n-linked glycosylation and reticuloendotheliosis retrovirus envelope glycoprotein function

VIROLOGY 179, 648-657 (1990)

N-Linked Glycosylation and Reticuloendotheliosis Retrovirus Envelope Glycoprotein Function

ERIC L. DELWART AND ANTONITO T. PANGANIBAN’

McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin 53706

Received April 12, 1990; accepted July 27, 1990

Different properties of the spleen necrosis virus (SNV) envelope glycoprotein were analyzed following biosynthesis in the presence of glycosylation inhibitors. Tunicamycin, which inhibits all asparagine N-linked glycosylation, prevented intracellular processing and translocation to the cell surface of the envelope protein. In contrast, castanospermine or deoxymannojirimycin, which block glycosidase trimming of the early high-mannose chains and subsequent complex type N-glycosylation, did not inhibit proteolytic cleavage or cellular translocation. The ability of unglycosylated and partially glycosylated envelope protein to bind the viral receptor was assayed using an infection interference assay. Tunicamycin abrogated SNV envelope glycoprotein-induced receptor interference, whereas the trimming glycosidase inhibitors had no effect on interference. Similarly, tunicamycin but not the glycosidase inhibitors reduced the titers of released virus 1 OO-fold. We conclude that carbohydrate trimming and complex N-glycosylation are not essential for envelope glycoprotein translocation, proteolytic cleavage, receptor binding, or infectivity, whereas cotranslational high-mannose N-glycosylation is essential for all of the SNV envelope glycoprotein properties tested. Syncytia formation can be induced following transfection into Di 7 cells of an envelope glycoprotein expression plasmid. Unlike virus particle infectivity, cell fusion is strongly inhibited by the glycosidase inhibitors. o isso Academic press, IW.

INTRODUCTION

The envelope glycoprotein (env-gp) of spleen necrosis virus (SNV), a member of the type C avian reticuloendotheliosis virus group (REV) (Witter, 1984) shows a high degree of sequence similarity with the glycoproteins of type D simian retroviruses (SRV) and other members of that interference group (Sommerfelt and Weiss, 1990). For example, when the amino acid sequence of the SNV envelope gene (Kewalramani and Panganiban, unpublished data) is compared to that of the simian Mason-Pfizer retrovirus (MPMV) (Sonigo er a/., 1986) there is close alignment of potential N-glycosylation sites: seven of the eight SNV N-linked glycosylation sites are found within three amino acids of MPMV N-glycosylation sites. Furthermore, 21 of the 23 cys- teine residues of the SNV env-gp are also found in the corresponding position in MPMV.

The mature, proteolytically processed SNV surface envelope glycoprotein is highly modified, owing almost half of its molecular weight to N-linked carbohydrate groups. Analogous to other systems N-linked glycosylation begins concurrently with the translation of the viral envelope polypeptide chain into the endoplasmic reticulum (ER) (Hubbard and Ivatt, 1981; Pfeffer and Rothman, 1987). High-mannose sugar chains (Rob- bins et a/., 1977) are transferred to the canonical N-glycosylation acceptor sites [Asn-X-Ser(Thr)] on the na-

’ To whom requests for reprints should be addressed.

scent polypeptides (Rothman and Lodish, 1977; Glabe et al., 1980). The high-mannose N-linked glycans may then be trimmed during glycoprotein translocation to the cell surface by glycosidase enzymes residing in the ER and Golgi (Hubbard and Ivatt, 1981; Fuhrmann et a/., 1985). Glycosyl transferases then add different sugar molecules to the correctly trimmed carbohydrate core, resulting in large, complex side groups with multiple possible sugar configurations (Fuhrmann et a/., 1985).

The importance of cotranslational glycosylation for the proper function of some polypeptides has been demonstrated using the drug tunicamycin, which prevents N-linked glycosylation by inhibiting the synthesis of the sugar donor group (Gibson et a/., 1980; Elbein, 1987). The effects of later post-translational glycosylation modifications can be studied using transition state analogue inhibitors of the trimming glycosidases (Fuhr- mann et al., 1985). These inhibitors cause the carbohydrate side chains to remain in the untrimmed or partially trimmed high-mannose configurations which can be detected by their sensitivity to cleavage by endoglycosidase H.

