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Association of murine leukemia virus gag antigen with extracellular matrices in productively infected mouse cells
VIROLOGYll6,306-317
Association
(1982)
of Murine Leukemia Virus gag Antigen with Extracellular Matrices in Productively Infected Mouse Cells
STEVEN The Tumor
A. EDWARDS,l
Virology Laboratory,
YING-CHIH
LIN,
The Salk Institute, P.O. Box
Received June
AND HUNG
85800,
San
FAN’
Diego, Califin-nia
92138
15, 1981; accepted September 2, 1981
The gag gene of murine leukemia virus (MuLV) encodes a nonstructural glycosylated polyprotein which appears at the cell surface, in addition to the polyprotein precursor for the virion internal structural proteins. The surface localization of gag nonstructural protein is reported here. Immunofluorescent staining of unfixed monolayers of Moloney MuLV-infected NIH/3T3 cells using anti-p30 serum as the primary antibody revealed an unusual pattern of gag antigen at the attached cell surface: highly organized cable-like structures. Stained cable-like structures were also observed in regions lacking cells or cell processes, which suggested that extracellular gag antigen may be associated with extracellular matrices. This was supported by the fact that detergent treatment of cell monolayers in a manner designed to removed intact cells but preserve extracellular matrices did not affect the cable-like anti-p30 staining patterns. Immunofluorescent staining with anti-gp70 serum revealed a different pattern than the anti-p30 staining, which indicates that surface gag antigen and envelope glycoprotein are not physically associated at the cell surface. Similar staining patterns were observed in mouse cells productively infected with a different strain of MuLV (AKR), and in mink cells productively infected with a mink cell focus-inducing (MCF) derivative of Rauscher MuLV.
culture medium (Edwards and Fan, 1979; Ledbetter et ah, 1978). In comparison to the intracellular gag precursor polyprotein for the virion internal structural proteins (Pr658”9), glycosylated gag polyprotein contains carbohydrate side-chains and additional amino-terminal peptide sequences (Schultz and Oroszlan, 1978; Edwards and Fan, 1979, 1980). A biological function has not yet been assigned to glycosylated gag polyproteins. Recent work from our laboratory suggests a correlation between surface gag expression and virus production (Edwards and Fan, 1981), although somewhat different conclusions were reached by other workers using a different host cell (Fitting et ah, 1981). Similar glycosylated gag polyproteins have been observed in cells infected with feline leukemia virus (Neil et al, 1980) and baboon endogenous virus (Whitely and Naso, 19Sl), and cells infected with avian sarcoma virus may contain minor amounts of surface gag polyprotein (Buetti and Diggelmann, 1980a). Other major classes
INTRODUCTION
Murine leukemia virus (MuLV), like other nondefective retroviruses, contains three genes within the RNA genome, gag, pal, and env, which encode the internal structural proteins, reverse transcriptase, and envelope glycoprotein, respectively (Baltimore, 1974). All of these genes are initially translated as polyprotein precursors which are subsequently cleaved to the mature viral proteins (Eiseman and Vogt, 1978). An unusual feature of MuLV is that its gag gene codes for a second, nonstructural polyprotein which is glycosylated and either associated with the cell surface (Tung et ab, 19’7’7; Ledbetter and Nowinski, 1977; Evans et al., 1977) or shed into the 1 Present address: Peptide Biology Laboratory, The Salk Institute, P.O. Box 85800, San Diego, Calif. 92138. ZTo whom requests for reprints should be addressed. Present address: Department of Molecular Biology & Biochemistry, University of California, Irvine, Calif. 92717. 0042-6822/82/010306-12.$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
306
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of retroviruses appear to lack an analogous protein. During the course of these experiments, we observed an unusual location of extracellular gag antigen by indirect surface immunofluorescence using anti-MuLV p30 serum: Most of the detectable antigen was in the extracellular matrix of the infected cells. These observations are described in the results reported here. MATERIALS
AND
METHODS
Cells. M-MuLV alpha, M-MuLV alpha clone 19, and M-MuLV(anti-p30) clone 12 are all NIH/3T3 mouse fibroblasts infected with Moloney MuLV (M-MuLV). The infection, cloning, and maintenance of these lines have been described previously (Edwards and Fan, 1981). AKR P2 cells are a line of embryonic NIH mouse fibroblasts productively infected with AKR strain MuLV, and were provided by Dr. Robert Nowinski. E&G2 cells are leukemia cells induced by AKR MuLV, and are the serological typing line for the Gross cell surface antigen (GCSA, Old et al, 1965; Aoki et al., 1972); they were also provided by Dr. Nowinski (Ledbetter and Nowinski, 1977). Mink lung fibroblasts productively infected with a “mink cell focus-inducing” (MCF) derivative of Rauscher MuLV were supplied by Dr. Marguerite Vogt (van Griensven and Vogt, 1980). Antisera. Rabbit antiserum to M-MuLV p30 was prepared in this laboratory (Mueller-Lantsch and Fan, 1976) and has been used extensively in previous investigations (Edwards and Fan, 1979,1980,1981). Goat antiserum to SCRF 60A MuLV (Lerner et ab, 1972) was a gift from Dr. Stephen Kennel. Goat anti-rabbit IgG and pig anti-goat IgG were purchased from Cappel Laboratories. Indirect immunojhwescence. Indirect immunofluorescence was performed on sparse cultures growing on glass coverslips. Details of this procedure have been previously reported (Edwards and Fan, 1981). Preparation of cell-free matrices. The procedure used was that of Chen et al. (1978). Subconfluent cultures growing on
