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Expression of cell surface friend virus gp70 does not block reinfection by ecotropic murine leukemia viruses
VIROLOGY
115,
I%-129
(1981)
Expression of Cell Surface Friend Virus gp70 Does Not Block Reinfection by Ecotropic Murine Leukemia Viruses B. CHESEBRO’,
J. K. COLLINS,
K. WEHRLY,
J. NISHIO,
AND M. CLOYD
U. S. Department of Health and Human Services, Public Health Service, National Institutes of Health, National Institute of Allergy and Iv&ctious Diseases, Laboratory of Persisttmt Viral Diseases, Rocky Mountain Laboratories, Hamilton, Montana 598.40 Received Apn’l 7, 1981; accepted June 25, 1981 The effect of cell surface gp70 expression on reinfection by murine leukemia virus was studied in Friend virus (FV)-induced erythroleukemia cell clones. A clone (2C) which released gp70-positive infectious virions and expressed large amounts of cell surface ecotropic FV gp70 was not superinfectable by ecotropic viruses, whereas another clone (7C) which expressed similar amounts of cell surface FV gp70 but released gp70-deficient virions showed no resistance to superinfection. The lack of interference to superinfection of 7C cells suggested that newly budded gp70-positive virions, rather than cell surface gp70 molecules, were responsible for the interference to superinfection seen in 2C cells which released infectious virus. INTRODUCTION
Acute exposure of uninfected cells to one oncornavirus can interfere with subsequent infection by a second related oncornavirus (Steck and Rubin, 1966a). This interference was shown to occur at the level of virus-specific cell surface receptors involved in the infection process (Vogt and Ishizaki, 1965; Steck and Rubin, 1966b). These receptors appear to recognize viral envelope glycoproteins. This was suggested by the observation that oncornaviruses can be divided into interference groups which appear to correlate with relatedness of the viral envelope glycoproteins (Vogt and Ishizaki, 1966). Furthermore, exposure of uninfected cells to large amounts of purified viral envelope glycoprotein, gp70, has been shown to cause interference (Hunsmann et al., 1974). Interference has also been frequently observed in chronically infected cells; such cells cannot be superinfected by viruses from related interference groups. However, the mechanism of this interference ’ To whom reprint requests should be addressed.
has not been adequately addressed. It is possible that since oncornavirus-releasing cells have large amounts of viral envelope glycoprotein on their cell surfaces, production and/or release of this protein could interfere with superinfection by related viruses by receptor blockade similar to that seen in uninfected cells. We have recently studied a Friend virus-induced erythroleukemia cell line which releases gp70-deficient virus particles but expresses amounts of cell surface gp70 similar to productively infected cells (Collins and Chesbro, 1980a, b; Collins et al., 1981). Surprisingly, this cell line can be easily superinfected with homologous ecotropic virus suggesting that cell surface expression of viral envelope glycoproteins per se does not lead to the interference with superinfection seen in chronically infected cells. MATERIALS
AND METHODS
Cells and viruses. Clones 2C, 7C, and 6C derived from the Y57 erythroleukemia line induced by B-tropic FV complex in a (C57BL/lO X A.BY)F1 mouse were de125
0042-6822/81/150125-05$02.00/O Copyright Q 1981 by Academic Press. Inc. All rights of reproduction in any form reserved
126
CHESEBRO
scribed previously (Collins and Chesebro, 1980a). F-MuLV was assayed on the D56 clone of mouse SL cells (Bassin et al., 1971). C243 and 3T3FL cells (Bassin et al., 1970) were kindly provided by Dr. Alan Rein, Frederick Cancer Research Center, Frederick, Maryland. Moloney sarcoma virus (MSV) pseudotypes were made by superinfection of C243 (SL) cells with FMuLV. MSV was assayed on 3T3FL cells. Friend murine leukemia helper virus (FMuLV) was obtained by limiting dilution infection of SC-1 cells (Hartley and Rowe, 1975) with a FV-induced leukemic spleen homogenate from a (BIO.A X A)Fi mouse. Peptide mapping. Cells from the 2C and 7C clones were surface labeled with ‘%I, lysed, precleared, immunoprecipitated, and digested with V3 protease as described (Collins and Chesebro, 1980b). Digests were separated by SDS-PAGE using 10% acrylamide gels, and were analyzed by autoradiography with intensifying screens. Membrane immunofluorescence. Monoclonal antibodies specific for F-MuLV were obtained from culture supernatants of hybridomas. These reagents were used in indirect membrane immunofluorescence tests on live cells with FITCconjugated goat anti-mouse Ig serum. The origin of the hybridomas and the details of the fluorescence assay were described previously (Chesebro et al., 1981). RESULTS
F-MuLV Reirlfection of an F-MuLV Positive Cell Line
gp70-
Previous results indicated that two variant FV-induced erythroleukemia cell clones, 6C and 7C, did not produce infectious MuLV as detected by infectious center assay on mouse S+L cells; however, both of these clones could be reinfected with ecotropic F-MuLV (Table 1) or MuLV derived from a B/T-L virus-induced leukemia cell line (Collins and Chesebro, 1980a). In the case of clone 7C, reinfectability with ecotropic F-MuLV was an unexpected finding because these cells expressed cell surface F-MuLV gp70 in amounts equal to that on a virus-producing clone, 2C (Collins et al., 1980). In contrast, clone 6C was negative for cell sur-
ET AL. TABLE REINFECTION
1
OF ERYTHROLEUKEM~A WITH F-MuLV Infectious
Erythroleukemia clone 6C 7C 2c
CELL CLONES
centers/lo6 cells”
Preinfection
Postinfeetion”
40 40 3.8 x lo4
3.6 x lo4 3.4 x lo4 5.4 x lo4
’ Mouse S+L- assay on D56 cells. * l-2 X lo6 cells were incubated 20 min at 37” with DEAE-dextran (0.005 mg/ml), then cells were washed once with buffer and infected with F-MuLV at a multiplicity of 2 PFU/cell. Cells were cultured overnight and then assayed as infectious centers.