In a previous study evidence was presented that expression of an SNV env-gp deletion mutant that is blocked in translocation in the ER could still confer resistance to superinfection (Delwart and Panganiban, 1989). The sugar groups of these mutant env-gp molecules were of the high-mannose, endo H-sensitive type, as expected of ER resident glycoproteins. This

0042-6822/90 53.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

648

N-LINKED GLYCOSYLATION AND env-gp FUNCTION 649

observation led to the proposal that retrovirus-induced resistance to superinfection may be due to the complexing of the cellular viral receptor with partially N-glycosylated wt env-gp in the ER. In order to determine whether the normal envelope protein might similarly be able to interact with the receptor in the absence of complex glycosylation, several env-gp functions were examined following biosynthesis in the presence of various glycosylation inhibitors. In this communication results consistent with intracellular receptor binding are described, indicating that complex N-glycosylation is dispensable for wt SNV env-gp-receptor recognition. The role of cotranslational high-mannose glycosylation in other env-gp functions and the requirement for complex N-glycosylation in cells undergoing SNV env-gp- induced syncytia formation are also analyzed.

MATERIALS AND METHODS

Plasmids

pED75 is identical to pSW253 (Chen et al., 1981) (a provirus clone of Rev-A) except that it contains an SNV substitution between a unique Xhol site and an Avrll site adjacent to the polypurine tract. This fragment [derived from pPB101 (Bandyopadhyay and Temin, 1984)] encodes all of the SNV envelope sequence except three carboxy-terminal amino acids. pED75 encodes a Rev-A/SNV chimera virus, which is a better helper for JD2 14Hy than Rev-A (data not shown). pJD214Hy was kindly provided by I. Dougherty (Dougherty and Temin, 1986). To construct the env protein expression construct pED27 the Sacll-to-Sac1 envelope gene fragment of pPB101 was treated with Sl nuclease, and HindIll linkers were ligated. This fragment was inserted into the HindIll site of pRSVcat (Gorman et a/., 1982) to give pED23. The region of the DNA that encodes the 3’ untranslated portion of the mRNA was shortened by deleting a Pvull-to-Hpal fragment to give pED24. The M/&to-&t1 fragment carrying the Rous sarcoma virus long terminal repeat, envelope gene, and poly(A) addition site from pED24 was treated with Klenow enzyme to generate a blunt-ended molecule and inserted into the similarly treatedxbal site of ~220.2 (kindly provided by Dr. B. Sugden) to give pED27. ~220.2 was derived from ~201 (Yates et a/., 1985) by insertion of a poly- linker sequence from pUC12 into the Narl site (J. L. Yates, personal communication). The orientation of the SNV env gene was the same as that of the EBNAl gene.

Cells, viruses, and assay for superinfection interference

The canine osteosarcoma cell line D17 (from the American Tissue Culture Collection, Rockville, MD),

SNV chronically infected D17 cells, and the previously described cell line 2.1 expressing the SNV env-gp were grown as previously described (Delwart and Pangani- ban, 1989). The REV stock was generated by cotrans- fecting chicken embryo fibroblast (CEF) by the polybrene/dimethyl sulfoxide technique (Kawai and Nishi- zawa, 1984) with 5 pg of pJD214Hy and 0.5 pg of pED75. Culture supernatant was collected 5 days later and frozen in aliquots at -70”. The assay for superinfection interference has been described previously (Delwart and Panganiban, 1989).

Glycosylation inhibitors

The same concentration of inhibitors was used in all experiments. Tunicamycin was from Sigma (T-7765) and was used at 1 pg/ml. Castanospermine and deoxymannojirimycin (dMM) were both from Boehringer- Mannheim and used at 0.66 and 1 mn/l respectively. Castanospermine and deoxymannojirimycin toxicity was measured by treatment of D17 cells for 3 days followed by trypsin digestion and passage of treated cells into normal cell media. Cloning efficiency was not affected by pretreatment with the trimming glycosidase inhibitors under the conditions used here (data not shown).

lmmunofluorescence and immunoprecipitation

lmmunofluorescence and immunoprecipitation were performed as described previously (Delwart and Pan- ganiban, 1989). Surface immunofluorescence staining was done by fixing the cells on a glass coverslip with 3% paraformaldehyde (pH 7.4, preheated at 37’) for 10 min at room temperature. Antibody reaction and rinses were done in the presence of 100 mM glycine. The immunofluorescence signals seen in Figs. 2A-H are representative of the fluorescence seen in multiple mi- croscopic fields seen in two experiments. Little varia- tion is seen in the cellular distribution of the envelope protein between cells treated with the same inhibitor.

Syncytia formation

D17 cells were plated at a density of 1 O6 tells/60- mm-diameter plates on polystyrene tissue culture dishes marked with measuring grids. One day later 10 pg of pED27 or ~220.2 plasmid was introduced into cells by transfection using the polybrene/DMSO technique (Kawai and Nishizawa, 1984). Trimming glycosidase inhibitors were added at the beginning of the 7-hr DNA/polybrene incubation period; after a 3.5-min 30% DMSO shock, media containing glycosylation inhibitors were added to the plates. Cells were incubated for 4 days and fixed with 0.5% violet blue in 50% methanol. Syncytia including at least four nuclei were counted by 156X magnification over an area of 2 cm’.

