ARCHIVES

OF BIOCHEMISTRY

AND BIOPHYSICS

Vol. 290, No. 1, October, pp. 248-257,

1991

gp160 of HIV-I Synthesized by Persistently Infected Molt-3 Cells Is Terminally Glycosylated: Evidence That Cleavage of gp160 Occurs Subsequent to Oligosaccharide Processing Roberta K. Merkle,* Dag E. Helland,t Jacqueline L. Welles,t Ali Shilatifard,* William A. Haseltine,? and Richard D. Cummings * p1 *Departmentof Biochemistry and the Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602; and t Division of Human Retrovirology, Dana-Farber Cancer Center, Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115

Received May 29, 1991, and in revised form July 10, 1991

The envelope glycoprotein of HIV-I in infected, cultured human T cells is synthesized as a precursor of apparent 1M, 160 kDa (gp160) and is cleaved to two glycoproteins, gp120 and gp41, which are the mature envelope glycoproteins in the virus. Neither the temporal and spatial features of glycosylation nor the oligosaccharide processing and proteolytic cleavage of the envelope glycoprotein are well understood. To understand more about these events, we investigated the glycosylation and cleavage of the envelope glycoproteins in the CD4+ human cell line, Molt-3, persistently infected with HIV-I (HTLV 111s). The carbohydrate analysis of gp160 and gp120 and the behavior of the glycoproteins and glycopeptides derived from them on immobilized lectins demonstrate that both of these glycoproteins contain complex- and high-mannose-type Asn-linked oligosaccharides. In addition, the N-glycanase-resistant oligosaccharides of gp120 were found to contain N-acetylgalactosamine, a common constituent of Ser/Thr-linked oligosaccharides. Pulse-chase analysis of the conversion of [3SS]cysteine-labeled gp160 showed that in Molt-3 cells it takes about 2 h for gp120 to arise with a halftime of conversion of about 5 h. At its earliest detectable occurrence, gp120 was found to contain complex-type Asn-linked oligosaccharides. Taken together, these results indicate that proteolytic cleavage of gp160 to gp120 and gp41 occurs either within the trans-Golgi or in a distal compartment. 0 1991 Academic PEWS. Inc.

Human T cells infected with HIV-I2 synthesize the envelope glycoproteins gp120 and gp41 from a precursor glycoprotein designated gp160 ( 1). The glycosylation of these proteins, like that known for other viral glycoproteins, is under control of the glycosylation machinery of the host cell. The oligosaccharide side chains on recombinant gp120 produced in Chinese hamster ovary cells have been studied ( 2,3), as have the oligosaccharides on gp120 produced by infected human T cells (4, 5). The oligosaccharide structures of the precursor glycoprotein gp160 have been only indirectly studied by biosynthetic radiolabeling, endoglycosidase sensitivity, and glycosylation inhibition experiments (6, 7). There is evidence that gp160 is synthesized as a precursor having highmannose-type Asn-linked oligosaccharides ( 1,8,9), and that some of the oligosaccharides of gp120 and gp41 have features of complex-type Asn-linked oligosaccharides (4)) implying that the oligosaccharides are modified by processing and terminal glycosylation within the Golgi apparatus. Although it is clear that gp160 is eventually cleaved within the cell to form gp120 and gp41 (6)) it is less clear where or when this cleavage occurs relative to the glycosylation status of the proteins. The relationship of these oligosaccharide processing events to proteolytic cleavage of gp160 has been explored but the results are equivocal. Interference with the oligosaccharide processing events by the application of specific glycosidase inhibitors, such as deoxynojirimycin, prevents maturation of gp160 to the mature envelope gly-

i To whom correspondence should be addressed at Department of Biochemistry, University of Georgia, Athens, Georgia 30602. Fax: (404) 542-1738.

’ Abbreviations used: HIV-l, human immunodeficiency virus-l; SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; endo H, endo-l\‘acetylglucosaminidase H; RCA-I, Ricinus communis agglutinin I; Con A, concanavalin A; ER, endoplasmic reticulum; GC, gas chromatography.

248

0003-9861/91 Copyright

0

1991 by Academic

All rights of reproduction

$3.00

Press, Inc.

in any form reserved.

COMPLEX-TYPE

OLIGOSACCHARIDES

coproteins (10, 11). Some investigators have proposed that proteolytic processing of gp160 takes place within the cis- or medial-Golgi apparatus, while the protein has high-mannose-type Asn-linked oligosaccharides, and that oligosaccharide processing occurs subsequent to cleavage (7,11). Other studies have demonstrated that only a small percentage of gp160 is eventually proteolytically cleaved by the cell and that this occurs after or closely associated with oligosaccharide processing and terminal glycosylation (6, 12). Most of the experiments to address this problem have relied on the sensitivity of the metabolically radiolabeled viral glycoproteins to endoglycosidases. An alternative approach to address this problem, which we have used, is to metabolically radiolabel the viral glycoproteins produced by infected human T cells with radioactive sugar precursors and to examine the types of oligosaccharides and monosaccharides present on the isolated glycoproteins. Our studies indicate that cellular gp160 can be fully processed and terminally glycosylated, and that it has a glycosylation status similar to that of gp120 and gp41. These results suggest strongly that cleavage of gp160 occurs concomitant with or subsequent to terminal glycosylation. EXPERIMENTAL

