Oligomerization of friend spleen focus-forming virus (SFFV) env glycoproteins

VIROLOGY 177,312-316 (1990)

Oligomerization of Friend Spleen Focus-Forming Virus (SFFV) env Glycoproteins

YANYAN YANG,*,’ ARINOBU TOJO,* NAOKO WATANABE,t AND HIROSHI AMANUMAt

*Department of Genetics, Institute of Medical Science, University of Tokyo, 4-6- 1 Shirokanedai Minato-ku, Tokyo 108, and tLaboratory of Gene Technology and Safety, Tsukuba Life Science Center, Institute of Physical and Chemical Research, Tsukuba, lbaraki 305, Japan

Received January 2, 1990; accepted March 15, 1990

The glycoprotein gp55 and its processed form gp65, which are encoded by the env gene of Friend spleen focus- forming virus (SFFV), have been implicated in the initiation of the murine acute erythroleukemia induced by Friend virus (FV). Analyses of these glycoproteins by chemical crosslinking and nonreducing/reducing two-dimensional electrophoresis showed that both gp55 and gp65 exist as monomer and disulfide-bonded dimer and trimer. These oligomers could be detected in various FV-infected etythroleukemia cell lines, as well as in the spleen cells of FV-induced ery-throleu- kemic mice, suggesting that oligomerization is an intrinsic feature of SFFV env glycoproteins. o 1990 Academic PWSS, IW.

INTRODUCTION

The env gene of SFFV has been identified as the only viral gene necessary for the initiation of FV-induced murine erythroleukemia (Ostertag et al., 1987, for review; Wolff and Ruscetti, 1988). Two env gene products can be detected in SFFV-infected cells: the major product gp55 and its processed from gp65. Like other murine retroviral envelope glycoproteins, gp55 is syn- thesized in the rough endoplasmic reticulum (ER), where the first step of glycosylation occurs. Further processing follows during its transport to the plasma membrane via the Golgi apparatus. However, unlike the gp7Os, 5-l 0% of gp55 is expressed on the cell surface as gp65 which is not incorporated into virions (Ruscetti and Wolff, 1984, for review). The pathogenic mechanism of gp55 and gp65 in FV-induced erythroleukemia is still unclear.

One possible mechanism for gp55 and gp65 to in- duce erythroblastosis is that they could interact with some cellular protein(s) involved in the proliferation and differentiation of erythroid progenitor cells. To investigate this possibility, we examined the possible com- plexformation of gp55 and gp65 by chemical crosslinking and nonreducing/reducing two-dimensional electrophoresis. We found that gp55 and gp65 form disulfide-bonded dimers and trimers, and that oligomerization seems to be an intrinsic feature of SFFV env glycoproteins.

MATERIALS AND METHODS

Cell lines

Murine erythroleukemia cell lines T3Kl (Ikawa et al., 1978), F5-5 (Ikawa et a/., 1976), F4-6 (Anders et al.,

’ To whom requests for reprints should be addressed.

1985), and B8-3 (Dube et al., 1975) were induced by the polycythemia strain of FV (FVp), while TSA8 was induced by the anemia strain (FVa) (Shibuya and Mak, 1983). FVTCT originated from an FVp-induced trans- plantable reticulum cell sarcoma (Fieldsteel et al., 1969).

Mice and viral infection

The NB-tropic strain of FVp was maintained in qur laboratory by continuous passage in BALB/c mice. The 100/o (v/v) spleen cell homogenate in a-medium was prepared from mice intraperitoneally infected with FVp 10 days before, and stocked at -70”.

Male DDD mice (Fv-,?“), 4-8 weeks of age, were in- jected intraperitoneally with five-fold-diluted FV stock. Spteen cells were obtained 9 days later and prepared for protein analysis.

Antibodies

Rat anti-gp55 monoclonal antibody 7C-10 (Ab 7C- 10) was generously supplied by Dr. S. Ruscetti (Na- tional Cancer Institute, Bethesda, MD; Wolff et al., 1982; Li et al., 1987). Rabbit anti-rat IgG (Wako) was used as the second antibody for Ab 7C-10. Both of them were used at 1 :lOOO dilution.

Chemical crosslinking of proteins

Crosslinking reagent 3,3’-dithiobis (sulfosuccinimidyl propionate) (DTSSP, Pierce; Staros, 1982) was added to lo6 T3Kl cells suspended in 1 ml phosphate- buffered saline (PBS) at a final concentration of 0.5 mll/l. After incubation for 15 min on ice, the reaction was quenched by addition of 2 mM glycine (Kreis and Lo- dish, 1986). The extracts from these cells were analyzed

0042-6822/90 $3.00 CopyrIght 0 1990 by Academic Press, Inc. All rights of reproduction I” any form reserved.