face F-MuLV gp70, although the cells did express a xenotropic MuLV gp70 (Collins and Chesebro, 1980b). Reinfectability of this line by F-MuLV was not surprising since ecotropic and xenotropic MuLV belong to different interference subgroups (Besmer and Baltimore, 1977). Comparison 6C
of gp70 frcmz Clones 2C, 7C, and
Since the quantitation of gp70 molecules on clones 2C and 7C was based on a radioimmunoassay, further data were sought to determine if small antigenic or peptide sequence differences were present which could explain the different reinfection properties of the two cell clones. The clones were analyzed by membrane immunofluorescence with a panel of monoclonal antibodies specific for F-MuLV gp70 and p15 (Table 2). Clone 7C and 2C cells reacted with each of the monoclonal antigp70 antibodies, and the patterns of fluorescence appeared indistinguishable. Since each monoclonal anti-gp70 antibody detected a unique antigenic determinant on the gp70 molecules (Chesebro et al., 1981), these data indicated that all gp70 determinants detected by this antibody panel were present on both 2C and 7C cells. In contrast, clone 6C did not react with any anti-F-MuLV gp70 monoclonal antibodies. Anti-p15 monoclonal antibodies
REINFECTION
OF gp’70-POSITIVE
TABLE 2 ANALYSIS OF CELL SURFACE F-MuLV ANTIGENS ON ERYTHROLEUKEMIA CLONES USING MONOCLONAL ANTIBODIES Membrane immunofluorescence on erythroleukemia clones” Hybridoma 48 273 55 350 59 66 47 307 34 257 None
Specificity
2C
7C
6C
Anti-gp’70 Antigp70 Anti-gp70 Anti-gp70 Anti-gp70 Anti-gp70 Antigp70 Anti-gp70
+ + + + + + + +
+ + + + + + + +
-
+ + -
-
+ + -
Anti-p15 Anti-p15 -
127
CELLS
with F-MuLV envelope (F-MuLV pseudotype) was used. Infection by MSV could be measured on 3T3FL cells even in the presence of MuLV infection. When 6C, 7C, and 2C cells were infected with various dilutions of MSV (F-MuLV), the 7C and 6C cells appeared identical and loo-fold more infectious centers were seen with these clones than with the 2C clone infected at the same multiplicity (Fig. 2). Thus, in spite of similar quantitative expression of cell surface F-MuLV gp70 in 7C and 2C cells, these clones differed markedly in reinfectability by MSV (FMuLV). DISCUSSION
The present experiments demonstrated that expression of cell surface F-MuLV 2c 1234
7c 5678
u (+) Greater than 95% of cells positive; (-) less than 1% of cells positive.
reacted with clones 2C and 6C, but failed to react with clone 7C which was shown previously to express reduced amounts of gag cell surface antigens detected by conventional antisera (Collins et al., 1980). In order to confirm the identity of the gp’70 molecules found on 7C and 2C cells, these gp7Os were compared by peptide mapping. Anti-gp70 immunoprecipitates from ‘?-surface-labeled cells were digested with V8 protease for various times and then compared by SDS-PAGE. The results indicated that these gp7Os were indistinguishable from each other (Fig. 1). Previous results with this technique indicated that xenotropic MuLV gp70 from clone 6C differed markedly from that on clone 2C (Collins and Chesebro, 1980b). Infection of SC, XT, and 2C Cells by MSV (F-MuL V) Since 2C cells were already high producers of MuLV detectable on S+L- cells, these cells could not be tested for interference using F-MuLV alone. Therefore, to compare the infectability of 7C and 2C clones, which expressed the same cell surface gp70, Moloney sarcoma virus (MSV)
FIG. 1. V8 protease digestion of ‘?-gp70 from 2C and 7C cells. Tracks 1 and 5, undigested, tracks 2 and 6, digested 46 min; tracks 3 and 7, digested 90 min; tracks 4 and 8, digested 180 min. Analysis by SDSPAGE in 10% acrylamide.