650 DELWART AND PANGANIBAN

A Deoxymann Castano Tunlcamycin ojlrlmycln spermine

A B A BA B Endo H + 1-l - I + I I

+, - 1 - ,+ +, - 1 - ,+

kibitor

A B

+ I- I-1 + kd

11 -22kd

FIG. 1. Biosynthesis and immunoprecipitation of [35S]methionrne-labeled putative SNV SU and TM envelope proteins. Cells of the D17 2.1 cell line expressing SNV env-gp were treated with tunicamycin for 24 hr or castanospermine and deoxymannojirimycin for 3 days prior to the addition of label. Inhibitor concentrations are given under Materials and Methods. Radiolabeling was done in the continued presence of inhibitors. Lanes A: 1 O6 cells were labeled for 1 hr and immediately immunoprecipitated. Lanes B: Cells were labeled for 1 hr and chased with cold media for 4 hr before immunoprecipitation. Endo H: Endoglycosidase H digestion of immunoprecipitated samples was done by overnight incubation with 0.01 U. (A) Samples were boiled in loading buffer and run on a 7% polyactylamide gel. (B) Same as (A) but samples were run on a 10% polyactylamide gel for analysis of the TM protein. The same immunoprecipitated samples were loaded on gels (A) and (B). Only a small section of the autoradiograph is shown in (B) since the remainder was identical to that of (A).

The glycosidase inhibitors did not affect transfection efficiency as determined by the number of hygromycin- resistant D17 clones obtained after polybrene/DMSO transfection with pJD214Hy.

RESULTS

Complex N-glycosylation of the SNV envelope protein is not required for proteolytic cleavage

A D17 cell line expressing the SNV env-gp (clone 2.1) (Delwart and Panganiban, 1989) was pretreated with tunicamycin for 24 hr or with castanospermine and dMM for 3 days. In the continued presence of inhibitors, cells were pulse-labeled for 1 hr with y5S]me- thionine and the label was then chased for 4 hr. Env-gp was immunoprecipitated using rabbit antisera raised against an Escherichia co/i synthesized envelope protein antigen. In untreated cells the initial translation product after a 1 -hr label was the endo H-sensitive precursor glycoprotein, gPr75 (Fig. 1A: no inhibitor A-,+) (Tsai et a/., 1986; Tsai and Orozslan, 1988; Delwart and Panganiban, 1989). After the 4-hr label chase the mature endo H-resistant surface gp90 (SU) and the transmembrane gp20 (TM) (Leis et al., 1988) could be detected by immunoprecipitation (Fig. 1A: no inhibitor B-,+, and Fig. 1B: no inhibitor). Glycoproteins gp90 and gp20 are the mature envelope product found on virus particles. Previous studies have shown that a short-lived, endo H-resistant gPr1 15 is the immediate precursor of gp90 and gp20. gPrl15 is derived from gPr75 following complex N-glycosylation and is endo H-resistant (Tsai and Orozslan, 1988; data not shown). The mature virion SU and TM glycoproteins are derived by proteolytic cleavage of gPr1 15 by a cellular endopeptidase.

When all N-linked glycosylation was blocked with tunicamycin, an endo H-insensitive protein of the same MW as enzymatically deglycosylated gPr75 (Fig. 1A:

tunicamycin A-,+) was synthesized. No envelope- specific label was detected following the label chase, indicating that the stability of the unglycosylated envelope protein was reduced (Fig. 1A: tunicamycin B).

Treatment of D17 2.1 cells with castanospermine, an inhibitor of glucosidase I (Fuhrman et al., 1985) resulted in the biosynthesis of an endo H-sensitive env-gp with a MW larger than that synthesized in the same time period without the inhibitor (Fig. 1A: castanospermine A-). This larger MW presumably results from the inhibition of ER resident glucosidase I, which in untreated cells removes the terminal glucose residue from the initial high-mannose chains and renders other sugar residues accessible to further trimming. The MW of the initial translation product in cells treated with dMM was lower than that in cells treated with castanospermine (Fig. 1A: deoxymannojirimycin A-, castanospermine A-). This result is consistent with glucosidase trimming activity and the absence of subsequent complex glycosylation due to the inhibition of mannosidases with dMM.

