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Virrology

Arch Virol (1992) 126:11-20

© Springer-Verlag 1992 Printed in Austria

An O-linked carbohydrate neutralization epitope of HIV-1 gp 120 is expressed by HIV-1 e n v gene recombinant vaccinia virus J. E. S. Hansen1, H. Clausen 2, S. L. Hu 3, J. O. Nielseni, and S. Olofsson1 1Department of Infectious Diseases, Hvidovre Hospital, Hvidovre, 2Department of Oral Diagnostics, Royal Dental College, Copenhagen, Denmark 3Bristol-Myers Squibb, Seattle, U.S.A. Accepted February 20, 1992

Summary. Previous studies have disagreed about the presence of O-linked carbohydrate epitopes on gp 120 of HIV, although antibodies against short-chain O-linked glycans neutralize HIV infection and block syncytium formation in vitro. To settle this question, we analysed the O-linked glycans of gp 120 by chemical methods using purified HIV-1 gp 120 from cells infected with recombinant vaccinia virus solely expressing gp 160 or gp 120. Alkaline borohydride degradation of recombinant gp 120 released monosaccharides and also slightly larger structures (di/trisaccharides) by a 13-elimination, confirming the presence of simple O-linked oligosaccharides. The functional activity as neutralisation epitopes of the O-linked oligosaccharides expressed on recombinant gp 120 was preserved, since fusion between uninfected CD 4 + cells and cells infected with recombinant vaccinia was blocked by monoclonal antibodies to the O-linked oligosaccharides of gp 120. Although the mechanism for HIV induction of Olinked oligosaccharide neoantigens is unknown, these results indicate that the O-linked neutralization epitopes are inherent to the glycoprotein itself, and that the unusual appearance of simple O-linked oligosaccharides on gp 120 is independent of any interaction between the host cell and retroviral genes other than e n v . Introduction Recently, carbohydrate epitopes of the extracellular envelope glycoprotein gp 120 of HIV have been shown to be targets for neutralizing antibodies in vitro. Antibodies against N-linked high mannose oligosaccharides [18], histoblood group A antigen and LeY [8], as well as the O-linked sialosyl-Tn [-8] and Tn antigens I-9] block HIV infection of susceptible target cells in vitro. The Tn epitope (Gal NAc-Ser/Thr) is especially interesting as all investigated HIV

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J.E.S. Hansen et al.

strains propagated in transformed cell lines as well as normal lymphocytes are neutralized by anti-Tn antibody [9], and as this antigen does not usually occur in normal human, non-malignant tissues [ 11, 25]. These properties suggest that Tn-active immunogens could be considered as a component in a future HIV vaccine. Until very recently, the data indicating O-glycosylation on gp 120 have solely been based in monoclonal antibodies (MAbs) specific for O-linked oligosaccharides. In this study we have therefore analysed gp 120 by alkaline borohydride degradation, and we present evidence that gp 120 indeed do express Tn and other simple O-linked carbohydrate structures. To date, little is known about the origin of these antigens in HIV. As glycosylation of animal viruses is normally carried out by glycosyltransferases specified by the host cell [for a review, see 5], appearance of the Tn antigen is not immediately expected. Although formation of the Tn-structure is the first step in O-glycosylation, additional saccharides will normally be added to form extended oligosaccharide structures. Viral infection has previously been found to be able to alter the glycosylation processes in host cells, and till now the only clue to the mechanism behind this phenomenon is that certain viral oncogenes (e.g. ras) are able to change the activity of specific glycosyltransferases and alter both N- and Olinked oligosaccharide structures, among other changes leading to production of truncated O-glycans [2, 3]. We therefore found it pertinent to examine whether Tn neoantigen expression subsequent to HIV infection depended on some interaction of the retroviral genome other than env with the cell. In the present paper, we show that HIV gp 120, expressed by recombinant vaccinia virus without involvement of any other HIV genes, preserves the Tn antigen defined by monoclonal antibodies (MAbs) as a target for interference with gp 120-mediated syncytium formation. Materials and methods Monoclonal antibodies (MAbs)