MuLV
gag ANTIGEN
307
glass coverslips were washed three times with phosphate-buffered saline solution, followed by washing three times with buffer containing 0.1 M Na2P04, 2 mM MgC12, and 2 mM EGTA (pH 9.6). Coverslips were then covered with 2 ml of lysis buffer containing 8 mMNa2HP04, 1% NP40, pH 9.6 for 20 min at 0”. The lysis buffer was aspirated, and then cells were incubated with fresh lysis buffer for an additional 60 min. Cultures were then washed five times with 0.3 MKCI, 10 mMNa2HP04 (pH 7.5) and then five times with distilled water. Examination of the coverslips by phase-contrast microscopy indicated eomplete removal of intact cells and cell structures. RESULTS
Pattern of gag Surface cence
Immuno$uores-
The extracellular location of MuLV gag protein was investigated by immunofluorescence microscopy as shown in Fig. 1. For these experiments, M-MuLV alpha clone 19 cells were used. These cells are a cloned line of M-MuLV infected NIH/3T3 cells, and were previously found to show high levels of gag surface protein (Edwards and Fan, 1981). Indirect surface immunofluorescence was performed using rabbit antiserum to M-MuLV p30 and FITC-conjugated goat anti-rabbit IgG, the major gag protein in virus particles. All operations were performed in the presence of 10 mlM sodium azide to minimize rearrangement of surface proteins. Under the conditions used, uninfected NIH/3T3 cells showed virtually undetectable immunofluorescence with anti-p30 serum, and normal rabbit serum did not react with infected cells. As shown in the figure, two types of surface immunofluorescence were observed in the infected cells. When the plane of focus was on the upper (unattached) surface of the cell, fluorescence was in irregular patchy structures, possibly protrusions from the cell surface (Fig. 1D). However, the majority of immunofluorescence was observed when the plane of focus was at the attached surface of the cell (Fig. lA-C): A striking pattern
308
EDWARDS,
LIN,
AND
FAN
EXTRACELLULAR
of overlapping arrays or cable structures was observed. The location of these structures at the attached surface as well as the highly organized pattern suggested that the gag antigen may have been associated with the extracellular matrix, the network of protein and carbohydrate by which cells attach to the tissue culture dish (Chen et al., 1978; Ruoslahti et aZ., 1981). This suggestion was further strengthened when fluorescent and transmission light micrographs of the same field of stained cells were compared. As shown in Fig. 2, immunofluorescent cablelike arrays were observed in regions lacking cells or cell processes, as well as in regions where the cells were. ImmunoJluorescent lular Matrix
Staining
of Extracel-
Preparations
To directly test the extracellular nature of the gag fluorescent staining patterns, experiments were performed on cell-free extracellular matrix preparations. A procedure described by Chen et al. (1978) for preparation of chick extracellular matrices was used. Briefly, this procedure consisted of lysis of cell monolayers in Nonidet P-40 at pH 9.6 followed by extensive washing with 0.3 M KC1 and water. Phasecontrast microscopy of such preparations showed the removal of all intact cells as well as subcellular structures. Anti-p30 immunofluorescent staining of extracellular matrix preparations from M-MuLV alpha cells is shown in Fig. 3A and B. The staining patterns were essentially the same as observed at the attached surface of intact cells, confirming the association of gag antigen with extracellular structures. Figure 3C shows immunofluorescent staining of extracellular matrix preparations from M-MuLV (anti-p30) clone 12 cells. These cells were immunoselected
MuLV
gag ANTIGEN
309
from M-MuLV alpha cells for lack of cell surface gag antigen (Edwards and Fan, 1981). As shown, extracellular matrices from these cells show no gag immunofluorescence. Metabolic labeling with [YS]methionine in a parallel experiment indicated that approximately 0.4% of total cell protein was retained in the extracellular matrix preparations from M-MuLV (anti-p3O) clone 12 as well as from the MMuLV alpha and M-MuLV alpha clone 19 cells. Thus, the lack of immunofluorescent staining of the M-MuLV (anti-p30) clone 12 extracellular matrices was not due to the absence of such structures from these cells. Previous experiments (Edwards and Fan, 1981) indicated that the intracellular levels of protein immunoreactive with anti-p30 serum in M-MuLV-infected cells did not result from an artifactual leakage of intracellular gag antigens during the preparation and staining procedures. Attempts to characterize the gag antigen associated with the extracellular matrix by immunoprecipitation and gel electrophoresis of solubilized 35S-labeled matrices were unsuccessful. This could be due to the fact that (1) the gag antigen might represent a small fraction of the solubilized material, or that (2) the gag antigen might have been denatured during solubilization such that it was not recognized by anti-p30 serum. ImmunoJluorescent env Protein
Staining
of M-MuLV
To determine if the surface immunofluorescence pattern observed for gag antigen was unique, cells were also examined for the surface distribution of M-MuLV env glycoproteins using goat anti-MuLV gp70 serum and FITC conjugated pig anti-goat IgG. Anti-gp’70 fluorescence of MuLV alpha and M-MuLV (anti-p30) clone 12 cells is shown in Fig. 4. The gp70 antigenicity
FIG. 1. Localization of the gag surface antigen. Several views of surface immunofluoreseence using rabbit anti-p30 serum as the primary antibody and FITC-goat-anti-rabbit serum on M-MuLV alpha clone 19 cells. (A-C) Immunofluorescence at the attached surface. (D) The upper surface. The bar indicates 50 pm.