PROCEDURES

Materials. Concanavalin A-Sepharose was purchased from Pharmacia. Ricinus communis agglutinin I (RCA-I) -agarose was obtained from Vector Laboratories. Gel electrophoresis molecular weight standards (i4C-radiolabeled) were purchased from BRL. CNBr, lactose, (Ymethylmannoside, and ol-methylglucoside were purchased from Sigma Chemical Co. Pronase (Grade B) was obtained from Cal-Biochem and Arthrobacter ureafaciens neuraminidase was obtained from Boehringer Mannheim. SDS, glycine, and Tris were purchased from Bio-Rad. Centricon-10 microconcentrators were obtained from Amicon. IgG Sorb was purchased from The Enzyme Center (Malden, MA). The standard Nacetyl- [ 6- 3H] glucosamine was prepared by N-acetylation of [ 63H]glucosamine (27 Ci/mmol; ICN) by standard procedures (13). N[ 4- ‘% ] Acetylneuraminic acid was prepared by neuraminidase digestion of CMP-N[4-“C] acetylneuraminic acid (1.8 mCi/mmol; NEN), using 10 milliunits of enzyme in 50 ~1 of 0.1 M sodium acetate, pH 4.8, for 16 h at 37°C. The standard N- [1-14C] acetylgalactosaminitol was prepared by reduction with NaBH, of N- [l- i4C ] acetylgalactosamine (60 mCi / mmol; Amersham) as described ( 13). Maintenance of cultured Molt-3 cells and infection with HTLV IZIB. Molt-3 cells persistently infected and producing the IIIB strain of HIV-1 were obtained from Dr. M. Essex (Harvard University, School of Public Health, Boston) and cultivated in RPM1 1640 medium supplemented with 20% fetal calf serum. Uninfected Molt-3 cells were used as controls. Metabolic radiolabeling and isolation of HIV glycoproteins. Cells (5 X 106) in 5 ml medium were labeled with [ 35S] cysteine ( 1029 Ci/mmol; NEN), D-[6-3H]galactose (25 Ci/mmol; NEN), D-[2-3H]mannose (30 Ci/mmol; NEN), or D- [6-3H]glucosamine (27 Ci/mmol; NEN) at a concentration of 1 mCi per milliliter for 24 h. The cells were collected by centrifugation, washed in phosphate-buffered saline, and resuspended in RIPA buffer (10 mM Tris-HCl, pH 7.4 (25’C), 0.5% aprotinin, 1% NP-40, 2 mM EDTA, 0.15 M NaCl, and 1% deoxycholate). Free virus was pelleted by centrifugation for 1 h at 14,000 rpm in a microcentrifuge and resuspended in 0.5 ml RIPA buffer. One-tenth volume of 10X RIPA was added to the supernatant. The viral glycoproteins were obtained by immunoprecipitation using an AIDS patient-derived high-titer serum known to immunoprecipitate the viral envelope proteins. The immunoprecipitations were performed in the presence of an excess of unlabeled