MAb 1E3 (IgG2a) specific for Tn (Clausen et al., unpubl.), MAb B 72.3 (IgG1) specific for sialosyl-Tn [19], and negative control MAb AGPA 10E 10 specific for glycophorin A (Clausen, unpubl.) were dialyzed supernatants from mouse hybridomas. MAb 110-4 [15] against gp 120 was purchased from Genetic Systems Corp., Seattle, WA, U.S.A. Virus

The following recombinant vaccinia constructs were used: v-J 1, expressing gp 160 (strain BRU) under the 7.5 promoter, and v-11 K-120, expressing gp 120 (strain BRU) as a secreted protein under the 11 K promotor [1, 12, 13]. A vaccinia field isolate, designated C577 [17], was used as a negative control. Production of recombinant gp 120

Vero cells (ATCC CCL 81) were infected with vaccinia vectors at a MOI of t0. Virus was allowed to adsorb to cells in monolayer for 90 min at 37 °C. After adsorption, Dulbecco's

O-linked oligosaccharides of HIV

13

modified Eagle medium, containing 75 gCi/ml of [1,6-3H]-glucosamine (GlcN, Amersham; specific activity 26 Ci/mmol), was added to the cells. Cells were harvested 72 h post infection and lysed in PBS containing 1% Triton X-100 and 0.2 mM PMSF.

Purification o f gp 120 Purification of gp 120 was carried out as described [6]. MAb 110.4 (Genetic Systems, Seattle, WA) was coupled to CNBr-activated Sepharose (Pharmacia Fine Chemicals, Uppsala, Sweden) according to the manufacturer's instructions at a density of 2 mg of antibody per gram of dry CNBr-activated Sepharose. Radiolabelled culture supernatants from cells infected with vaccinia vector v-120, secreting gp 120 into the culture medium, was applied to the affinity column (approximately 5 ml gel) and the column was extensively washed (1 ml/min) with PBS. Specifically adsorbed glycoprotein was eluted under reversed flow by 0.5M formic acid. The fractions were immediately neutralized by addition of 2 M Tris base. Finally, the purified gp 120 was desalted against PBS, on Sephadex G 25 (Pharmacia Fine Chemicals, Lurid, Sweden).

Alkaline borohydride treatment of gp 120 13-Elimination of O-glycans was carried out by the method of Carlson [4] as modified for viral glycoproteins by Lundstrrm etal. [16]. Purified gp 120 was desalted against 0.1 M ammonium acetate and lyophilized. The lyophilisate was dissolved in 1 M NaBH4 in 0.05 M NaOH, and incubated at 45 °C for 16h. After incubation, the samples were neutralized with 1 M acetic acid and dried under a stream of nitrogen. Excess of borate was evaporated as methylborates by repeated dissolving and evaporation with methanol in the presence of acetic acid. The liberated glycans from alkaline borohydride treatment were separated from gp 120 by two methods: (0 For determination of the total amount of small O-glycans, the evaporated methanol-treated reaction mixture was subjected to gel filtration on a short disposable PD 10 Sephadex G 25 column (Pharmacia Fine Chemicals, Uppsala, Sweden), equilibrated with PBS. (ii) For size determination of small glycans, the evaporated methanoltreated reaction mixture was passed through a disposable Sep-Pak C 18 cartridge (Waters, Richmond, CA), thoroughly washed with methanol and equilibrated with water. The radioactivity from the flow-through and subsequent water wash fractions was considered to contain glycans and this material was subjected to gel filtration on Bio-Gel P 2 (Bio-Rad Laboratories, Richmond, CA) in a 0.9 × 120 cm column, equilibrated with PBS, and with tritiated GlcN as a monosaccharide marker.