Endo H deglycosylation of the major early translation products from untreated cells or from cells treated with either castanospermine or deoxymannojirimycin yielded products of the same MW as the unglycosylated product synthesized in the presence of tunicamy- tin (Fig. 1A: tunicamycin A, no inhibitor A+, castanospermine A+, deoxymannojirimycin A+). This result indicates that the differences in the major early translation products are due to different forms of endo H-sensitive high-mannose groups. The smaller “ladder-like” bands are derived from endo H digestion of the chase products (Fig. 1 A: castanospermine B- and B+, deoxymannojirimycin B- and B+). Following the 4-hr label chase of castanospermine-treated cells, two envelope peptides of similar MW become more appar- ent (Fig. 1A: castanospermine B-). Like the normally synthesized gp90 (released by cleavage of gPr1 15)


(Tsai and Orozslan, 1988) these two glycoproteins have a MW approximately 20 kDa smaller than the high-MW initial translation products synthesized in the presence of castanospermine. This change in MW could have resulted from the proteolytic cleavage of the untrimmed initial translation product to release the TM and SU. To determine whether TM proteins were generated in the presence of the glycosidase inhibitors, the immunoprecipitation products were examined under gel conditions suitable for resolution of the TM peptides (Fig. 1 B). From castanospermine-treated cells a transmembrane glycoprotein doublet of approximately 22 kDa could indeed be immunoprecipitated (Fig. 1 B: castanospermine). The env-gp chase pattern observed with dMM-treated cells was the same as that observed for castanospermine-treated cells. The ap- parent MWs of the SNV env-gps synthesized with dMM were smaller than those of the env-gps synthesized with castanospermine, as expected from the removal of the outer glucose residues from the high- mannose chains in the presence of dMM (Fig. 1A). The label chase patterns, the appearance of a major band approximately 20 kDa (the size of the TM) smaller than the major initial translation product, and the appearance of TM glycoproteins lead us to believe that proteolytic cleavage of SNV env-gp can take place in the absence of complex N-glycosylation.

After a labeling time of only 1 hr large levels of putative SU and TM are detected in cells treated with glycosidase inhibitors, whereas at this time they are mostly absent from untreated cells (Fig. 1A: deoxymannojirimycin A-, castanospermine A-, no inhibitor, A-; Fig. 1 B: deoxymannojirimycin A, castanospermine A, no inhibitor, A). This may indicate that translocation of the cleavage precursor to the cellular site of proteolytic processing takes place faster for the SNV env-gp-bearing high-mannose sugars than for the more heavily modified gPR1 15. It may also be that partially N-glycosylated env-gp is a better substrate for proteolytic cleavage.

The high-mannose chains found on the initial translation products were all efficiently cleaved by endo H, yielding a single sharp band of the same MW (Fig. 1A: A+). This is unlike the endo H digestion pattern of the putative SU glycoprotein synthesized in the presence of trimming glycosidase inhibitors, which instead of yielding a single band produced a ladder-like series of bands (Fig. 1 A: deoxymannojirimycin B+, castanospermine B+). Such a ladder-like series of glycoproteins may represent modification of some of the untrimmed (castanospermine) or partially trimmed (dMM) high-mannose chains that may occur during glycoprotein translocation to the cell surface. Such aberrant oligosaccharide modification could account for the partial resistance to endo H digestion of the putative

SU. Nonetheless, the large reduction in the MW of the putative SNV SU proteins synthesized in the presence of either trimming glycosidase inhibitor (Fig. 1A: B-) versus the MW of gp90 attests to the efficiency of these inhibitors in preventing proper complex, high- MW N-glycosylation in D17 cells.

Cellular distribution of envelope glycoprotein in the presence of N-glycosylation inhibitors

Cells expressing SNV env-gp were treated with tunicamycin for 24 hr or with castanospermine and dMM for 3 days. These cells were then analyzed by immunofluorescence labeling using anti-SNV envelope protein antibodies (Fig. 2). The distribution of fluorescence seen in permeabilized cells was largely unaffected by the trimming glycosidase inhibitors (Figs. 2A, E, G). As in untreated cells the env-gp antigen was found mostly in the Golgi and the plasma membrane (Delwar-t and Panganiban, 1989). In contrast, tunicamycin treatment resulted in the distribution of envelope protein in vesi- cles distributed through the cytoplasm with some concentration in a Golgi-like perinuclear location (Fig. 2C).

We next examined the plasma membrane distribution of the env-gp by immunofluorescence analysis of paraformaldehyde-fixed cells. Tunicamycin-treated cells were negative for envelope antigen on their surface (Fig. 2D) with the exception of a subpopulation comprising about 1% of the cells. These apparently tunicamycin-insensitive cells appeared brightly positive (Fig. 21). Cells treated with the trimming glycosidase inhibitors were positive for surface env-gp but the fluorescence signal intensity was slightly weaker than that seen on untreated cells (Figs. 2B, F, H). This immunofluorescence analysis shows that tunicamycin treatment blocked the translocation of unglycosylated envelope protein to the cell surface. On the other hand, blocking complex N-linked glycosylation did not abol- ish SNV env-gp translocation to the cell surface.