ON

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gp160

249

lysates from noninfected Molt-3 cells to reduce nonspecific precipitation of labeled cellular proteins. Following precipitation of immune complexes using IgG Sorb, the complexes were dissociated in SDS and mercaptoethanol and the proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The radiolabeled glycoproteins were visualized by fluorography. Pulse-chase labeling of the envelope glycoproteins. Virus-producing Molt-3 cells (10’ cells) in exponential growth phase were harvested by centrifugation, washed once in prewarmed PBS ( 37’C ) , and resuspended in 15 ml prewarmed (37’C) cysteine-free RPM1 1640 medium containing 10% dialyzedcalf serum and 15 mCi [35S]cysteine (600 Ci/mmol). The cells were incubated in this labeling medium for 10 min at 37°C and then centrifuged at 1OOOg for 2 min. The chase was initiated by resuspension of the cell pellet in 35 ml normal RPM1 1640 (420 aM cysteine = 168fold excess of unlabeled cysteine compared to the pulse label) containing 10% fetal calf serum, and 5-ml samples were collected at the chase times indicated, harvesting the cells by centrifugation. The cell pellets were resuspended in 1 ml RIPA buffer containing 1% Na deoxycholate, and 0.5 ml 10X RIPA buffer was added to the supernatants. The envelope glycoproteins were isolated by immunoprecipitation as described above. After separation of the [?S Jcysteine-radiolabeled viral glycoproteins by SDS-PAGE, the dried gel was scanned for radioactivity using a Model 200 imaging scanner from Bio-Scan. Preparation of uiral glycopeptides. Viral glycoproteins identified by their mobility on SDS-PAGE following fluorography were excised from the gel and treated with 1 ml of 0.1 M Tris-HCl, 1 mM CaCl,, pH 8.0, containing Pronase (10 mg/ml) at 60°C for 24 h, as described previously for studies on the low-density lipoprotein receptor and other glycoproteins ( 13, 14). Ekctroelution of gp160. Gel pieces containing [ 35S] cysteine-labeled gp160 were excised from an SDS-polyacrylamide gel and transferred to a glass tube of a Model 422 electroeluter ( Bio-Rad) . Electroelution was carried out in elution buffer (25 mM Tris base, 192 mM glycine, 0.1% SDS) at 10 mA for 8 h, as described by the manufacturer. Cyanogen bromide digestion of glycoproteins. Gel pieces containing [35S]cysteine-labeled gp160 or gp120 were excised from a dried SDSpolyacrylamide gel. The excised bands were rehydrated in 500 ~1 of 70% formic acid for 20 min and then 200 pg of CNBr was added (15). The digestion mixture was incubated at 25°C in an argon atmosphere in the dark for 24 h, and the reaction was stopped by the addition of 10 vol of water. The glycopeptides released from the gel by this treatment were lyophilized. The glycopeptide fragments remaining in the gel piece after CNBr treatment were electroeluted as described above. Glycine was removed from the electroeluted peptides by ultrafiltration using a Centricon 10 microconcentrator and diluting with 2 ml of TBS before each concentration. Excess SDS was precipitated at 4°C and then removed by centrifugation. Lectin affinity chromatography. Radiolabeled glycopeptides were fractionated on columns of concanavalin A (Con A)-Sepharose and RCA-I-agarose as described previously ( 16). The intact glycoprotein gp160 was electroluted from a gel as described above, and Triton X-100 was added to a final concentration of 0.1%. The electroeluted protein was applied to a 2-ml column of RCA-I-agarose in phosphate-buffered saline ( PBS-NaNs, 6.7 mM KH,PO,, 150 mM NaCl, 0.02% NaN,) containing 0.1% Triton X-100. Because the glycoprotein bound so tightly that it could not be efficiently eluted with lactose, the resin was removed from the column, mixed with an equal volume of scintillation fluor, and counted directly in a liquid scintillation counter. The CNBr-produced, electroeluted glycopeptides from gp160 and gp120 were applied to a l-ml column (0.3 X 15 cm) of RCA-I-agarose in Tris-buffered saline (TBS-NaN,, 0.01 M Tris, pH 8.0, 0.15 M NaCl, 1 mM CaCl,, 1 mM MgCl,, and 0.02% NaN3) as described previously. After addition of 10 ml TBS and collection of l-ml fractions, the resin was transferred to a tube, and 1 ml of boiling TBS containing 0.5 M lactose was added. This mixture was then heated at 1OO’C for 5 min and transferred to a column (0.8 X 4 cm), and the elution buffer was collected. Elution was continued by batchwise addition of l-ml aliquots of 1OO’C TBS containing 0.5 M lactose.

250

MERKLE

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Treatment of glycopeptide by neuraminidase. Glycopeptides were dried by evaporation under reduced pressure and suspended in 49 al of 0.1 M sodium acetate buffer (pH 4.5). A. ureufaciens neuraminidase ( 10 mU) was added and the mixture was incubated at 37°C for 16 h. Release of monosaccharides by acid hydrolysis. [ 3H] Galactoseand [ sH] glucosamine-labeled glycopeptides were treated with 4 M HCl for 4 h at 1OO’C to hydrolyze glycosidic linkages and liberate monosaccharides. [sH] Mannose-labeled glycopeptides were treated with 2 N HCl for 2 h at 100°C to liberate monosaccharides. Descendingpaper chromatography. The monosaccharides mannose and fucose were separated by descending paper chromatography in the solvent system ethyl acetate:pyridine:water (8:2:1) for 24 h as previously described ( 17). The released sialic acid from neuraminidase digestion was separated by descending paper chromatography in the solvent system ethyl acetate:pyridine:glacial acetic acidwater (5:5:1:3) for 16 h, as described previously ( 17). GlcNAc and GalNAc were separated by descending paper chromatography using borate-impregnated Whatman No. 1 filter paper in the solvent system n -butyl alcohol:pyridine:water (6:4:3) ( 17). The distribution of radioactivity on the chromatograms was assayed by liquid scintillation counting of l-cm sections of the paper. N-Glycanase treatment of gp120. [ 3H] Glucosamine-labeled viral envelope glycoproteins were immunoprecipitated and subjected to SDSPAGE as described above. gp120 was identified by fluorography, excised from the dried gel, and hydrated for 30 min in 250 gl of 50 mM sodium phosphate buffer, pH 7.5. N-Glycanase (1 unit) was added to the tube, and it was incubated at 37’C under a toluene atmosphere for 24 h. A total of 2 more units of N-glycanase was added in two aliquots at 24-h intervals. The incubation mixture was then heated at 100°C for 5 min. The liquid was removed, and the glycoprotein remaining in the gel piece was subjected to acid hydrolysis in 4 N HCl and reacetylation as described previously ( 17). Alditol acetate derivatives of both the reacetylated monosaccharides and authentic, nonradiolabeled GlcNAc and GalNac were prepared as described by York et al. ( 18). Gas chromatography. The alditol acetate derivatives of the [ 3H ] glucosamine-labeled gp120 were analyzed by gas chromatography. Analysis was performed on a Hewlett-Packard Model 5710A gas chromatograph using a fused-silica DB-1 column (J & W Scientific) at an initial temperature of 175’C. After 2 min, the temperature was increased at a rate of B”C/min to a final temperature of 25O’C. The carrier gas used was helium at a flow rate of 1.5 ml/min. The radiolabeled amino sugar derivatives were collected manually by condensation into chilled (-20°C) scintillation vials using a flow adaptor with the flame ionization detector on during the collection. Sample collection was begun 15 min after injection, and samples were collected every 10 s for 5 min.