310
EDWARDS,
LIN,
AND
FAN
FIG. 2. Comparison of fluorescent and transmission micrographs. Surface immunofluorescence performed on M-MuLV alpha cells using anti-p30 serum as the primary antibody. (a) Fluorescence. (b) Phase contrast light micrograph of the same field as (A). The bar indicates 50 Wm.
was clustered in patches that were diffuse over the cell surface, and no significant differences were observed between MMuLV and M-MuLV (anti-p30) clone 12 cells in either pattern or intensity of fluorescence. No correlation between the fluorescence patterns obtained using anti-
gp70 and anti-p30 sera were noted, and anti-gp70 staining did not show extracellular fluorescence. Thus, there is apparently no physical association between extracellular gag antigen and surface gp70. It should be noted that gp70 is the major surface component of MuLV virus parti-
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MuLV
gag ANTIGEN
FIG. 2. Continued.
cles. The different staining patterns obtained with anti-gp70 and anti-p30 indicate that the anti-p30 staining does not result from binding of virus particles to the extracellular matrix. Immunojibrescent fected with
Other
Staining of Cells InMuLVs
Immunofluorescence microscopy of other lines of M-MuLV-infected NIH/3T3 fibro-
blasts showed the same staining patterns for surface gag proteins as described above, and it was of interest to know if cells infected with other strains of MuLV show a similar binding of gag antigen to extracellular matrices. Figures 5a and b show immunofluorescent staining with anti-p30 serum of NIH mouse embryo fibroblasts productively infected with AKR strain MuLV, and of mink lung fibroblasts in-
312
EDWARDS,
LIN,
AND
FAN
FIG. 3. Immunofluorescence of cell-free matrices. Cells were removed from coverslips by a combination of NP40 lysis at pH 9.6 and many washes with 0.3 M KC1 and HaO. Indirect immunofluorescence was performed as in Fig. 1 using anti-p30 serum. Fluorescent structures were essentially identical to those seen at the attached surface when cells were not removed. (A) M-MuLV alpha. (B) M-MuLV alpha clone 19. (C) M-MuLV (anti-p30) clone 12 cells. The bar indicates 50 pm.
fected with an “MCF” derivative of Rauscher MuLV (van Griensven and Vogt, 1980). In both of these cell lines, association of extracellular matrices and gag antigen was observed, although in the latter case the staining was less pronounced. These results indicate that the binding of gag antigen to extracellular matrices occurs for many (if not all) strains of MuLV, and that the extracellular matrices can be derived from species other than mouse. Figure 5c shows anti-p30 surface immunofluorescent staining of E8G2 lymphocytes (Ledbetter and Nowinski, 1977). These cells are interesting because they are the original serological typing line for the Gross cell surface antigen (GCSA).
EXTRACELLULAR
MuLV gag ANTIGEN
FIG. 4. Surface expression of gp70. Indirect immunofluorescence was performed using goat an1tigp70 serum and FITC-conjugated pig anti-goat IgG. (A) M-MuLV alpha cells; (B) M-MuLV (am ti~30) clone 12. Patchy fluorescence was seen over the entire surface of the cells, with no obvio lls difference between the upper and attached surface. The bar indicates 50 pm.
GCSA antigen was the first cell surface dY cosylated gag protein identified (Tung et cch, 1977; Ledbetter and Nowinski, 1977). TheBstaining pattern showed highly localizecd regions of gag staining, but no cablelikt : structures were observed. The lack of
cable-like structures might not bbe expected, since lymphocytes do not pr aoduce extracellular matrices. The highly localized staining pattern might represen t sublocalization of gag antigen in the E$G2 surface membrane, or it might rep] resent
EDWARDS,
FIG.
LIN,
AND
FAN
4. Continued.
“pa .tching” of antigenic determinants during the staining procedure (even though out in sodium Pro cedures were carried azicde and at 4”). Somewhat more diffuse, altl lough still localized, patterns of fluores cence were observed under the same con ditions of immunofluorescent labeling Wh en the same cells were studied using
antisera for two normal lymphocyte surface proteins, Thy-l and T-200 (not she bwn). DISCUSSION
The results reported here demonst rate the association of MuLV gag antigen with extracellular structures (presumably the
EXTRACELLULAR
FIG. 5. Surface gag antigen on other virus-infected cell lines. Indirect surface immunofluorescence using anti-p30 serum as primary antibody on (a) AKR P2 cells, an embryonic fibroblast line from AKR AKR mice, (b) mink lung cells infected with Rauscher MCF virus, and (c) EdG2 cells, a lymphoblast line which is the serological typing strain for the Gross cell surface antigen. The bar indicates 50 urn.
extracellular matrix) of infected fibroblasts. This appears to be a common phenomenon for different MuLV strains, and for infected cells derived from different
MuLV
gag ANTIGEN
315
species. The matrix-associated gag antigen did not result from attached virus particles, since the immunofluorescence patterns using anti-gp70 serum did not resemble the anti-p30 pattern. Furthermore, extensive washing of extracellular matrix preparationswith detergent and 0.3 MKCl (which would disrupt and dissociate virus particles) did not remove the antigen. It should be noted that the gag matrix-associated antigen does not display an obvious spatial relationship to intracellular gag protein. When cells are first permeabilized with acetone prior to immunofluorescent staining, a diffuse pattern of cytoplasmic gag antigen lacking structure or regularity is observed (Edwards and Fan, 1981). All of the characterizations of the extracellular gag antigen described in this report were performed using anti-p30 serum as the primary antibody. It is thus unclear at the present time which other determinants of the gag gene are present in the matrix-associated gag antigen. It should also be noted that while the major forms of cell surface and extracellular gag antigen are glycosylated (Ledbetter et al., 1978; Edwards and Fan, 1979), there is no direct evidence that the gag antigen associated with the extracellular matrix is glycosylated.