Complex glycosylation of the envelope glycoprotein is not required for superinfection interference

Chronically infected D17 cells and a cell line expressing the SNV env-gp are resistant to infection by viruses of the same interference group (Delwat-t and Panganiban, 1989). This phenomenon presumably results from the complexing of the cellular viral receptor with the endogenously synthesized SNV env-gp (Steck and Rubin, 1966a,b). This specific binding is thought to result in superinfection interference due to the reduction of functional virus receptor on the cell surface. In this model the ability of endogenously synthesized

FIG. 2. lmmunofluorescence staining of the SNV envelope protein. Effect of glycosylation Inhibitors on cellular distribution of SNV envelope proteins and pED27-induced syncytium. (A-H) immunofluorescent staining of the 2.1 D17 cell line expressing SNV env-gp (magnification X586). Left-hand pictures and J show the staining pattern following permeabilization of cells with methanol; the right-hand prctures (except J) depict surface staining following paraformaldehyde membrane fixing. (A, B) No inhibitor; (C, D) tunrcamycin; (E, F) castanospermine; (G. H) dMM; (I) tunicamycin on cells chronically infected with SNV (magnification X 186); (J) D17 cell syncytrum induced by SNV env-gp expression.

652


TABLE 1

RESISTANCE TO REV INFECTION AFTER TREATMENT WITH DIFFERENT N-GLYCOSYLATION INHIBITORS

Relative superinfectiona

Inhibitorb

None Tunicamycin Castanospermrne dMM

017 D17(SNV)’ D17(2.1)d

1 1 x 1o-4 3 x 1o-4 0.3 0.12 0.28 1 4 x 1om4 6 x 1O-4 1 1 x 1o-4 3 x 1o-4

’ Relatrve supennfection IS normalized to the level of infection ex- habited by D17 cells infected in parallel. A value of 1 .O indicates 0.8 X lo6 Hyg’ colony transforming units/ml. Infections were done in duplrcate to obtain the average titer. Infections were done twice and the average values are presented.

b Inhibitor concentrations used are given under Materials and Methods. Cells were pretreated wrth tunicamycin for 24 hr and with castanospermrne and dMM for 3 days. Cells were kept in the presence of castanospermine and dMM for 24 hr following Infection.

’ D17 (SNV) are D17 cells chronically infected with SNV virus. d D17 (2.1) is a D17 cell line expressing the SNV env-gp (Delwart

and Panganrban, 1989).

env-gp to induce resistance to infection reflects its ability to reach and complex the cell receptor.

A population of virus particles containing a REV vector expressing the gene for hygromycin resistance and associated REV helper virus was generated by co- transfection of pJD214Hy and pED75 into CEFs (see Materials and Methods). This virus stock was used to determine the level of resistance to REV infection for cells expressing the SNV env-gp following treatment with the glycosylation inhibitors. Chronically infected cells and the env-gp expressing cell line were treated with tunicamycin for 24 hr or with the trimming glycosidase inhibitors for 3 days. Treated cells were infected with the hygromycin resistance REV virus stock, and the number of hygromycin-resistant colonies that arose was used to measure their level of resistance to infection (Table 1).

Tunicamycin was somewhat toxic to cells, as evi- denced by a slight reduction in the number of hygromycin-resistant colonies following infection of inhibitor-treated D17 control cells. However, tunicamycin almost completely abrogated the superinfection interference of SNV env-gp expressing cells, as evi- denced by an increase in infection efficiency and gener- ation of Hyg’ colonies. In contrast, castanospermine and dMM treatment was not toxic to D17 cells (see Materials and Methods) and did not decrease the high level of resistance to REV infection. Thus the trimming of the oligosaccharides and their subsequent modification to high-MW, complex type carbohydrate chains are dispensible for the formation of the SNV env-gp receptor binding site.