312

OLIGOMERIZATION OF SFFV env GLYCOPROTEINS 313

by electrophoresis under nonreducing or reducing conditions

DTT ! 2 3 4 + - +

lmmunoprecipitation

Cells (1 O”/ml) were labeled with 50 $Cilml L-[35S]methionine (Amersham) for 2 or 18 hr and lysed with 0.1 o/o SDS buffer (0.1 o/o SDS, 1% sodium deoxycholate in 1% Triton X-100 buffer, see Electrophoresis) without any reducing reagent. The lysates were reacted with Ab 7C-10 and rabbit anti-rat IgG, and precipitated by protein A-Sepharose CL-4B. The precipitated proteins were resuspended in the sample buffer with or without 50 mM reducing reagent dithiothreitol (DTT) and boiled

200KD-

116KD-

97KD-

67KD-

43KD-

FIG. 1. DTSSP-mediated crosslinking of SFFV eny glycoproteins. T3Kl cells (1 06) were treated with 0.5 mM DTSSP for 15 min at 4”.

for 5 min before electrophoresis.

Enzymatic digestions

L-[35S]Methionine-labeled gp55 monomers and complexes were recovered from the dried gel, digested with 10 pg Staphy/ococcus aureus \/8 protease per lane (Machida et al., 1984), and analyzed on a 15% acrylamide gel as described previously (Cleveland et al., 1977).

(Western blotting). The blot was then reacted with a rat anti-gp55 monoclonal antibody 7C-10, the secondary antibody rabbit anti-rat IgG, and visualized by ‘251-protein A. Lanes 1 and 2, DTSSP (-); lanes 3 and 4, DTSSP (+). Arrowheads indicate the 4 extra bands at 97, 116,170,and200kDa.

Then the extracts were treated with (+) or without DTT (-) before electrophoresis, followed by protein transfer onto a membrane

Electrophoresis

One- or two-dimensional gel electrophoresis under nonreducing or reducing conditions was performed ac- cording to the method of Phillips and Agin (1977). Pro- teins were extracted with 1% Triton X-l 00 buffer (1% Triton X-l 00, 50 mM Tris-HCI, pH 7.4, 150 mM NaCI, 20 mM EDTA, and 1 %Trasylol), treated with or without DlT before the first-dimensional electrophoresis on a 7.5% acrylamide gel. Gel strips containing the samples of interest were cut out, treated with or without DlT, and analyzed by second-dimensional electrophoresis. The L-[35S]methionine-labeled gel samples were prepared for Western blotting, dried, and exposed to film at -70”.

Western blotting

Western blotting was performed as previously described (Towbin et a/., 1979). The transferred mem- branes (blots) were reacted with Ab 7C-10 and rabbit anti-rat IgG, then with ‘251-protein A and exposed to film at -70”.

Quantitative analysis of gp55/gp65 monomers and oligomers

RESULTS

Since DTSSP-mediated crosslinking can be cleaved by reducing reagents such as DTT or 2-mercaptoetha- nol, the proteins of T3Kl cells were crosslinked with DTSSP and analyzed by electrophoresis under reducing or nonreducing conditions, and then by Western blotting with Ab 7C-10 (Fig. 1). In addition to the bands at 55 and 65 kDa, four major extra bands at 97, 116, 170, and 200 kDa were generated by DTSSP-mediated crosslinking under nonreducing conditions and disap- peared under reducing conditions (Fig. 1, lanes 3 and 4). Surprisingly, under nonreducing conditions, the four extra bands also appeared when the proteins were not crosslinked (Fig. 1, lanes 3 and 4). This result suggested that gp55/gp65 may be in association with some protein(s) by disulfide bonds. Besides these, it can be seen from Fig. 1 that the nonreduced monomeric gp55 and gp65 migrated faster than the reduced forms, suggesting that they contain at least one intra- molecular disulfide bond, which would affect their con- formations and change their mobility in electrophoresis (Pinter and Honnen, 1984). Also, the intensity of the radiolabeled bonds under nonreducing conditions was stronger than that under reducing conditions, probably due to the relatively higher affinity of Ab 7C-10 to the nonreduced SFFV env glycoproteins.

To investigate the properties of the four extra bands, proteins of T3Kl cells were analyzed by two-dimensional gel electrophoresis under nonreducing and reducing conditions, followed by Western blotting with Ab 7C-10. When both dimensions were under nonreducing conditions, gp55, gp65, and the four extra

The radioactivity of each gp55/gp65 band on an lz51- protein A-labeled Western blot was determined with the Ambis radioanalytic imaging system (Tack el a/., 1988) and the ratio of monomer, dimer, and trimer was calculated.

314 YANG ET AL.

A. 1st. +, Nonreducing

' SP65

- LIP55

1 st. + , Nonreducing

116kd-

97kd-

67kd-

?