Vaccinia infection o f MT-4 cells Dilution series of C 577 or v-J1 stock virus were incubated with 1 × 10 6 MT-4 cells [10] for 3 h at 37 °C in 1 ml RPMI with 10% heat-inactivated fetal calf serum, 2 gM glutamine, 100 IU/ml penicillin, 20 ~tg/ml gentamicin, and 100 IU/ml streptomycin (growth medium). After washing, the cells were transferred to 24-well cell culture plates and cultured in duplicates for 7 days. After 2, 4, and 7 days the cultures were evaluated for syncytia (giant cell with > 5 nuclei and balooning cytoplasm) and live cell counts were obtained by trypan blue exclusion. Presence of HIV env DNA in cells infected with the recombinant vaccinia virus (v-J1) was checked at day 7 by PCR as previously described [8].

Syncytium assay 5 x 10 6 MT-4 cells in 7ml growth medium were inoculated with wild type (C 577) or env recombinant vaccinia (v-J1) and incubated for 24h. After washing, 10.000 infected MT-4 cells were incubated with 2 gg MAb against Gal NAc-Ser/Thr (MAb 1E3), against sialosyl-

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Gal NAc-Ser/Thr (MAb B 72.3), against the V 3-loop ofgp 120 (positive control: MAb F 58) or against an irrelevant antigen (negative control: MAb AGPA 10 E 10). Then 50.000 uninfected H 9 cells [-24] were added, and after coculture for 24 h the number of syncytia (giant cells with > 5 nuclei and "balooning" cytoplasm) in duplicate cocultures was determined by microscopy (x 100).

Results Inhibition o f recombinant gp 160-induced syncytia formation by anti-Tn M A b Infectivity of the recombinant vaccinia virus, expressing H I V gp 160, in h u m a n lymphocytic cells was examined by inoculating C D 4 + M T - 4 cells with a dilution series of virus. A n identical dose response effect as evaluated by cytopathic effect was f o u n d for wild type and recombinant vaccinia. However, syncytia were only found in cultures infected with H I V env r e c o m b i n a n t vaccinia virus (Fig. 1). Functional integrity of the O-linked oligosaccharides on gp 120 expressed by vaccinia-gp 160 infected cells was examined using M A b s against sialosylG a l N A c - S e r / T h r ( M A b B 72.3) and G a l N A c - S e r / T h r ( M A b 1E3), which have previously been found to block syncytium f o r m a t i o n between H I V infected and uninfected cells [8, 9]. MT-4 cells were infected with vaccinia, incubated with M A b and uninfected H 9 cells were added. After 2 4 h syncytia appeared in

live cells % control

C577 v-J1

1004

•

[]

80

60

40

20

virus inoculum, ~1 syncytia:

C577 v-J1

0

0,02 0.2 -

+

2

20

200

++

+++

+++

Fig. l. Infectability and HIV env expression in a CD 4 + lymphocytic cell line (MT-4) inoculated with wild type vaccinia (C 577) or env recombinant vaccinia (v-Jl). MT-4 cells were inoculated with a dilution series of each virus. Presence of syncytia was evaluated after 2 days (+ < 5, + + < 20, + + + > 20 syncytia per well), and the number of live cells at day 7 in percent of mock-treated cells was determined by trypan blue exclusion. The cultures inoculated with 20 t~1virus were tested for HIV env DNA by polymerase chain reaction and gel etectrophoresis: v-J1 inoculated cultures were positive and C 577 cultures were negative

O-linked oligosaccharides of HIV

15

control-cultures where the MT-4 cells had been infected with recombinant vaccinia (v-J 1) and incubated with irrelevant MAb AGPA (Table 1). No syncytia were detected in cocultures with wild type vaccinia (C 577). MAbs against both Gal NAc-Ser/Thr (MAb 1E3) and sialosyl-Gal NAc-Ser/Thr (MAb B 72.3) completely blocked or inhibited syncytium formation as did control-MAbF 58 against a peptide sequence in the V 3-region of gp 120 (Table 1).