RESULTS

Radiolabeling of viral glycoproteins in Molt-3 cells infected with HTLVIII,. Molt-3 cells infected with HTLV IIIa synthesize gp160, gp120, and gp41, which are immunoprecipitated by human antisera to HIV. These glycoproteins can be metabolically radiolabeled over 24-36 h with either [ 35S]cysteine (Fig. 1A) or the monosaccharide precursor [ 3H] glucosamine, [ 3H] mannose, or [ 3H] galactose. The observation that gp160 was radiolabeled with [ 3H] galactose (Fig. 1B) led us to examine the possibility that its oligosaccharides are processed and terminally glycosylated. Demonstration that gp160 glycopeptides contain complex-type and high-mannoselhybrid-type Asn-linked oligosaccharides. Glycopeptides were prepared from the [ 3H] glucosamine, [ 3H] galactose, and [ 3H] mannose metabolically radiolabeled gp160, gp120, and gp41 and applied to a column of concanavalin A-Sepharose. It is

gpl60gp120-

p66 -

-

gPlm gp120

-

gP41

p55 p51 gP4’-

FIG. 1. Electrophoresis of immunoprecipitated viral proteins from metabolically radiolabeled Molt-3/HTLV 111s cells. Persistently infected Molt III cells were metabolically radiolabeled with [ %] cysteine (A) or [ 3H] galactose (B) as described under Experimental Procedures. After solubilization of the cells, viral proteins were immunoprecipitated and subjected to electrophoresis on SDS-polyacrylamide gels under reducing conditions, and the radiolabeled proteins were visualized by fluorography. Lane 1, cell-derived proteins; lane 2, viral-derived proteins; lane 3, viral isolation supernatant-derived protein. The positions of the viral proteins (~66, ~55, and ~51) and glycoproteins (gp160, gp120, and gp41) are indicated by lines.

known that this lectin interacts strongly with high-mannose/hybrid-type Asn-linked oligosaccharides, which require 100 mM a-methylmannoside for elution; interacts less strongly with biantennary complex-type Asn-linked oligosaccharides, which require 10 mM a-methylmannoside for elution; and does not interact appreciably with either tri- and tetraantennary or bisected biantennary complex-type Asn-linked oligosaccharides; it also does not bind mucin-type oligosaccharides ( 19-21). Seventy to 75% of the [ 3H] mannose-labeled glycopeptides from gp160 and gp120 behaved as high-mannoselhybrid-type Asn-linked oligosaccharides on this column, whereas 15 20% were not bound (Figs. 2A, 2B). In contrast, 60-65% of the [ 3H] glucosamine-labeled glycopeptides from gp160 and gp120 were not bound by Con A-Sepharose (Figs. 2D, 2E). For [ 3H] mannose-labeled gp41, 38% of the radioactivity was not bound to the immobilized lectin (Fig. 2C) . For gp41, a majority (86% ) of the [ 3H] glucosaminelabeled glycopeptides were not bound by Con A-Sepharose (Fig. 2F). These results indicate strongly that gp160, gpl20, and gp41 contain both complex-type and highmannose / hybrid-type Asn-linked oligosaccharides. Greater than 95% of the [ 3H] galactose-labeled glycopeptides from gp160 and gp120 were not bound by Con ASepharose (Figs. 2G, 2H). The lack of [ 3H] galactose in the glycopeptides bound by Con A-Sepharose suggests that there are only low levels of hybrid-type Asn-linked oligosaccharides.

COMPLEX-TYPE

OLIGOSACCHARIDES

ON

HIV-I

251

gp160

3 H-Glucosamlne

3 H-Mannose

3H-Galactose 100

500 4w

!3P 160

52 Y

2

‘I-

SO

-

300

c

-

200 -

G

gp160

A

i

50

t 100 -

t

\1/

60 60

ii

-

Y (?

5

Y (?E

5 7 E

40 20 * 0

0 0

10

20

30

300 SP

B

120

200 -

0

10

20

300 c3P 120

300

30

H

LIP 120

200 200

loo

100 4

0 0

IO

20

0

30

10

3cor gP

.

9P

C

41

2w

50 -

F

41

-

100 -

l

. 0

0

30

t

100 -

0

20

10

20

30

0

10

20

30

FRACTION FIG. 2. Chromatography on Con A-Sepharose of [“HI mannose-, [3H]gIucosamine-, and [3H] galactose-labeled glycopeptides gp160, gp120, and gp41. Infected cells were metabolically radiolabeled with [ 3H] mannose (A-C), [ 3H] glucosamine (D-F), and (G, H), and viral glycoproteins were isolated by immunoprecipitation and SDS-PAGE as described under Experimental Procedures. were prepared by treatment of the radiolabeled glycoproteins with Pronase and directly applied to columns of Con A-Sepharose. Bound were eluted sequentially with 10 mM oc-methylglucoside and 100 mM a-methylmannoside as indicated by the arrows. Portions of were subjected to liquid scintillation counting, and the profile of radioactivity is shown for each column.