%

$ f. Icl +gp65

T T T

2dOkd ilbkd’97kd k7kd

FIG. 2. Nonreducing/reducing two-dimensional electrophoresis and Western blotting of SFFV env glycoproteins. Unlabeled proteins from 1 O6 T3Kl cells were electrophoresed without Dn in the first dimension. The gel strips were cut out, treated with (B) or without DlT (A) before electrophoresis in the second dimension, and then analyzed by Western blotting as described in the legend to Fig. 1. Molecular weights below indicate the corresponding positions in the first dimension, and those on the left indicate the corresponding positions in the second dimension. Arrowheads indicate the electrophoretic direction from (-) to (+). Four off-diagonal spots can be seen in B; two are on the same horizontal level as gp55 and two are on the same level as gp65.

spots lay on a straight diagonal (Fig. 2A), whereas,

when the second dimension was under reducing conditions, the four extra spots migrated to the same horizontal level as gp55 or gp65 (Fig. 28). This indicated that the bands of 97 and 170 kDa contained the disulfide-bonded gp55 complexes and the bands of 116 and 200 kDa, the gp65 complexes.

To identify the protein(s) complexed with gp55, the L-[35S]methionine-labeled proteins of T3Kl cells were immunoprecipitated with Ab 7C-10 before two-dimensional gel electrophoresis under nonreducing and reducing conditions. If gp55 associated with another protein(s), an extra spot(s) should appear over or under the reduced gp55 spots. Figure 3 shows the result of a 2- hr labeling. Only two off-diagonal spots (S-2 and S-3) were seen on the same horizontal level as gp55 monomers (S-l), one was from the 97-kDa nonreduced complex and the other, from the 170-kDa complex. No other spots were observed over or under them. The 18- hr labeling gave the same result (data not shown). These results suggested that the complexes were the homooligomers of gp55. To exclude the possibility that gp55 forms complexes with a protein that is identical to gp55 in its electrophoretic behavior, gp55 monomer and the two extra spots were analyzed by enzymatic

s-3 s-2 S-l

FIG. 3. lmmunoprecipitation of L-[35S]methionine-labeled SFFV env glycoproteins. T3Kl cells (106) were labeled with L-[%]methionine for 2 hr. The extracts were reacted with Ab 7C-10. rabbit anti-rat IgG, precipitated with protein A-Sepharose, and then analyzed by nonreducing/reducing two-dimensional electrophoresis. Arrows indicate the electrophoretic direction from (-) to (+). Two off-diagonal spots S-2 and S-3 can be seen to the left and on the same horizontal level as S-l, the gp55 monomers.

digestion with S. aureus 1/8 protease. Digestion of the monomeric gp55 produced 2 primary fragments V-l (33 kDa) and V-2 (23 kDa) (Fig. 4, lane l), consistent with a previous report (Machida eta/., 1984). Compari- son of the cleavage patterns of the two extra spots with that of gp55 monomers showed no significant differ- ence among them (Fig. 4, lanes 2 and 3). Thus, the bands at 97 and 170 kDa are dimers and trimers of gp55, respectively. This result, together with the one shown in Fig. 2, suggests that the complexes of gp65

97KD-

66KD-

43KD-

31 KD-

22KD-

14KD-

+3P55

-VI

-v2

FIG. 4. Peptide mapping of Staphylococcus aureus V8 protease- digested gp55 monomers and its complexes. L@%]Methionine-labeled spots S-l, S-2, and S-3 were recovered from the dried gel in Fig. 4 and incubated with S. aureus V8 protease (10 pg) for 30 min in the stacking gel before electrophoresis on a 15% acrylamide gel. Lane 1, S-l ; lane 2, S-2; lane 3, S-3. V-l and V-2 are the two primary proteolytic fragments with apparent molecular masses of about 33 and 23 kDa, respectively.

OLIGOMERIZATION OF SFFV em GLYCOPROTEINS 315

may also be its dimers and trimers. The ratio of monomer, dimer, and trimer of gp55 is 73:23:4 and that of gp65 is 57:37:6.

In addition to T3K1, the oligomeric gp55/gp65 could also be detected in several other FVp-infected cell lines such as F4-6, 68-3, FVTCT, and in the spleen cells of FV-induced erythroleukemia mice. In an FVa-infected erythroleukemia cell line TSA8, the gp55 of the anemia strain was also detected as oligomers. These results suggested that oligomerization may be an intrinsic feature of SFFV env glycoproteins.

Furthermore our examination of the products of two nonpathogenic SFFV env mutants (Amanuma et al., 1989; Watanabe eta/., in press) revealed that the oligomers were also formed, but in a less efficient manner apparently than those of the wild-type gp55 (data not shown). This result indicated that loss of pathogenicity cannot be linked to a deficiency in oligomerization of mutant env products, although further quantitative analysis would be needed to determine whether efficient oligomerization of the SFFV env glycoproteins is related to leukemogenesis or not.