316
EDWARDS,
The molecular nature of the gag antigen binding to extracellular matrices was not investigated in these experiments. Several possibilities are suggested by previous work which identified molecular forms of cell surface and extracellular gag polyproteins. Lymphocytes and fibroblasts infected with AKR strain MuLV contain two glycosylated cell surface gag polyproteins of 85,000 and 95,000 daltons (gP85Q”Q) and gP9.!jgW) (Tung et ab, 1977; Ledbetter and Nowinski, 1977), and similar glycoproteins have been identified at the surface of Rauscher-MuLV infected fibroblasts (Buetti and Diggelmann, 1980b). It is possible that one of these polyproteins might represent the matrix-associated gag antigen. Other candidates for the matrixassociated gag antigen might be two gP95QaQ cleavage products of 55,000 and 40,000 daltons (gP55gW and gP40QW), which are released into the culture fluid of infected cells (Edwards and Fan, 1979; Ledbetter et al., 1978). It is possible that one or both of these proteins subsequently associates with extracellular matrices after release from the cell. Alternatively, cellsurface glycosylated gag polyprotein could associate with extracellular matrices as they are laid down, followed by release from the matrix of cleavage fragments by cellular or serum proteases. The nature of the attachment of gag antigen to extracellular matrices is currently under investigation. Extracellular matrices contain fibronectin, laminin, collagen, proteoglycans, and possibly cytoskeletal proteins such as myosin, actin, and tubulin (Ruoslahti et al., 1981; Chen et ab, 1978). It is possible that the gag antigen could associate with any one of these components. Recent preliminary experiments with antisera specific for fibronectin and laminin suggest that gag antigen is not directly associated with either of these two components of the extracellular matrix (H. Fan, Y. C. Lin, and E. Ruoslahti, unpublished). The staining patterns of this report could also formally be explained by the self-association of extracellular gag antigen into cable-like structures, rather than the association of the antigen with extra-
LIN,
AND
FAN
cellular matrices. Indeed, mature p30 protein can self-associate in solution (Burnette et al., 1976). However, this selfassociation gives rise to octomeric, globular structures, and not linear arrays. The association of MuLV gag antigen with extracellular matrices was an unexpected finding, and it is unclear if this protein has any biological effect. Extracellular matrices are important in maintaining the flattened morphology of fibroblasts (Ali et ah, 1977), and they are important in cell motility (Iqbal and Hynes, 1978). Thus, a protein which interacts with these matrices might alter the growth state of the cell. However, it should be noted that viruses such as M-MuLV do not morphologically transform fibroblasts. It should also be noted that in vuivo, the biological target cells of MuLV infection are lymphoid cells (Jaenisch et al, 1976), which are not known to elaborate extracellular matrices. It is possible that the presence of cell-surface gag antigen on a leukemic lymphocyte might confer upon this cell affinity for other cells or structures which normal lymphocytes lack. REFERENCES ALI, I. U., MAUTNER, V., LANNA, R., and HYNES, R. 0. (19’77). Restoration of normal morphology: Adhesion and cytoskeleton in transformed cells by addition of a transformation sensitive surface protein. Cell 11, 115-126. AOKI, T., HERBERMAN, R. B., JOHNSON, P. A., Lru, M., and STURM, M. M. (1972). Wild-type gross leukemia virus: Classification of soluble antigens. J. viral 10,1208-1219. BACHELER, L. T., and FAN, H. (1979). Multiple integration sites for Moloney murine leukemia virus in productively infected fibroblasts. J. Viral. 30, 657-667. BALTIMORE, D. (1974). Tumor viruses: 1974. Cold Spring Harbor Symp. Quant. Biol. 39, 1187-1200. BUETTI, E., and DIGGELMANN, H. (1980a). Avian oncovirus proteins expressed on the surface of infected cells. virology 102, 251-261. BUETTI, E., and DIGGELMANN, H. (1980b). Murine leukemia virus proteins expressed on the surface of infected cells in culture. J. viral 33, 936-944. BURNETTE,W.N.,HOLLADAY, L. A., and MITCHELL, W. M. (1976). Physical and chemical properties of Moloney murine leukemia virus p30 protein: A major core structural component exhibiting high
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helicity and self-association. J. Mol. BioZ. 107,131143. CHEN, L. B., MURRAY, A., SEGAL, R. A., BUSHNELL, A., and WALSH, M. L. (1978). Studies on intercellular LETS glycoprotein matrices. Cell 14,377-391. EDWARDS, S. A., and FAN, H. (1979). Gag-related polyproteins of Moloney murine leukemia virus: Evidence for independent synthesis of glycosylated and unglyeosylated forms. J. Viral. 30, 551-563. EDWARDS, S. A., and FAN, H. (1980). Sequence relationship of glycosylated and unglycosylated gag polyproteins of Moloney murine leukemia virus. J. viroz. 35, 41-51. EDWARDS, S. A., and FAN, H. (1981). Immunoselection and characterization of Moloney murine leukemia virus-infected cell lines deficient in surface gag antigen expression. virolog?d, 113,95-108. EISENMAN, R., and VOGT, V. (1978). The biosynthesis of oncovirus proteins. Biochim. Biophys. Acta 473, 187-239. EVANS, L. H., DRESSLER, S., and KABAT, D. (1977). Synthesis and glycosylation of polyprotein precursor to the internal core proteins of Friend murine leukemia virus. J. viral. 24, 865-874. FITTING, T., RUTA, M., and KABAT, D. (1981). Mutant cells that abnormally process plasma membrane glycoproteins encoded by murine leukemia virus. Cell 24, 847-858. IGBAL, U. A., and HYNES, R. 0. (1978). Effect of LETS glycoprotein on cell mobility. Cell 14, 439-446. JAENISCH, R., FAN, H., and CROKER, B. (1975). Infection of preimplantation mouse embryos and of newborn mice with leukemia virus: Tissue distribution of viral DNA and RNA and leukemogenesis in the adult animal. Proc. Nat. Aead Ski. USA 72, 4008-4012. LEDBETTER, J. A. (1979). Two-dimensional analysis of murine leukemia virus gag gene polyproteins. Virology 95, 85-98. LEDBETTER, J., and NOWINSKI, R. C. (1977). Identification of the Gross cell surface antigen associated with murine leukemia virus infected cells. J. I%ol. 23, 315-322. LEDBETTER, J. A., NOWINSKI, R. C., and EISENMAN, R. N. (1978). Biosynthesis and metabolism of viral proteins expressed on the surface of murine leukemia virus-infected cells. virology 91, 116-129.