Because [ 3H] mannose can be metabolized by cells to [ 3H] fucose, it is possible to assess the degree of fucosylation of the viral glycopeptides. The [3H]mannose-labeled glycopeptides derived from both gp160 and gpI20 that were not bound by Con A-Sepharose were hydrolyzed and the radiolabeled monosaccharides were recovered and analyzed by descending paper chromatography. Approximately one-quarter of the radioactivity was recovered as [ 3H] fucose and the remainder as [ 3H] mannose (data not shown). The recovery of [ 3H] fucose in glycopeptides from both gp160 and gp120 and the behavior of these glycopeptides on Con A-Sepharose are consistent with the idea that they contain complex-type Asn-linked oligosaccharides. It is typically found that complex-type Asn-linked

derived from [ 3H] galactose Glycopeptides glycopeptides each fraction

oligosaccharides have three mannose residues and have one fucose residue attached to GlcNAc in the chitobiosyl core. The fact that glycopeptides from gp160 are fucosylated to the same extent as those from gp120 makes it likely that the oligosaccharides on gp160 are highly processed. Demonstration that glycopeptides from gp160 contain galactosyl and sialyl residues. To look for the presence

of terminal galactose on the viral glycopeptides, the [ 3H] mannose-labeled glycopeptides from both gp160 and gp120 not bound by Con A-Sepharose were passed over columns of RCA-I-agarose. This lectin binds with high affinity to oligosaccharides containing terminal 61-4 galactosyl linkages and elution requires the addition of 100

252

MERKLE

ET

AL.

mM lactose ( 22 ) . Approximately

40% of the radioactivity gp120 contains O-linked oligosacchrides. We considin glycopeptides from both gp160 and gp120 was bound ered the possibility that in addition to Asn-linked oligoby RCA-I-Sepharose (Figs. 3A, 3C). Because the presence saccharides, gp120 might also contain O-linked oligosacof terminal sialic acid can block the binding of glycopep- charides, a common constituent of which is GalNAc linked tides to RCA-I-agarose, the glycopeptides were treated to peptide. Animal cells are able to efficiently metabolize with neuraminidase and then applied to the immobilized [ 3H] glucosamine to GlcNAc, GalNAc, and sialic acid. lectin. After neuraminidase treatment the amount of Direct hydrolysis of [ 3H] glucosamine-labeled gp120 gen[ 3H] mannose-labeled glycopeptides that bound to RCAerated large amounts of [ 3H] GlcNAc and a small amount I-agarose increased to approximately 60% for both gp160 of [ 3H] GalNAc, as evidenced by descending paper chromatography (data not shown). Because gpl20 contains and gp120 (Figs. 3B, 3D). These results demonstrate that gp160 and gp120 contain glycopeptides with both terminal greater than 20 N-linked oligosaccharides which, in total, /3-galactosyl residues and P-galactosyl residues penultihave a large number of GlcNAc residues, it could make mate to sialic acid. the identification of GalNAc residues problematic if there To directly investigate the possibility that gp160 con- were only a few GalNAc residuespresent. To remove most tains sialic acid, the [ 3H] glucosamine-labeled glycopep- of the Asn-linked oligosaccharides and thereby enrich our sample for O-linked oligosaccharides, we treated the tides from gp160 that did not bind to Con A-Sepharose [ 3H] glucosamine-labeled gp120 with N-glycanase. After were treated with neuraminidase and the treated glycothis treatment, which released 91% of the radiolabel, the peptides were analyzed by descending paper chromatogresidual glycoprotein was hydrolyzed in strong acid and raphy. It is well known that [3H]glucosamine is a prethe released sugars were reacetylated and then were sepcursor in animal cells to N-acetylglucosamine, Nacetylgalactosamine, and sialic acid. Approximately 10% arated both by descending paper chromatography on boof the [ 3H] glucosamine-derived radioactivity in gp160 rate-impregnated paper and by gas chromatography. The GC analysis of this sample demonstrates that the majority glycopeptides was released by neuraminidase treatment and identified by its chromatographic mobility as sialic of the radioactivity recovered from this N-glycanaseacid (data not shown). treated gp120 was GalNAc (data not shown). This hy-

After

Neuraminidase

Treatment

140

A

c3P120

cJP120

120

100

-

100 80 60

25

40

20

10

After 120. 100

Neuraminidase

Treatment

120 . -

C

w160

0

10

100

. -

so

-

60

_

20

0

D

a'160

40%

10

20

FRACTION FIG. 3. Chromatography on treatment. [ 3H] Mannose-labeled to a column of RCA-I-agarose glycopeptides were eluted with and the profile of radioactivity

RCA-I-agarose of [3H]mannose-labeled glycopeptides from gp160 and gp120 before and after neuraminidase glycopeptides derived from gp120 and gp160 that were not bound by Con A-Sepharose were desalted and applied before (A, C) or after neuraminidase treatment (B, D) as described under Experimental Procedures. Bound 0.1 M lactose as indicated by the arrow. Portions of each fraction were subjected to liquid scintillation counting, is shown for each column.