Demonstration of O-glycans in recombinant gp 120 Secreted gp 120 was purified from culture supernatants of Vero cells infected with a recombinant vaccinia virus, encoding only the gp 120 portion of gp 160. The supernatant was passed through a column containing MAb 110.4-Sepharose. The eluted fraction appeared in SDS-PAGE (Fig. 2) as one major band with an apparent molecular weight of l l0k, comprising 85-90% of the total 3H-GlcN label, and one minor band, containing a proteolytically degraded gp 120 fragment. These results were in accordance with those previously published for expression of gp 120 in G M K cells [1]. The purified glycoprotein was subjected to alkaline borohydride treatment to specifically release O-linked glycans by a 13-elimination reaction [4, 16]. To quantitate possible small O-glycans we subjected the reaction mixture to gel filtration on short Sephadex G 25 columns (Fig. 3). Less than 4% of the total 3H-GlcN label (which is metabolized to 3H-GlcNAc and 3H-Gal NAc during the experimental conditions used [20]) was found in the totally included volume (Fig. 3), indicating that the amount of O-glycans was very small compared with N-glycans. To determine the size distribution of the small O-glycans, we subjected the reaction mixture to Bio-Gel P 2 gel filtration. Prior to this analysis, excess borate was eliminated as described in Materials and methods and hydrophobic materials were removed by passing the reaction mixture through a Table 1. Inhibition of recombinant gp 160-induced syncytia by anti-carbohydrate antibodies v-J1 (txl)

C577 (~tl)

20

50

20

50

34 20 12.5 22

0 0 0 0

0 0 0 0

Control MAb AGPA 10 E 10 20 Control MAb F 58 3 MAb 1E3 0 MAb B 72.3 5

MT-4 cells were infected with wild type vaccinia (C 577) or recombinant vaccinia expressing HIV gp 160 (v-J1). Infected cells were then incubated with antibody prior to coculture with uninfected CD 4 + H 9 cells. Results are expressed as number of syncytia (mean of two determinations) after 24 h

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Fig. 2. SDS-PAGE of purified recombinant 3HGlcN-labelled gp 120. Purified gp 120, diluted 1/5 in SDS-PAGE sample buffer (A) and 14C-labelled commercial molecular weight markers (B) were run in parallel and subjected to fluorography. The molecular weights (kDa) of the marker proteins are indicated

CPM 9000

6000

3000

10 Fraction

20

Fig. 3. Sephadex G25 (PD-10) gel filtration of the reaction mixture from alkaline borohydride treatment. Prior to analysis the mixture was cleared for excess borates. Positions of the void volume (V) and the totally included volume (/) are indicated

Sep-Pak C t8 cartridge. M o s t o f the radioactivity was f o u n d in the void volume (Fig. 4), representing gp 120 n o t retained by the Sap-Pak C 18 cartridge. T w o distinct O-glycan peaks, designated A and M respectively, were partially included by Bio-Gel P 2. Peak M comigrated with the 3H-GlcN marker, and peak A migrated as a tri- or disaccharide.

O-linked oligosaccharides of HIV

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CPM

1500

1000

500

20

40 Fraction number

60

Fig. 4. Bio-Gel P 2 gel filtration of the reaction mixture from alkaline borohydride treatment. Prior to analysis the mixture was cleared for excess borates and passed through a Waters SepPak C 18 cartridge. Positions of the void volume (If), the di-/trisaccharide region (A), and the monosaccharide region (M) are indicated