infectivity of released virus is independent of complex glycosylation

We next determined whether complex glycosylation of the env-gp was needed for virus infectivity. D17 cells were transfected with pJD2 14Hy and selected for hygromycin resistance. Hygromycin-resistant colonies were pooled and infected with REV virus derived from pED75-transfected CEF. These chronically infected, hygromycin-resistant D17 cells were then treated with tunicamycin for 24 hr or with castanospermine and dMM for 3 days. The virus titer produced by these cells was then measured by two methods (Table 2). First, D17 cells were infected with the culture supernatant and 24 hr later selected for resistance to hygromycin. Resistant colonies were then counted to determine the titer of the defective virus vector packaged by the helper REV. In addition, replication-competent virus was titered by serial dilution and infection of CEF. The presence of infectious virus in the diluent was determined by immunofluorescence staining of the infected CEF after 4 days. Tunicamycin reduced both virus titers to about 2% of controls (Table 2). When chronically infected cells treated with tunicamycin were analyzed for cell surface expression of SNV env-gp by immunofluorescence analysis, about 1% still appeared brightly positive (Fig. 21). The continued, although much reduced, production of infectious SNV may be due to those few D17 cells that are resistant to the action of tunicamycin. In contrast, castanospermine and dMM treatment did not reduce the virus titer (Table 2). Thus, it appears that the SNV env-gp functions required for the formation of infectious viral particles are not dependent on complex type N-glycosylation.

Complex glycosylation is required for syncytia formation

Transfection of D17 cells, plated at a high density, with an SNV env-gp-expressing plasmid (pED27) in-

TABLE 2

VIRUS TITERS FROM CELLS TREATED WITH DIFFERENT N-GLYCOSYLATION INHIBITORS

lnhrbrtor Hyg’CFU” TCIDb

None 1.1 x lo5 10” Tunicamycrn 2.6 x 10” 1 o4 Castanospermine 0.4 x lo5 10” dMM 1.3 x lo5 1 o6

a Hyg’ CFU: hygromycin resrstance colony transformrng units per milliliter of supernatant as determined by Infection of D 17 cells.

’ TCID/ml: tissue culture infectrous dose per mrlliliter of supernatant as determrned by lo-fold dilutrons and infection of CEF. Pres- ence of vrrus was determined by tmmunofluorescence analysis for viral antrgen.


TABLE 3

SYNCMIA FORMATION IN THE PRESENCE OF DIFFERENT N-GLYCOSYLATION INHIBITORS

SyncytiaB

Inhibitor*

None Castanospermine dMM control

Expt 1

193 4

20 3

Expt 2

83 16

2 3

Expt 3

112 15 11 10

a Syncytia formation described under Materials and Methods. ’ Inhibitor concentrations given under Materials and Methods.

duces the formation of multinucleated giant cells (Ta- ble 3). A syncytium labeled by immunofluorescence staining is shown in Fig. 2J. In a manner analogous to HIV-1 env-gp-induced cell fusion (Lifson et al., 1986; Sodroski et al., 1986) syncytia formation is presumably initiated by the intercellular recognition of the retroviral receptor by env-gp expressed on the surface of a neighboring cell. Such syncytia cannot be induced in preinfected cells or in cells already expressing SNV env-gp, as their viral receptors are already complexed by the endogenously synthesized env-gp (Delwart and Panganiban, 1989). Syncytia formation was attempted in the presence of the trimming glycosidase inhibitors. Tunicamycin was not tested because it proved too toxic over the time period required for the assay. Cas- tanospermine and dMM did not affect the transfection efficiency in D17 cells or cell survival during the 4 days required for the experiment (see Materials and Meth- ods). In the presence of these trimming inhibitors SNV env-gp-induced syncytia formation was strongly inhibited (Table 3). It therefore appears that SNV env-gp-induced cell fusion is dependent on proper complex N- glycosylation in cells participating in syncytia formation.

DISCUSSION

N-linked oligosaccharides have different effects on glycoprotein function depending on the particular polypeptide. Some glycoproteins are at least partially functional when N-glycosylation acceptor sites are re- moved by mutagenesis (Bedinger et a/., 1988) or when N-glycosylation is blocked by tunicamycin (Gibson et a/., 1980; Keating et a/., 1989). For other glycoproteins such as the influenza hemagglutinin, specific N-glycosylation sites can affect folding (Gething et al., 1986), antigenicity (Skehel et a/., 1984) and proteolytic cleavage (Deshpande et a/., 1987). The primary role of N- glycosylation for many viral glycoproteins appears to be in assisting proper folding and in maintaining intra-

cellular solubility (Gibson et a/., 1980; Fennie and Lasky, 1989). Preventing N-glycosylation of various retroviral env-gps with tunicamycin can block their translocation to the cell surface (Bassin et al., 1982; Polonoff et a/., 1982; Rein eta/., 1982) reduce the yield of infectious virus (Schwarz et al., 1976) and abrogate superinfection interference (Bassin eta/., 1982; Rein et a/., 1982; Kai et a/., 1986).