DISCUSSION

Previous studies on wild-type and mutant oligomeric proteins, such as the hemagglutinin of influenza virus (Gething eta/., 1986) and the G protein of vesicular stomatitis virus (VSV) (Kreis and Lodish, 1986) have suggested that oligomerization is a prerequisite for their intracellular transport. As for SFFV env glycoproteins, Kilpatrick et al. (1988) have investigated the folding of SFFV envglycoproteins using sucrose density gradient centrifugation and demonstrated that gp65 formed oligomers and was transported to the cell surface, whereas gp55 was defective in oligomerization and re- mained as a monomer in the ER. On the other hand, studies of Gliniak and Kabat (1989) using nonreducing/ reducing two-dimensional electrophoresis showed that gp55 exists as both monomer and disulfide- bonded dimer. Using ‘251-mediated cell surface labeling, they also showed that only the dimer of gp55p (the processed form of gp55) could be detected on the cell surface. However, our studies clearly showed that both gp55 and gp65 could be detected as disulfide- bonded monomer, dimer, and trimer in the whole cell lysate. This was obtained not only from the results of nonreducing/reducing two-dimensional electrophoresis, in which iodoacematide (50 mM) was used to pre- vent the formation of any spurious disulfides, but also from the results of chemical crosslinking with DTSSP and an uncleavable crosslinking reagent DSS (disuc- cinimidyl suberate; Sayer eta/., 1987.) which crosslink proteins before lysis of cells (data not shown).

These observations, together with the fact that only gp65 but not gp55 can be detected on the cell surface, indicate that gp55 does oligomerize but is defective in transport to the cell surface. Gliniak and Kabat (1989) interpreted the retention of gp55 in ER as due to the formation of heterogeneous dimers and suggested that only one “properly folded” homodimer is compe- tent for export from the ER, since on their nonreducing/ reducing two-dimensional electrophoresis gel, the mo- bilities of dimeric gp55 seem different. However, this may result from the large amount of gp55 or the experi- mental conditions. In our opinion, oligomerization is prerequisite, but it alone may not be sufficient for the intracellular transport. Previous studies on the G proteins of VSV have shown that some mutant G proteins, which were identical to the wild-type G proteins in folding and trimerizing, were either transported to the cell surface slowly or not at all (Doms et al., 1988). Also, the proteolysis or early glycosylation that occurs in ER may be involved in the transport of some proteins such as the gp70 of F-MuLV, whose transport is temporally linked with the proteolysis of its precursor PrENV (Srini- vas et a/., 1982). Thus it seems likely that besides oligomerization, another additional transport signal(s) could be involved in governing the retention of gp55 in the ER and the transport of SFFV env glycoproteins. An argu- ment in favor with this view is the observation that enzymatic analysis of gp55p and gp55”(secreted gp65) with S. aureus V8 protease showed that the C-termini of gp55p and gp55’ have undergone some modifications (Gliniak and Kabat, 1989). Investigating whether a proteolytic event occurs before gp55 is exported from the ER may be of interest.

It has been suggested that the gp65 on the cell surface is essential for SFFV-induced pathogenesis. Gli- niak and Kabat (1989) found that only dimeric gp65 could be detected on the cell surface, and the present studies showed that besides the dimer, both monomer and trimer of gp65 were detected in the whole cell lysates. It seems likely that after the oligomeric gp55 was exported from ER, the oligomerfell apart and gp55 was glycosylated into gp65 separately in the Golgi apparatus. Then the gp65 was reoligomerized and the dimeric one was transported to cell surface.

Although no cellular protein associated with gp55/ gp65 was found in this study, it is still possible that the interaction of gp55/gp65 with some cellular proteins might be detected by a method other than that which we used. A recent study by Li et a/. (1990) using double immunoprecipitation with anti-gp70 Ab and anti-EPO- receptor Ab has shown that gp55 binds to the EPO receptor in fibroblasts and hemopoietic cells. In the present study, several high-molecular-weight bands were detected when [35S]methionine-labeled proteins from

316 YANG ET AL.

T3Kl cells were immunoprecipited with 7C-10 Ab (Fig. 3). However further studies have not been performed to characterize them. Since each T3Kl cell expresses less than 1 X 1 O3 EPO receptors, it would be difficult to detect the association of gp55 with the EPO receptor in this cell line. It is of interest to investigate whether this association also occurs in EPO receptor-positive, FV- induced erythroleukemia cells, or in the erythroblasts of FV-infected mice.

ACKNOWLEDGMENTS

We thank Dr. S. Ruscetti for the generous gifts of anti-gp55 monoclonal antibody 7C-10 and Dr. Takeshi Odaka for discussions and reading of the manuscript. This work was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Education, Science and Culture of Japan.

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Oligomerization of friend spleen focus-forming virus (SFFV) env glycoproteins