COMPLEX-TYPE

OLIGOSACCHARIDES

drolyzed material was also subjected to descending paper chromatography and GalNAc and GlcNAc were observed (data not shown). Virtually all molecules of gp160 contain complex-type Asn-linked oligosaccharides. Taken together the above results demonstrate that gp160 and gp120 synthesized by Molt-S/HTLV 111s cells contain both complex-type and high-mannose-type Asn-linked oligosaccharides. The results above do not clearly indicate, however, the percentage of gp160 molecules that contain complex-type oligosaccharides. For this estimation the [ 35S] cysteine-labeled gp160 synthesized by the Molt-3/HTLV IIIa cells was electroeluted directly from gels following SDS-PAGE and fluorography. The radiolabeled glycoprotein was treated with neuraminidase and passed directly over a column of RCA-I-agarose (Fig. 4). All of the applied radioactivity was bound by the immobilized lectin and could not be released by elution either with the hapten 0.1 M lactose or with low pH buffer. A portion of the column was mixed with scintillation fluor and counted directly, and all applied radioactivity was recovered. Considering that gp160 probably contains several dozen Asn-linked oligosaccharides and that a high percentage of these are complextype Asn-linked oligosaccharides interactive with RCAI-agarose (see Fig. 3)) it is not surprising that intact gp160 binds with such high avidity to RCA-I-Sepharose and that it cannot be easily dissociated. As a control, [ 35S] cysteine-labeled gp160 was applied to the RCA-Iagarose column in the presence of 0.5 M lactose. Under this condition none of the radiolabel bound, indicating that the interaction with RCA-I-agarose was dependent on attached carbohydrate. Pulse-chase analysis of glycosylution status of gpl60 and gp120. In the above experiments the oligosaccharides of the envelope precursor and product were analyzed from gp160 and gpl20 that had been produced in cells that were equilibrium-labeled for a 24-h period. To establish kinetically that proteolytic cleavage of the precursor gp160 occurs after terminal processing of its oligosaccharides, we analyzed the glycosylation of both the envelope precursor and its product synthesized during a pulse-labeling experiment. Molt-3 cells were pulse-labeled with [35S]cysteine for 10 min and then chased in unlabeled medium for varying amounts of time as described under Experimental Procedures. As shown in Fig. 5A, gp160 is detectably radiolabeled at the end of the lo-min pulse (0 time of chase), and declined in amount throughout the 16-h chase period. The individual lanes of the gel shown in Fig. 5A were scanned for radioactivity as described under Experimental Procedures. Since the total amount of radioactivity recovered differed in the samples at each chase point, the results were expressed as a change in the percentage distribution of radioactivity between the precursor gp160 and its product gp120 throughout the chase period (Fig. 5B). Although visually detectable at 1 h of chase time, gp120 was first detectably radiolabeled by gel

ON

5 k

400

-

200

-

0-------0

HIV-I

100% 253

gp160

4

10

20

FRACTION FIG. 4. Chromatography of intact [35S]cysteine-labeled gp160 on RCA-I-agarose. [ %S] Cysteine-labeled gp160 was electroeluted from an SDS-polyacrylamide gel and applied to a column (3 ml) of RCA-Iagarose as described under Experimental Procedures. After elution of the column with 0.2 M lactose the column was unpacked and the resin was directly subjected to liquid scintillation counting.

scanning at the 2-h time of chase, and increased throughout the 16-h chase period with a half-time of occurrence at between 5 and 6 h (Figs. 5A and 5B). In results not shown, radiolabeled gp120 was recovered in the media of this experiment, beginning at the 5.5-h chase point, thus indicating that most of the gp160 was efficiently processed to cell- and viral-associated gp120. This, therefore, accounts for the decrease in the intensity of the radiolabeled bands in the lanes during the chase period (Fig. 5A). To examine the glycosylation status of these proteins during the course of the pulse-chase, we determined the interaction of glycopeptides derived from gp160 and gp120 with immobilized RCA-I-agarose. CNBr cleavage was used to generate a large, water-soluble glycopeptide fragment containing the majority of the glycosylation sites of the envelope proteins. A pulse-chase experiment similar to the one described above was performed, and bands corresponding to gp160 at chase times of 0 and 16 h and bands corresponding to gp120 at 2 and 16 h were excised from the gel and digested with CNBr. Approximately 50% of the radioactivity was released by treatment with CNBr. Based on the published amino acid sequence of gp160/ gp120, CNBr cleavage results in the production of 10 fragments, 1 of which is a large, 322-amino-acid fragment (Met 103-Met 425) containing 16 glycosylation sites that would not be expected to be released from the gel matrix. This large fragment was therefore released from the gel by electroelution. After removal of glycine and excess SDS from the electroeluted glycopeptides, they were applied to a column of RCA-I-agarose (Fig. 6). After the pulse, 59% of the radioactivity of the electroeluted glycopeptides from gp160 bound to RCA-I-agarose, and after 16 h of chase this binding increased to 80% (Figs. 6A, 6B). In contrast, greater than 80% of the radioactivity from gpl20 glycopeptides at both the 2-h and the 16-h chase times

254

MERKLE

SIP160 gPla

ET

AL.

c’

4’

l’

.’