Discussion

Antibodies to simple O-linked carbohydrate antigens have previously been found to neutralize HIV infection and syncytium formation between gp 120bearing infected and CD 4-bearing uninfected cells [8, 9]. In this study syncytium formation between MT-4 cells expressing the envelope glycoproteins of HIV through infection with a vaccinia vector and uninfected C D 4 + H 9 cells was blocked or inhibited by antibodies to short-chain O-linked carbohydrate epitopes. This suggests that preservation of simple O-linked carbohydrate neutralization epitopes, which are only expressed on lymphoid cells after HIV infection or malignant transformation [8, 25], is inherent to gp 120 itself. Although O-linked glycans are found on other retroviruses [23], indications have previously been found that gp 120 expressed in Chinese hamster ovary cells does not contain O-linked glycans [14]. However, using MAbs specific for O-linked oligosaccharides gp 120 and gp 160 have been found to contain short-chain O-glycans, which function as neutralization epitopes on HIV [8, 9]. Recent results have demonstrated PNGase F resistent sialylation taking place prior to proteolytic cleavage of gp 160 in cis Golgi, which also suggests the existence of small amounts of sialylated O-linked glycans [26]. In the present study, alkaline [}-elimination, which specifically releases O-linked oligosaccharides, resulted in release of two classes of small 3H-labelled molecules, corresponding to a di-/trisaccharide and a monosaccharide respectively as indicated by their elution position in Bio-Gel P2 gel filtration. The monosaccharide identifies the Tn structure, since the Tn structure (Gal NAc-Ser/Thr) consists of only one hexosamine. The results of the present study therefore confirm that O-linked oligosaccharides are indeed present on gp 120, and that the Tn structure is among these oligosaccharides.

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Compared with other O-glycosylated viral glycoproteins, such as the HSVspecified glycoprotein gC-1 [16, 2 0 2 2 ] gp 120 contains a very restricted amount of small O-linked glycans. Our analysis indicated that not more than 4% of the total gp 120 GlcN label was found in the totally included region of a Sephadex G 25 column after alkaline borohydride treatment, and this radiolabel was associated both with a di-/trisaccharide peak and a monosaccharide peak in the P 2 gel filtration. These results suggest that the number of Gal NAc-Ser/ Thr (i.e., Tn) units of gp 120 should not exceed 4. This number is quite low, considering the nature of the Tn antigen as a sensitive and very broadly reacting neutralization epitope of HIV [9]. Certain oncogenes and protooncogenes, such as r a s may induce alterations in oligosaccharide structure after transfection into mammalian cells [2]. Moreover, H - r a s was found to be directly correlated to altered glycosylation in MadinDarby canine kidney cells after transformation by murine sarcoma virus [3]. Although it is uncertain whether HIV contains oncogenes in a strict sense [7], the alteration of glycosylation in HIV infected cells which results in expression of the primitive Tn antigen could be caused by, e.g., the regulatory genes of HIV. However, our results showing that the Tn antigen was expressed on recombinant g 120 produced in both MT-4 cells and Vero cells, indicate that acquisition of the Tn antigen is solely dependent on the e n v gene. We therefore suggest that expression of incompletely elongated short-chain O-linked glycans on gp 120 may be caused by the quartenary structure ofgp 160/gp 120 oligomers, restricting the accessibility for subsequent glycosyltransferases in the normal elongation of O-glycans in Golgi.

Acknowledgements We wish to thank Ms. Katja Bergholdt for excellenttechnical assistance, and Mr. Lennart ~kerblom for supplying the monoclonal antibody F 58. This study was supported by The Danish Insurance Association, The University of Copenhagen and The Hartmann Foundation and by grants to S. O. from the Swedish Cancer Society (Grant 2962-B89-01XA), The Swedish Medical Research Council (Grant 9083) and The National Swedish Board for Technical Development (Project 8%0256P).

