase, washed, and antibody binding was detected using diaminobenzidine as substrate for the peroxydase reaction. Lane 1: viral pellet from H9 cells infected with LAV. Lane 2: viral pellet from HTLV-III producing H9 cells. Lane 3: viral pellet from LAV producing CEM cells. Lane 4: control pellet from noninfected H9 cells. Arrows indicate, from top to bottom: gpll0, ~70, ~40, ~34, ~25, ~18.

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FIG. 5. SDS-PAGE of immunoprecipitates of [asS)cysteine-labeled viral lysate immunoprecipitated with different sera. Lane 1, 3: adult AIDS patients. Lane 6: l&month-old girl, AIDS. Lane 7: father. Lane 3~ mother of patient 6, both healthy. Lane 9: LAS. Lanes 2, 4, and 5: healthy donors. Lanes 10 to 14: AIDS patient, bone marrow grafted. Lane 10: ‘7 days before graft. Lane 11: 15 days after graft. Lane 12: 2 months later. Lane 13: 7 months later. Lane 14: 24 months later (onset of AIDS).

Sequence data indicate a potential proteolytic site at the second third of the env gene, so that the 70K and the 40K proteins may indeed be cleavage products, the 40K being the transmembrane portion (3&,38). The increased detection of these fragments by immunoblotting may be due to the fact that the virus used in this technique was a 4% to 72-hr harvest from cultures of infected cells, whereas virus used for immunoprecipitation was labeled with [“S]cysteine for less than a day. Moreover, quantitative transfer of gpll0 on immunoblots proved to be difficult. Such a high molecular weight envelope protein was a general property of the LAV/HTLV-III group: HTLV-III produced by the H9 cell line (a gift of Dr. Gallo) exhibited a similar high molecular weight protein precipitated by the same sera which were positive for LAV gp (Figs. 1 and 4) (see also Ref. (37)). A preliminary survey of patients’ sera for antibodies against the gpll0 indicate that almost all patients with AIDS have antibodies against the gpll0, even those which were negative for antibodies against the core proteins (Fig. 5 and Table 1). Only two exceptions were found, that of

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1

RELATIONSHIP BETWEEN CLINICAL PRESENTATION OF SUBJEIXS WITH ANTIBODIES AGAINST LAV AND SEROREACTIVITY PATTERNS TOWARD LAV PROTEINS~ Seropositivity

Clinical

pattern

presentation

~13

~25

BP119

AIDS

LAS/ARC

No symptom

+ +

+ + -

+ + + +

3 (17%) 4 (24%) 10 (59%) 0

10 (72%) 1(7%) 2 (14%) 1(7%)

5 (50%) 3 (30%) 2 (20%) 0

17 (100%)

14 (100%)

10 (100%)

-

Total a Number

and percentage

inside each clinical

subgroup. ARC = AIDS-related

two infants who were presumably infected by their mothers (serologically positive) and could not mount an immune response after birth. In some cases where serial samples were made available, variation of antibodies against the core proteins (~25 and ~18) was observed with time (Fig. 5). In some cases, these antibodies tend to disappear at the onset of fullblown AIDS, while antibodies against the gpll0 were constantly found. Interestingly, patients with lymphadenopathy syndrome had more frequently antibodies against all viral proteins than AIDS patients (Table 1). We surmise that antibodies against the core proteins reflect a recent expression in the host of whole virus followed by lysis of virions or of infected cells, whereas antibodies against the envelope protein are determined by a constant expression of the latter at the surface of infected lymphocytes, even in the absence of whole virus release. This partial expression may be a major factor in AIDS pathogenicity. We have recently shown that the T4 molecule is acting as a receptor for LAV at the surface of T4 lymphocytes (30). Similar results were obtained independently by A. G. Dalgleish et al. (31). The binding of the viral glycoprotein, either in a free state or incorporated in virions, may mask the T4 molecule, and impair its function. Indeed, we had previously observed that the peak of LAV production by T4 lymphocytes of a healthy carrier was accompanied by a decrease of the T4 marker (7). In this hypothesis, the cyto-

complex.