4’

PB

-

gP1w

-a--

gpml

Chase Time (h) FIG. 5. Electrophoresis of immunoprecipitated viral proteins from pulse-chase metabolically radiolabeled Molt-3/HTLV 111s cells. Persistently infected Molt-3 cells were radiolabeled for 10 min with [?S]cysteine, and then chased in complete medium for the time periods indicated as described under Experimental Procedures. (A) After solubilization of the cells, viral proteins were immunoprecipitated and subjected to electrophoresis on SDS-polyacrylamide gels under reducing conditions and the radiolabeled proteins were visualized by fluorography. (B) The dried gel was scanned for radioactivity, and the relative percentages of immunoprecipitated radioactivity in gp160 and gp120 are plotted with respect to chase time.

bound to RCA-I-agarose (Figs. 6B, 6C). Since the radiolabel used in these experiments, [ 35S]cysteine, labels the protein moiety, the unbound radiolabeled material may represent nonglycosylated peptides. These results demonstrate that, at its earliest detectable appearance, gp120 contains complex-type N-linked oligosaccharides. DISCUSSION

Our results demonstrate that both gp160 and gp120 acquire complex-type Asn-linked oligosaccharides and retain some high-mannose-type Asn-linked oligosaccharides. Although our studies have focused on processing of the Asn-linked oligosaccharides that are sensitive to Nglycanase, we obtained evidence for the occurrence of Nglycanase-resistant oligosaccharides containing GalNAc, a typical constituent of O-linked oligosaccharides (23). In contrast, Kozarsky et al. ( 12) found that recombinant gp160 synthesized by Chinese hamster ovary cells lacks detectable GalNAc. These results might indicate that Oglycosylation of gp120 could be cell-type specific. Recently, Hansen et al. (24) reported that a monoclonal antibody reactive with a carbohydrate epitope commonly found in O-linked oligosaccharides was reactive with gp120 produced by infected MT-4 cells, a human CD4+ T-cell line. Stein and Engleman (7) found that N-glycanase was unable to remove all the sialic acid-containing oligosaccharides from gp120, also suggesting that gp120 might contain O-linked oligosaccharides. Our results directly demonstrate the presence of GalNAc in N-glycanase-resistant oligosaccharides on gp120.

With regard to the Asn-linked oligosaccharides, it is known that both correct glycosylation and oligosaccharide processing of gp160 are essential for its exit from the ER/ Golgi complex and for maturation and cleavage to gp120 and gp41. Gp160 is synthesized as a glycoprotein containing high-mannose-type Asn-linked oligosaccharides fully susceptible to endo H (7, 11, 25). Glycosidase inhibitors which interfere with glucose removal from oligosaccharides in the ER block the exit of gp160 from the ER and impair the functional activity of the envelope glycoproteins ( 10,26, 27), whereas inhibitors that interfere with mannosidases in the Golgi complex do not block proteolytic cleavage (11). Although gp160 itself can interact with CD4 (28, 29), cleavage of gp160 is essential for producing infective HIV-I (30). The proteolytic cleavage of gp160 in some cell types other than Molt-3 may not be an efficient process since only a small fraction of gp160 is converted to gp12O/gp41 and most of the gp160 is degraded intracellularly (6 ) . In other cell types it appears that a significant amount of gp160 is secreted in some form (28). In CHO cell lines transfected with the envelope gene of HIV-I a gp160 that is not processed, cleaved, or exported is produced (31) . Although the precise mechanism is not known, the intracellular cleavage of gp160 to gp120 and gp41 in some cell types occurs via an acid-dependent proteolysis event that is inhibitable by treating cells with NH&l (6). Other studies have shown that the proteolytic cleavage is effected by a non-acid-dependent protease in other cell types (7).

COMPLEX-TYPE

OLIGOSACCHARIDES

ON gpl60-16h

1~160 - 0 h chase

HIV-I

255

gp160

B

60

u

200.

gp120

- 2h

C

D 60

150 -

FRACTION FIG. 6. Chromatography on RCA-I-agarose of CNBr-produced glycopeptides from [ %] cysteine-labeled gp160 and gp120 at different pulsechase times. Gel pieces containing gp160 and gp120 that had been pulse-labeled with [ %] cysteine and chased for the indicated amounts of time were treated with CNBr as described under Experimental Procedures. The large glycopeptide remaining in the gel after digestion was electroeluted and applied to a column of RCA-I-agarose and eluted with 0.5 M lactose as indicated by the arrows. The top two panels (A, 8) show the chromatography of gpl60-derived glycopeptides at 0- and 16-h chase times, respectively. The bottom panels (C, D) show the chromatography of gpl20-derived glycopeptides at 2- and 16-h chase times, respectively.