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LERNER, R. A., JENSEN, F., KENNEL, S. J., DIXON, F. J., ROCHES, G. D., and FRANKE, U. (1972). Karyotypic, virologic and immunologic analyses of two continuous lymphocyte lines established from New Zealand black mice: Possible relationship of chromosomal mosaicism to autoimmunity. Proc. Nat. Acad
5%. USA 69,2967-2969.
MUELLER-LANTZSCH, N., and FAN, H. (1976). Monospecific immunoprecipitation of murine leukemia virus polyribosomes: Identification of p30 protein specific mRNA. Cell 9,579-588. NEIL, J. C., SMART, J. E., HAYMAN, M. J., and JARRETT, 0. (1980). Polypeptides of feline leukemia virus: A glycosylated gag-related protein is released into culture fluids. lrirology 105,250-253. OLD, L. J., BOYSE, E. A., and STOCKERT, E. (1965). The G (Gross) leukemia antigen. Cancer Rex 25, 813-819. OROSZLAN, S., HENDERSON, L. E., COPELAND, T. D., SCHLLJTZ,A. M., and RABIN, E. M. (1980). In “Biosynthesis, Modification and Processing of Cellular and Viral Polyproteins” (G. Koch and D. Richter, eds.). Academic Press, New York. RUOSLAHTI, E., ENGVALL, E., and HAYMAN, E. G. (1981). Fibronectin: Current concepts of its structure and function. Collagen Related Rex 1, 95-128. SCHULTZ,A. M., and OROSZLAN,S. (1978). Murine leukemia virus gag polyproteins: The peptide chain unique to Pr80 is located at the amino terminus. virology 91, 481-486. SCHULTZ, A. M., RABIN, E. H., and OROSZLAN, S. (1979). Post-translational modification of Rauscher leukemia virus precursor polyproteins encoded by the gag gene. J. viral. 30, 255-278. TUNG, J. S., YOSHIKI, P., and FLEISSNER, E. (1977). A core polyprotein of murine leukemia virus on the surface of mouse leukemia cells. Cell 9, 573-578. VAN GRIENSVEN, L. J. L. D., and VOGT, M. (1980). Rauscher “mink cell focus-inducing” (MCF) virus causes erythroleukemia in mice: Its isolation and properties. firology 101, 376-388. WHITELEY, S. A., and NASO, R. B. (1981). Comparative analysis: Intracellular precursor polyproteins of baboon endogenous retroviruses and human viral isolate HL23V. J. Vzrol. 37, 860-870.
Association
(1982)
of Murine Leukemia Virus gag Antigen with Extracellular Matrices in Productively Infected Mouse Cells
STEVEN The Tumor
A. EDWARDS,l
Virology Laboratory,
YING-CHIH
LIN,
The Salk Institute, P.O. Box
Received June
AND HUNG
85800,
San
FAN’
Diego, Califin-nia
92138
15, 1981; accepted September 2, 1981
The gag gene of murine leukemia virus (MuLV) encodes a nonstructural glycosylated polyprotein which appears at the cell surface, in addition to the polyprotein precursor for the virion internal structural proteins. The surface localization of gag nonstructural protein is reported here. Immunofluorescent staining of unfixed monolayers of Moloney MuLV-infected NIH/3T3 cells using anti-p30 serum as the primary antibody revealed an unusual pattern of gag antigen at the attached cell surface: highly organized cable-like structures. Stained cable-like structures were also observed in regions lacking cells or cell processes, which suggested that extracellular gag antigen may be associated with extracellular matrices. This was supported by the fact that detergent treatment of cell monolayers in a manner designed to removed intact cells but preserve extracellular matrices did not affect the cable-like anti-p30 staining patterns. Immunofluorescent staining with anti-gp70 serum revealed a different pattern than the anti-p30 staining, which indicates that surface gag antigen and envelope glycoprotein are not physically associated at the cell surface. Similar staining patterns were observed in mouse cells productively infected with a different strain of MuLV (AKR), and in mink cells productively infected with a mink cell focus-inducing (MCF) derivative of Rauscher MuLV.