Tunicamycin treatment reduced the stability of the SNV envelope protein and prevented its translocation to the cell surface. This could be due to increased proteolysis in the ER as a result of improper folding of the env-gp or because of a lack of protection from degrada- tive proteolysis by appended oligosaccharides. Im- proper oligomerization of the unglycosylated envelope protein may also, as for the influenza hemagglutinin (Gething et a/., 1986) prevent exit from the ER. Com- plexing of unglycosylated SNV envelope proteins with tunicamycin-induced, folding chaperone proteins in the ER could also prevent translocation to the cell surface (Flynn et al., 1989).

Superinfection interference is due to a block in retrovirus penetration (Steck and Rubin, 1966a,b) and is re- stricted to viruses belonging to the same immunological subgroup (Vogt and Ishizaki, 1966) implicating the viral env-gp of the preinfecting virus in the establish- ment of a receptor interference. Expression of REV retroviral env-gp is sufficient to induce receptor interference (Delwart and Panganiban, 1989; Federspiel eta/., 1989) and for HIV-1 env-gp expression results in down- regulation of the virus receptor from the cell surface (Stevenson et al., 1988). Superinfection interference prevents high levels of reinfection, a phenomenon associated with cytopathic infections (Temin eta/., 1980; Mullins et a/., 1986; Temin, 1988).

The almost total abrogation of superinfection intet-fer- ence seen in chronically infected D17 cells following tunicamycin treatment indicates that the unglycosylated SNV envelope protein can no longer sequester the cellular viral receptor. The susceptibility to REV infection induced by tunicamycin could be due to the inability of the unglycosylated envelope protein to translocate to the cell surface in order to complex the virus receptor at that location. However, since an SNV env-gp arrested in translocation within the ER still confers resistance to REV infection (Delwart and Pan- ganiban, 1989) and an intracellular form of the HIV-1 env-gp (gpl60) can be coimmunoprecipitated with the CD4 receptor (Hoxie et a/., 1986; Stevenson et a/., 1988) it is unlikely that the effect of tunicamycin on interference is caused simply by blocking env protein translocation. In further support of intracellular complexing a modified CD4 with an ER retention sequence was recently shown to bind the HIV-1 glycoprotein (Buonocore and Rose, 1990). Abrogation of interfer-


ence by tunicamycin could also be due to the reduced half-life of the unglycosylated envelope protein. How- ever, direct binding studies have shown that the unglycosylated HIV-1 envelope protein synthesized in the presence of tunicamycin (Fennie and Lasky, 1989) or in E. co/i (Putney et al., 1986) could not bind the CD4 receptor. Similarly SNV envelope protein synthesized in E. co/i also did not block REV infection of D17 cells (data not shown). Therefore we favor the hypothesis that as for the HIV-l env-gp, EGF, and insulin receptors (Fennie and Lasky, 1989; Slieker et a/., 1986; Olson and Lane, 1987) the effect of tunicamycin on the SNV env protein is caused by the inability of the polypeptide synthesized in the absence of cotranslational glycosylation to properly attain its ligand binding conformation.

The role of the trimming glycosidases in SNV replication was investigated using inhibitors of glucosidase I and mannosidase I enzymes. Castanospermine inhibits the removal of the distal glucose residue from the high-mannose group, Glc,Man,GlcNAc,, initially transferred to the growing polypeptides. dMM inhibits the mannosidase l-mediated trimming of the five outer mannose residues from the Man,GlcNAc, oligosaccharide side chains produced following glucosidase I and II trimming. In the presence of such inhibitors the label chase pattern of the SNV envelope gene products and the appearance of TM proteins indicate that correct proteolytic cleavage of env-gp is likely to be still taking place (Figs. 1A and B). The complex N-glycosylation normally found on the Rev-A cleavage precursor (Tsai and Orozslan, 1988) therefore appears dispensable for its processing by the host cell endopeptidase. Proteo- lytic cleavage of the HIV-l env-gp precursor synthesized in the presence of trimming inhibitors has also been demonstrated (Gruters et al., 1987; Montefiori et a/., 1988; Stein and Engleman, 1990). As with other retroviruses proteolytic cleavage is likely to be essential for viral infectivity (Perez and Hunter, 1987; Freed and Risser, 1987; McCune et a/., 1988) by rendering accessible a highly hydrophobic peptide domain at the amino terminus of the TM glycoprotein required for membrane fusion. The normal infectivity of REV released in the presence of the trimming inhibitors also indicates that correct proteolytic cleavage is likely to be taking place. As seen by immunofluorescence analysis, the ability of SNV glycoproteins bearing untrimmed oligosaccharides to translocate to the Golgi and the cell surface was not grossly affected, indicating that the high-MW complex form of the SNV env-gp carbohydrate chains is not essential as a signal for translocation.