Because of the many studies showing that intracellular forms of gp160 contain predominantly high-mannose-type Asn-linked oligosaccharides, it has been concluded that the proteolytic cleavage of gp160 occurs in a compartment proximal to the terminal oligosaccharide processing reactions in the Golgi apparatus (7, 11). Our results demonstrate, however, that the bulk of the gp160 in Molt-3/ HTLV-111s cells is in a form containing complex-type Asn-linked oligosaccharides. The reasons for these different observations are not certain but may lie in the nature of the unusual processing stages of gp160 within infected human T cells and in potential cell-type-dependent differences in the steps and rates of proteolytic cleavage and oligosaccharide processing. There is evidence that some retroviral envelope precursors, such as the envelope glycoprotein of avian reticuloendotheliosis virus and the influenza envelope precursor, HA,, are proteolytically cleaved after terminal glycosylation is completed (32, 33). However, there is evidence for other viral envelope precursors, such as the murine leukemia virus and the Rous sarcoma virus, that they are probably cleaved prior to terminal glycosylation (34-36). Whether there are specific cell-type-dependent differences in these proteolytic cleavage reactions versus oligosaccharide processing is not known.

The biosynthesis of gp160 and its conversion to gp120 are exceedingly slow in persistently infected Molt-3 cells, as evidenced by pulse-chase kinetics (Fig. 5 ) ; gp120 is not detectably radiolabeled until 2 h of chase time. The carbohydrate processing of gp160 must occur rapidly since the form appearing at the end of the lo-min pulse-labeling period already contained some complex-type Asn-linked oligosaccharides. In other cell types it appears that it can take several hours for gp160 with its high-mannose-type Asn-linked oligosaccharides intact to exit the endoplasmic reticulum (6, 7). The,results of our studies suggest the scheme shown in Fig. 7 for the possible glycosylation and processing pathway of the gp160 within Molt-3 cells. The precursor gp160 with only high-mannose-type chains is synthesized in the ER and exits to the Golgi complex. Within the Golgi complex its oligosaccharides are probably rapidly processed by mannosidase I and II and then terminally glycosylated by the addition of fucose, N-acetylglucosamine, galactose, and sialic acid. Our studies unequivocally reveal the presence of this fucosylated, sialylated, and galactosylated gp160 intermediate within persistently infected human T cells (Figs. l-3,5,6). Within the Golgi complex or in some other distal intracellular compartment, cell-derived proteases cleave the mature gp160 to

256

MERKLE

w160

gp120 LIP41 complex chains

high mannose chains

xi

ET

AL.

Thus, the rates for the steps shown in Fig. 7 may be dissimilar among different cell lines and this could result in distinct steady-state levels of the intermediates. Clearly, further experimentation will be required to understand the precise regulation of proteolytic cleavage and oligosaccharide processing of the viral envelope glycoproteins in different infected human T cells and cell lines.

proteolysis ACKNOWLEDGMENTS gp120 gP41 high mannose chains FIGURE

x

7

generate the final proteolytic products gp120 and gp41 as predicted by Willey et al. (6). Such a series of events has been postulated for the 150-kDa envelope glycoprotein precursor in the human immunodeficiency virus HIV-2 (37, 38). It could be predicted from the pathway shown in Fig. 7 that interference with proteolytic cleavage might cause the accumulation of the gp160 intermediate containing complex-type Asn-linked oligosaccharides. In support of this possibility, Guo et al. (39) analyzed the forms of gp160 accumulating in cells expressing a mutant envelope protein that cannot be proteolytically cleaved. They observed that endo H treatment of the mutant gave rise to heterogeneous forms of the glycoprotein, whereas treatment of the wild-type gp160 gave more uniform products. They suggestedthat this increased heterogeneity of the mutant gp160 might be due to oligosaccharide processing. In addition, when it was expressed in CHO cells, a chimeric gp160, in which the transmembrane domain and cytoplasmic tail were replaced with the corresponding sequences from herpes simplex virus, was found to be proteolytically cleaved within the Golgi complex after oligosaccharide processing (40). In contrast to our results, Stein and Engleman (7) reported that gp160 lacks fucose and that gp120 from infected VB cells contains fucose. However, they also reported that gp160 is partially sialylated based on its sensitivity to neuraminidase, which is consistent with our observation. In their experiments they incubated VB cells infected with the B-LAV isolate of HIV-I for 7 h with [ 3H] fucose and then analyzed the degree of fucosylation by fluorography of immunoprecipitated gp160 and gp120 after SDS-PAGE. The fluorographs indicated that only gp120 was radiolabeled. This variance may be due to several factors, including differences in cell lines resulting in different steady-state levels of the terminally glycosylated gp160 in VB relative to Molt-3 cells, different analytical approaches (i.e., fluorography versus quantitative isolation and characterization of incorporated monosaccharides), and cell-type-dependent differences between cells in the cellular proteases responsible for the cleavage.

This work was supported by NIH Program Project Grant NIH AI 27135. The authors thank Drs. Joseph Sodroski, Peter Albersheim, Alan Darvill, Herman van Halbeek, Gerald Beltz, and Jonathan Seals for advice during the course of this investigation. We thank Dr. Pedro Prieto for assistance with the gel scanner and Karen Howard for help with the preparation of the manuscript.

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