culture medium (Edwards and Fan, 1979; Ledbetter et ah, 1978). In comparison to the intracellular gag precursor polyprotein for the virion internal structural proteins (Pr658”9), glycosylated gag polyprotein contains carbohydrate side-chains and additional amino-terminal peptide sequences (Schultz and Oroszlan, 1978; Edwards and Fan, 1979, 1980). A biological function has not yet been assigned to glycosylated gag polyproteins. Recent work from our laboratory suggests a correlation between surface gag expression and virus production (Edwards and Fan, 1981), although somewhat different conclusions were reached by other workers using a different host cell (Fitting et ah, 1981). Similar glycosylated gag polyproteins have been observed in cells infected with feline leukemia virus (Neil et al, 1980) and baboon endogenous virus (Whitely and Naso, 19Sl), and cells infected with avian sarcoma virus may contain minor amounts of surface gag polyprotein (Buetti and Diggelmann, 1980a). Other major classes
INTRODUCTION
Murine leukemia virus (MuLV), like other nondefective retroviruses, contains three genes within the RNA genome, gag, pal, and env, which encode the internal structural proteins, reverse transcriptase, and envelope glycoprotein, respectively (Baltimore, 1974). All of these genes are initially translated as polyprotein precursors which are subsequently cleaved to the mature viral proteins (Eiseman and Vogt, 1978). An unusual feature of MuLV is that its gag gene codes for a second, nonstructural polyprotein which is glycosylated and either associated with the cell surface (Tung et ab, 19’7’7; Ledbetter and Nowinski, 1977; Evans et al., 1977) or shed into the 1 Present address: Peptide Biology Laboratory, The Salk Institute, P.O. Box 85800, San Diego, Calif. 92138. ZTo whom requests for reprints should be addressed. Present address: Department of Molecular Biology & Biochemistry, University of California, Irvine, Calif. 92717. 0042-6822/82/010306-12.$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
306
EXTRACELLULAR
of retroviruses appear to lack an analogous protein. During the course of these experiments, we observed an unusual location of extracellular gag antigen by indirect surface immunofluorescence using anti-MuLV p30 serum: Most of the detectable antigen was in the extracellular matrix of the infected cells. These observations are described in the results reported here. MATERIALS
AND
METHODS
Cells. M-MuLV alpha, M-MuLV alpha clone 19, and M-MuLV(anti-p30) clone 12 are all NIH/3T3 mouse fibroblasts infected with Moloney MuLV (M-MuLV). The infection, cloning, and maintenance of these lines have been described previously (Edwards and Fan, 1981). AKR P2 cells are a line of embryonic NIH mouse fibroblasts productively infected with AKR strain MuLV, and were provided by Dr. Robert Nowinski. E&G2 cells are leukemia cells induced by AKR MuLV, and are the serological typing line for the Gross cell surface antigen (GCSA, Old et al, 1965; Aoki et al., 1972); they were also provided by Dr. Nowinski (Ledbetter and Nowinski, 1977). Mink lung fibroblasts productively infected with a “mink cell focus-inducing” (MCF) derivative of Rauscher MuLV were supplied by Dr. Marguerite Vogt (van Griensven and Vogt, 1980). Antisera. Rabbit antiserum to M-MuLV p30 was prepared in this laboratory (Mueller-Lantsch and Fan, 1976) and has been used extensively in previous investigations (Edwards and Fan, 1979,1980,1981). Goat antiserum to SCRF 60A MuLV (Lerner et ab, 1972) was a gift from Dr. Stephen Kennel. Goat anti-rabbit IgG and pig anti-goat IgG were purchased from Cappel Laboratories. Indirect immunojhwescence. Indirect immunofluorescence was performed on sparse cultures growing on glass coverslips. Details of this procedure have been previously reported (Edwards and Fan, 1981). Preparation of cell-free matrices. The procedure used was that of Chen et al. (1978). Subconfluent cultures growing on
MuLV
gag ANTIGEN
307
glass coverslips were washed three times with phosphate-buffered saline solution, followed by washing three times with buffer containing 0.1 M Na2P04, 2 mM MgC12, and 2 mM EGTA (pH 9.6). Coverslips were then covered with 2 ml of lysis buffer containing 8 mMNa2HP04, 1% NP40, pH 9.6 for 20 min at 0”. The lysis buffer was aspirated, and then cells were incubated with fresh lysis buffer for an additional 60 min. Cultures were then washed five times with 0.3 MKCI, 10 mMNa2HP04 (pH 7.5) and then five times with distilled water. Examination of the coverslips by phase-contrast microscopy indicated eomplete removal of intact cells and cell structures. RESULTS
Pattern of gag Surface cence
Immuno$uores-
The extracellular location of MuLV gag protein was investigated by immunofluorescence microscopy as shown in Fig. 1. For these experiments, M-MuLV alpha clone 19 cells were used. These cells are a cloned line of M-MuLV infected NIH/3T3 cells, and were previously found to show high levels of gag surface protein (Edwards and Fan, 1981). Indirect surface immunofluorescence was performed using rabbit antiserum to M-MuLV p30 and FITC-conjugated goat anti-rabbit IgG, the major gag protein in virus particles. All operations were performed in the presence of 10 mlM sodium azide to minimize rearrangement of surface proteins. Under the conditions used, uninfected NIH/3T3 cells showed virtually undetectable immunofluorescence with anti-p30 serum, and normal rabbit serum did not react with infected cells. As shown in the figure, two types of surface immunofluorescence were observed in the infected cells. When the plane of focus was on the upper (unattached) surface of the cell, fluorescence was in irregular patchy structures, possibly protrusions from the cell surface (Fig. 1D). However, the majority of immunofluorescence was observed when the plane of focus was at the attached surface of the cell (Fig. lA-C): A striking pattern
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of overlapping arrays or cable structures was observed. The location of these structures at the attached surface as well as the highly organized pattern suggested that the gag antigen may have been associated with the extracellular matrix, the network of protein and carbohydrate by which cells attach to the tissue culture dish (Chen et al., 1978; Ruoslahti et aZ., 1981). This suggestion was further strengthened when fluorescent and transmission light micrographs of the same field of stained cells were compared. As shown in Fig. 2, immunofluorescent cablelike arrays were observed in regions lacking cells or cell processes, as well as in regions where the cells were. ImmunoJluorescent lular Matrix
Staining
of Extracel-
Preparations