pathic effect observed in vitro (3, 7) will not be the only factor to explain the impairment of T4-cell functions which is a main feature of AIDS. The depression of the absolute number of T4 cells may be explained along the same line by destruction of the infected T4 cells by a cytotoxic response, concomitantly to a direct cytopathic effect. Finally, it is noteworthy that among the world of animal retroviruses, only Lentiviruses have such high molecular weight envelope proteins: 135K for VisnaMaedi viruses (.28), 120K for the caprine arthritis encephalitis virus (CAEV) (29), 90K for EIAV (35). By contrast, HTLV glycoproteins have a much smaller molecular weight, in the range of 61K to 68K (24, 25, 27’). This is another important difference between these C-type retroviruses and the AIDS LAV retrovirus. This similarity, together with similar morphology and the sharing of a common antigenic epitope with gag protein of EIAV, suggest that LAV may be a member of the Lentiviruses subfamily of retroviruses. REFERENCES 1. BARR&SINOUSSI, et al, Science (Washing&n, D. C.) 220, 868 (1983). 2. MONTAGNIER, L., et o& In “Human T Cell Leukemia Lymphoma Viruses” (R. C. Gallo, M. E. Essex, and L. Gross, eds.), Vol. 1, p. 363. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y., 1934. 8. VILMER, E., et al, Lancet 2,753 (1934).

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,$. ELLRODT, A., et al, Len.& 1, 1383 (1984). 5. FEORINO, M. P., et aL, Science (Washington, D C.) 225, 69 (1984). 6. MONTAGNIER, L., et a& In “Prog. Immunodef. Res. Therapy” (C. Griscelli and J. Vossen, eds.), p. 367 (1984). 7. KLATZMANN, D., et al, Science (Washington, D. C.) 225, 59 (1984). 8. BRUN-V~ZINET, F., et al, Laneet 1. 1253 (1984). 9. KALYANARAMAN, V. S., et al, solace (Washington, D. C.) 225, 321 (1984). 10. POPOVIC, M., et aL, Science (Washington, D. C.) 224,497 (1984). 11. GALLO, R. C., et al, Science (Washington, D. C.) 224, 500 (1984). 12. LEVY, J. A., et al, Science (Washington, D. C.) 225, 840 (1984). IS. SARNGADHARAN, M. G., et a& solace (Washing@ D. C.) 224, 506 (1984). 14. SCH~~PBACH,J., et al, S&me (Washington, D. C!) 224,503 (1984). 1.5.LUCIW, P. A., et d, Nature (Imdon) 312, 760 (1984). 16. POIESZ, B. J., et al, Proc Nat1 Acad Sci USA 77,7415 (1980). 17. KALYANARAMAN, V. S., et al, Zbience (Washington, D. C.) 218, 571 (1982). 18. MONTAGNIER, L., et al, Ann Vird (Inst. Pasteur) 135E, 119 (1984). 19. ARYA, S. K., et al, Science (Washington, D. C.) 225, 927 (1984). 20. ALIZON, M., et aL, Nature (London) 312, 757 (1984). 21. MONTAGNIER, L., et al, Science (Washington, D. C.) 225, 63 (1984).

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22. BRUN-V&INET, F., et aL, Science (Washington, D. C.) 226, 453 (1984). 25. ESSEX, M. E., et aL, Science (Washington, D. C.) 220, 859 (1983). 24 YAMAMOTO, N., et aL, 2. Naturforsch 37c, 731 (1982). 25. HATPORI, S., et a& Gann 74, 790 (1983). 26. TARENTINO, A. L., and MALEY, F., J. Bid Chem. 249,811 (1974). 27. LEE, T. H., et d, Proc NatL Acad Sci USA 81, 3856 (1984). 29. SCOTT, J. V., et at, CeU 18, 321 (1979). 29. QU~RAT, G., et aL, .I. Vid 52, in press. $0. KLATZMANN, D., et al, Nature (London) 312, 767 . (1984). $1. DALGLEISH, A. G., et al, Nature (London) 312, 763 (1984). 32. KONO, Y., et d, Arch Ges ViruQbrsh 41, 1 (1973). 93. SHAW, G. M., et at, Science (Washington, D. C.) 226,1165 (1984). $4 WAIN-HOBSON, S., et a& Cell 40, 9-17 (1985). 35. PAREKH, B., et al, Virology 107, 520 (1980). 36. TOWBIN, H., et al, Proc. Nat1 Acad Sci USA 76, 4350 (1979). 37. KITCHEN, L. W., et aL, Nature (London) 312, 367 (1984). 38. MUESING, M. A., et d, Nature (London.) 313, 451 (1985). 39. RATNER, L., et aL, Nature (Lmdm) 313. 277 (1985). 40. SANCHEZ-PESCADOR, R., et at, Science (Washington, D. C.) 227,434-492 (1985). 41. MINOWADA, J., OHNUMA, T., and MOORE, G. E., .J. Nati Cancer Inst. 49,891 (1972).