As opposed to the effects seen with tunicamycin the resistance to REV infection of cells expressing SNV env-gp was not lowered by the trimming glycosidase inhibitors. Similarly the titers of REV released from in-

fected cells treated with the same inhibitors was not reduced. These results indicate that complex type oligosaccharide chain modification is dispensible for receptor binding and viral infectivity in D17 cells. It is possible that the complex oligosaccharides found on SNV glycoproteins are necessary to assist in infecting other cell types or to escape proteolytic degradation and immune neutralization during infection of an ani- mal host (Huso et al., 1988).

Unlike SNV, HIV-l released in the presence of different trimming glycosidase inhibitors shows a reduction in tissue culture infectious dose per milliliter ranging from one to five orders of magnitude (Gruters et a/., 1987; Walker et al., 1987; Karpas et a/., 1988; Monte- fiori et al., 1988; Pal et al., 1989). Like the SNV env-gp, the HIV-1 env-gp bearing untrimmed glycans remains capable of binding its cellular receptor (Walker et al., 1987; Fennie and Lasky, 1989). This has led to the hypothesis that HIV-l infectivity is blocked when complex N-glycosylation is prevented by interfering with another env-gp function. The strong antisyncytial activity of such inhibitors points toward a decrease in the HIV-l gpl20 membrane fusion activity (Gruters et al., 1987; Walker et al., 1987; Montefiori et a/., 1988; Pal et a/., 1989). Analogous to the influenza hemagglutinin, partially N-glycosylated HIV-1 env-gp may be unable to undergo a conformational change (Skehel et al., 1982; Wiley and Skehel, 1987) required to expose its hydrophobic membrane fusion domain. Alternatively, in the absence of complex N-glycosylation the HIV-1 env-gp may be unable to bind a hypothetical fusion domain receptor.

Castanospermine and dMM also efficiently blocked SNV env-gp-induced syncytia formation. This block in syncytia formation was unexpected in light of the normal infectivity of REV particles released from similarly treated cells. The pathway of SNV entry into D17 cells may occur through fusion of the virion’s membrane with the cell surface’s plasma membrane following receptor binding (as is the case for some other retroviral systems, including the D-type simian retroviruses (McCune et al., 1990) and HIV-l (Stein et al., 1987)), rather than being induced by a low pH in the endocytic pathway (Marsh, 1984) since ammonium chloride and chloroquine did not affect the ability of D17 cells to be infected by SNV (data not shown). The mechanism of SNV env-gp-induced cell fusion is also apparently pH independent, as pED27-transfection-induced syncytia formation occurs at a physiological pH and lowering the medium’s pH does not induce syncytia in a culture of cells expressing SNV env-gp (data not shown). Syn- cytia inhibition was not due to a decrease in biosynthesis or proteolytic processing of the env-gp since the SU and TM proteolytic product levels are not decreased in treated cells (Figs. 1A and B). Syncitia inhibition may


instead result from the slight decrease in the cell surface level of SNV env-gp in trimming glycosidase inhibitor-treated cells (Fig. 2). It may also be that other cell surface molecules need to be properly N-glycosylated for cells to come into close contact and fuse. As for HIV-1 a cell surface adhesion glycoprotein (Hildreth and Orentas, 1989; Valentin et a/., 1990) may play an auxiliary role in cell fusion. The inhibitory effect of trimming glycosidase on SNV env-gp-mediated cell-cell fusion while virus-cell membrane fusion required for virus entry remains apparently unaffected could reflect the lower density of viral glycoproteins on the cell surface than on the virion surface, where it is preferentially concentrated by interaction with viral core molecules during virus budding. The high viral glycoprotein concentration on virions may therefore make virus entry less dependent on the full fusogenic potential of the SNV envelope glycoprotein than cell-to-cell fusion and this may account for the differential effects of trimming glycosidases inhibitors in the two assays. A direct effect of the trimming inhibitors on the SNV receptor is also possible. The demonstration that the chimpanzee CD4 is unable to mediate cell-cell fusion with HIV-1 -infected human cells while still able to support HIV-1 infection (Camerini and Seed, 1990) may similarly reflect different receptor requirements for the two kinds of membrane fusion events.

ACKNOWLEDGMENTS

We thank Kevin Schultz and Howard Temin for critical review of this manuscript and Betty Sheehan for proofreading. This work was supported by Research Grant CA22443-10 from the NIH, support from the Shaw Scholars Fund, and a junior faculty research award (JFRA-147) from the ACS.

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n-linked glycosylation and reticuloendotheliosis retrovirus envelope glycoprotein function

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