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5. Datema R, Olofsson S, Romero PA (1987) Inhibitors of protein glycosylation and glycoprotein processing in viral systems. Pharmacol Ther 33:221-286 6. Fenouillet E, Gluckman J-C, Bahraoui E (1990) Role of N-linked glycans of envelope glycoproteins in infectivity of human immunodeficiencyvirus type 1. J Virol 64: 28412848 7. Guy B, KJeny MP, Riviere Y, Le Peuch C, Dott K, Girard M, Montagnier L, Lecoq J-P (1987) HIV F/3' orf encodes a phosphor3qated GTP-binding protein resembling an oncogene product. Nature 330:266-269 8. Hansen J-ES, Clausen H, Nielsen C, Teglba~rg LS, Hansen LL, Nielsen CM, Dabelsteen E, Mathiesen L, Hakomori S, Nielsen JO (1990) Inhibition of human immunodeficiency virus (HIV) infection in vitro by anti-carbohydrate monoclonal antibodies: peripheral glycosylation of HIV envelope glycoprotein gp 120 may be a target for virus neutralization. J Virol 64:2833-2840 9. Hansen J-ES, Nielsen C, Arendrup M, Olofsson S, Mathiesen LR, Nielsen JO, Clausen H (1991) Broadly neutralizing antibodies targeted to mucin-type carbohydrate epitopes of human immunodeficiency virus. J Virol 65:6461-6467 10. Harada S, Koyanagi Y, Yamamoto N (1985) Infection of HTLV-III/LAV in HTLVI-carrying cells MT-2 and MT-4 and application in a plaque assay. Science 229: 563566 11. Hirohashi S, Clausen H, Yamada T, Shimosato Y, Hakomori S-I (1985) Blood group A cross-reacting epitope defined by monoclonal antibodies NCC-LU-35 and -81 expressed in cancer of blood group 0 or B individuals: its identification as Tn antigen. Proc Natl Acad Sci USA 82:7039-7043 12. Hu S-L, Kosowski SG, Dalrymple JM (1986) Expression of AIDS virus envelope gene in recombinant vaccinia viruses. Nature 320:537-540 13. Klaniecki J, Dykers T, Travis B, Schmitt R, Wain M, Watson A, Sridhar P, McClure J, Morein B, Ulrich JT, Hu S-L, Lewis J (1991) Cross-neutralizing antibodies in rabbits immunized with HIV gp 160 purified from simian cells infected with a recombinant vaccinia virus. AIDS Res Hum Retrovirus 7:791-798 14. Kozarsky K, Penman M, Basiripour L, Haseltine W, Sodroski J, Krieger M (1989) Glycosylation and processing of the human immunodeficiency virus type 1 envelope protein. J AIDS 2:163-169 15. Linsley PS, Ledbetter JA, Kinney-Thomas E, Hu S-L (1988) Effects of anti-gp 120 monoclonal antibodies on CD 4 receptor binding by the env protein of human immunodeficiency virus type 1. J Virol 62:3695-3702 16. Lundstr6m M, Olofsson S, Jeansson S, Lycke E, Datema R, Mgmsson J-E (1987) Host cell-induced differences in O-glycosylation of herpes simplex virus gC-t. I. Structures of non-sialylated HPA- and PNA-binding carbohydrates. Virology 161:385-394 17. Marquardt J, Holm SE, Lycke E (1964) Preparation of a purified vaccinia virus freed from soluble antigens. Proc Soc Exp Biol Med 116:112-116 18. M/iller WEG, Schr6der HC, Reuter P, Maidhof A, Uhlenbruck G, Winkler I (1990) Polyclonal antibodies to mannan from yeast also recognize the carbohydrate structure of gp 120 of the AIDS virus: an approach to raise neutralizing antibodies to HIV-I infection in vitro. AIDS 4:159-162 19. Nuti M, Teramoto YA, Mariani-Constantini R, Hand PH, Colcher D, Schlom J (1982) A monoclonal antibody (B 72.3) defines patterns of a novel tumor-associated antigen in human mammary carcinoma cell populations. J Inst Cancer 29:539 20. Olofsson S, Jeansson S, Lycke E (1981) Unusual lectin-binding properties of a herpes simplex virus type 1-specific glycoprotein. J Virol 38:564-570 21. Olofsson S, Blomberg J, Lycke E (1981) O-glycosidic carbohydrate-peptide finkages of herpes simplex virus glycoproteins. Arch Virol 70:321-329 22. Olofsson S, Sj6blom I, Lundstr6m M, Jeansson S, Lycke E (1983) Glycoprotein C of

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Authors' address: J. E. S. Hansen, Department of Infectious Diseases, Hvidovre Hospital, DK-2650 Hvidovre, Denmark. Received October 16, 1991