To directly test the extracellular nature of the gag fluorescent staining patterns, experiments were performed on cell-free extracellular matrix preparations. A procedure described by Chen et al. (1978) for preparation of chick extracellular matrices was used. Briefly, this procedure consisted of lysis of cell monolayers in Nonidet P-40 at pH 9.6 followed by extensive washing with 0.3 M KC1 and water. Phasecontrast microscopy of such preparations showed the removal of all intact cells as well as subcellular structures. Anti-p30 immunofluorescent staining of extracellular matrix preparations from M-MuLV alpha cells is shown in Fig. 3A and B. The staining patterns were essentially the same as observed at the attached surface of intact cells, confirming the association of gag antigen with extracellular structures. Figure 3C shows immunofluorescent staining of extracellular matrix preparations from M-MuLV (anti-p30) clone 12 cells. These cells were immunoselected
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gag ANTIGEN
309
from M-MuLV alpha cells for lack of cell surface gag antigen (Edwards and Fan, 1981). As shown, extracellular matrices from these cells show no gag immunofluorescence. Metabolic labeling with [YS]methionine in a parallel experiment indicated that approximately 0.4% of total cell protein was retained in the extracellular matrix preparations from M-MuLV (anti-p3O) clone 12 as well as from the MMuLV alpha and M-MuLV alpha clone 19 cells. Thus, the lack of immunofluorescent staining of the M-MuLV (anti-p30) clone 12 extracellular matrices was not due to the absence of such structures from these cells. Previous experiments (Edwards and Fan, 1981) indicated that the intracellular levels of protein immunoreactive with anti-p30 serum in M-MuLV-infected cells did not result from an artifactual leakage of intracellular gag antigens during the preparation and staining procedures. Attempts to characterize the gag antigen associated with the extracellular matrix by immunoprecipitation and gel electrophoresis of solubilized 35S-labeled matrices were unsuccessful. This could be due to the fact that (1) the gag antigen might represent a small fraction of the solubilized material, or that (2) the gag antigen might have been denatured during solubilization such that it was not recognized by anti-p30 serum. ImmunoJluorescent env Protein
Staining
of M-MuLV
To determine if the surface immunofluorescence pattern observed for gag antigen was unique, cells were also examined for the surface distribution of M-MuLV env glycoproteins using goat anti-MuLV gp70 serum and FITC conjugated pig anti-goat IgG. Anti-gp’70 fluorescence of MuLV alpha and M-MuLV (anti-p30) clone 12 cells is shown in Fig. 4. The gp70 antigenicity
FIG. 1. Localization of the gag surface antigen. Several views of surface immunofluoreseence using rabbit anti-p30 serum as the primary antibody and FITC-goat-anti-rabbit serum on M-MuLV alpha clone 19 cells. (A-C) Immunofluorescence at the attached surface. (D) The upper surface. The bar indicates 50 pm.
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EDWARDS,
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FIG. 2. Comparison of fluorescent and transmission micrographs. Surface immunofluorescence performed on M-MuLV alpha cells using anti-p30 serum as the primary antibody. (a) Fluorescence. (b) Phase contrast light micrograph of the same field as (A). The bar indicates 50 Wm.
was clustered in patches that were diffuse over the cell surface, and no significant differences were observed between MMuLV and M-MuLV (anti-p30) clone 12 cells in either pattern or intensity of fluorescence. No correlation between the fluorescence patterns obtained using anti-
gp70 and anti-p30 sera were noted, and anti-gp70 staining did not show extracellular fluorescence. Thus, there is apparently no physical association between extracellular gag antigen and surface gp70. It should be noted that gp70 is the major surface component of MuLV virus parti-
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MuLV
gag ANTIGEN
FIG. 2. Continued.
cles. The different staining patterns obtained with anti-gp70 and anti-p30 indicate that the anti-p30 staining does not result from binding of virus particles to the extracellular matrix. Immunojibrescent fected with
Other
Staining of Cells InMuLVs
Immunofluorescence microscopy of other lines of M-MuLV-infected NIH/3T3 fibro-
blasts showed the same staining patterns for surface gag proteins as described above, and it was of interest to know if cells infected with other strains of MuLV show a similar binding of gag antigen to extracellular matrices. Figures 5a and b show immunofluorescent staining with anti-p30 serum of NIH mouse embryo fibroblasts productively infected with AKR strain MuLV, and of mink lung fibroblasts in-
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FIG. 3. Immunofluorescence of cell-free matrices. Cells were removed from coverslips by a combination of NP40 lysis at pH 9.6 and many washes with 0.3 M KC1 and HaO. Indirect immunofluorescence was performed as in Fig. 1 using anti-p30 serum. Fluorescent structures were essentially identical to those seen at the attached surface when cells were not removed. (A) M-MuLV alpha. (B) M-MuLV alpha clone 19. (C) M-MuLV (anti-p30) clone 12 cells. The bar indicates 50 pm.
fected with an “MCF” derivative of Rauscher MuLV (van Griensven and Vogt, 1980). In both of these cell lines, association of extracellular matrices and gag antigen was observed, although in the latter case the staining was less pronounced. These results indicate that the binding of gag antigen to extracellular matrices occurs for many (if not all) strains of MuLV, and that the extracellular matrices can be derived from species other than mouse. Figure 5c shows anti-p30 surface immunofluorescent staining of E8G2 lymphocytes (Ledbetter and Nowinski, 1977). These cells are interesting because they are the original serological typing line for the Gross cell surface antigen (GCSA).
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MuLV gag ANTIGEN
FIG. 4. Surface expression of gp70. Indirect immunofluorescence was performed using goat an1tigp70 serum and FITC-conjugated pig anti-goat IgG. (A) M-MuLV alpha cells; (B) M-MuLV (am ti~30) clone 12. Patchy fluorescence was seen over the entire surface of the cells, with no obvio lls difference between the upper and attached surface. The bar indicates 50 pm.
GCSA antigen was the first cell surface dY cosylated gag protein identified (Tung et cch, 1977; Ledbetter and Nowinski, 1977). TheBstaining pattern showed highly localizecd regions of gag staining, but no cablelikt : structures were observed. The lack of
cable-like structures might not bbe expected, since lymphocytes do not pr aoduce extracellular matrices. The highly localized staining pattern might represen t sublocalization of gag antigen in the E$G2 surface membrane, or it might rep] resent
EDWARDS,
FIG.
LIN,
AND
FAN
4. Continued.
“pa .tching” of antigenic determinants during the staining procedure (even though out in sodium Pro cedures were carried azicde and at 4”). Somewhat more diffuse, altl lough still localized, patterns of fluores cence were observed under the same con ditions of immunofluorescent labeling Wh en the same cells were studied using
antisera for two normal lymphocyte surface proteins, Thy-l and T-200 (not she bwn). DISCUSSION
The results reported here demonst rate the association of MuLV gag antigen with extracellular structures (presumably the
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FIG. 5. Surface gag antigen on other virus-infected cell lines. Indirect surface immunofluorescence using anti-p30 serum as primary antibody on (a) AKR P2 cells, an embryonic fibroblast line from AKR AKR mice, (b) mink lung cells infected with Rauscher MCF virus, and (c) EdG2 cells, a lymphoblast line which is the serological typing strain for the Gross cell surface antigen. The bar indicates 50 urn.
extracellular matrix) of infected fibroblasts. This appears to be a common phenomenon for different MuLV strains, and for infected cells derived from different
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315
species. The matrix-associated gag antigen did not result from attached virus particles, since the immunofluorescence patterns using anti-gp70 serum did not resemble the anti-p30 pattern. Furthermore, extensive washing of extracellular matrix preparationswith detergent and 0.3 MKCl (which would disrupt and dissociate virus particles) did not remove the antigen. It should be noted that the gag matrix-associated antigen does not display an obvious spatial relationship to intracellular gag protein. When cells are first permeabilized with acetone prior to immunofluorescent staining, a diffuse pattern of cytoplasmic gag antigen lacking structure or regularity is observed (Edwards and Fan, 1981). All of the characterizations of the extracellular gag antigen described in this report were performed using anti-p30 serum as the primary antibody. It is thus unclear at the present time which other determinants of the gag gene are present in the matrix-associated gag antigen. It should also be noted that while the major forms of cell surface and extracellular gag antigen are glycosylated (Ledbetter et al., 1978; Edwards and Fan, 1979), there is no direct evidence that the gag antigen associated with the extracellular matrix is glycosylated.
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The molecular nature of the gag antigen binding to extracellular matrices was not investigated in these experiments. Several possibilities are suggested by previous work which identified molecular forms of cell surface and extracellular gag polyproteins. Lymphocytes and fibroblasts infected with AKR strain MuLV contain two glycosylated cell surface gag polyproteins of 85,000 and 95,000 daltons (gP85Q”Q) and gP9.!jgW) (Tung et ab, 1977; Ledbetter and Nowinski, 1977), and similar glycoproteins have been identified at the surface of Rauscher-MuLV infected fibroblasts (Buetti and Diggelmann, 1980b). It is possible that one of these polyproteins might represent the matrix-associated gag antigen. Other candidates for the matrixassociated gag antigen might be two gP95QaQ cleavage products of 55,000 and 40,000 daltons (gP55gW and gP40QW), which are released into the culture fluid of infected cells (Edwards and Fan, 1979; Ledbetter et al., 1978). It is possible that one or both of these proteins subsequently associates with extracellular matrices after release from the cell. Alternatively, cellsurface glycosylated gag polyprotein could associate with extracellular matrices as they are laid down, followed by release from the matrix of cleavage fragments by cellular or serum proteases. The nature of the attachment of gag antigen to extracellular matrices is currently under investigation. Extracellular matrices contain fibronectin, laminin, collagen, proteoglycans, and possibly cytoskeletal proteins such as myosin, actin, and tubulin (Ruoslahti et al., 1981; Chen et ab, 1978). It is possible that the gag antigen could associate with any one of these components. Recent preliminary experiments with antisera specific for fibronectin and laminin suggest that gag antigen is not directly associated with either of these two components of the extracellular matrix (H. Fan, Y. C. Lin, and E. Ruoslahti, unpublished). The staining patterns of this report could also formally be explained by the self-association of extracellular gag antigen into cable-like structures, rather than the association of the antigen with extra-
LIN,
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FAN
cellular matrices. Indeed, mature p30 protein can self-associate in solution (Burnette et al., 1976). However, this selfassociation gives rise to octomeric, globular structures, and not linear arrays. The association of MuLV gag antigen with extracellular matrices was an unexpected finding, and it is unclear if this protein has any biological effect. Extracellular matrices are important in maintaining the flattened morphology of fibroblasts (Ali et ah, 1977), and they are important in cell motility (Iqbal and Hynes, 1978). Thus, a protein which interacts with these matrices might alter the growth state of the cell. However, it should be noted that viruses such as M-MuLV do not morphologically transform fibroblasts. It should also be noted that in vuivo, the biological target cells of MuLV infection are lymphoid cells (Jaenisch et al, 1976), which are not known to elaborate extracellular matrices. It is possible that the presence of cell-surface gag antigen on a leukemic lymphocyte might confer upon this cell affinity for other cells or structures which normal lymphocytes lack. REFERENCES ALI, I. U., MAUTNER, V., LANNA, R., and HYNES, R. 0. (19’77). Restoration of normal morphology: Adhesion and cytoskeleton in transformed cells by addition of a transformation sensitive surface protein. Cell 11, 115-126. AOKI, T., HERBERMAN, R. B., JOHNSON, P. A., Lru, M., and STURM, M. M. (1972). Wild-type gross leukemia virus: Classification of soluble antigens. J. viral 10,1208-1219. BACHELER, L. T., and FAN, H. (1979). Multiple integration sites for Moloney murine leukemia virus in productively infected fibroblasts. J. Viral. 30, 657-667. BALTIMORE, D. (1974). Tumor viruses: 1974. Cold Spring Harbor Symp. Quant. Biol. 39, 1187-1200. BUETTI, E., and DIGGELMANN, H. (1980a). Avian oncovirus proteins expressed on the surface of infected cells. virology 102, 251-261. BUETTI, E., and DIGGELMANN, H. (1980b). Murine leukemia virus proteins expressed on the surface of infected cells in culture. J. viral 33, 936-944. BURNETTE,W.N.,HOLLADAY, L. A., and MITCHELL, W. M. (1976). Physical and chemical properties of Moloney murine leukemia virus p30 protein: A major core structural component exhibiting high
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