128
CHESEBRO
UNDIL.
l/ID
VIRUS DILUTION l/100
1/1000
FIG. 2. Infection of 2C, ?C, and 6C cells with MSV (F-MuLV pseudotype) at various dilutions was followed by analysis of cells as MSV infectious centers on 3T3FL cells.
gp’70 on 7C erythroleukemia cells did not lead to interference with reinfection of these cells by F-MuLV or the F-MuLV pseudotype of MSV. This lack of interference was not due to a quantitative difference in gp70 expression on 7C cells since the amount of gp70 on 7C cells was similar to the gp70 on the 2C erythroleukemia clone, which did show interference to superinfection. Furthermore, the gp70 molecules on these two cell lines had identical peptide maps and demonstrated identical reactivity with monoclonal antibodies detecting different F-MuLV gp70-specific antigens. Therefore, any differences in these two gp7Os would most likely be quite minor. It would be surprising if differences so difficult to detect could give rise to an altered interference pattern because even obvious differences in gp70 antigenicity or peptide maps do not eliminate the cross-interference seen between AKR and FMR serogroups of ecotropic viruses (Sarma et al., 1967; Hartley and Rowe, 1976). However, these results do not completely exclude the possibility that the gp70 on 7C cells had some alteration or defect in a small portion of the molecule critical for interference. If so, then more detailed biochemical comparison of the gp7Os on 2C and 7C cells might provide an
ET AL.
indication as to which portion of the gp70 molecule is responsible for interference, and presumably also receptor specificity. Although 7C cells do not produce infectious MuLV, these cells do release noninfectious viral particles which incorporate [‘Hluridine into 70 S RNA and band at the usual MuLV buoyant density (Collins and Chesebro, 1981). The lack of infectivity in these particles is not surprising as they contain no active reverse transcriptase. Furthermore, they do not process the gag core polyprotein pr65 into its smaller components. Possibly as a result of this core defect, virions from 7C cells incorporate markedly reduced amounts of gp70 and p15E into their envelopes in spite of the abundance of gp70 on the cell surface (Collins and Chesebro, 1981). The reduced amounts of gp70 in 7C virions might account for the lack of interference to superinfection of 7C cells by nondefective viruses. This would be expected if interference in virus producer cells was medicated by blockade of cell surface receptors by virus particles just released from the same cell surface. Due to lack of multivalent binding, free soluble gp70 molecules or virus particles with reduced amounts of gp70 might have a decreased receptor binding affinity compared to virions with normal amounts of gp70. This mechanism of interference could also account for the decreased number of cell surface gp70 binding receptors reported on infected producer cells (DeLarco and Todaro, 1976) since many receptors might be blocked by released virions. Newly budded virions would have an advantage in reaching such receptors as they would be ‘in close proximity and in high concentations near the cell surface, in contrast to other extrinsically added virions. The present results do not easily account for the resistance to superinfection previously noted in some gp70-positive virus nonproducer cells (Bile110 et al., 1977; Besmer et al., 1979). In some cases virus release was measured by infectivity (Bilello et al., 1977), and the possibility remains that noninfectious envelope-positive particles were released and caused the interference. Alternatively, if free gp70 molecules were released in large enough
REINFECTION
OF gp70-POSITIVE
amounts, they might reach very high concentrations in the immediate vicinity of the cell surface. This could result in significant receptor occupancy with subsequent interference. The lack of interference in gp70-positive 7C cells indicated that 7C cells probably did not release free gp70 in high enough concentrations to cause interference by this mechanism. This would suggest that the quantity of released gp70 is not necessarily proportional to the amount on the cell surface, and that the former may be more important than the latter in resistance to reinfection. ACKNOWLEDGMENT The paring
authors thank the manuscript.
Mrs.
Helen
Blahnik
for
pre-
REFERENCES BASSIN, R. H., TUNE, N., and FISCHINGER, P. J. (1970). Isolation of murine sarcoma virus transformed mouse cells which are negative for leukemia virus from agar suspension cultures. Inst. J. Cancer 6, 95-107. BASSIN, R. H., TUTPLE, N., and FISCHINGER, P. J. (1971). Rapid cell culture assay technique for murine leukemia viruses. Nature (London) 229, 564566. BESMER, P., and BALTIMORE, D. (1977). Mechanism of restriction of ecotropic and xenotropic murine leukemia viruses and formation of pseudotypes between the two viruses. J. Viral 21,965-973. BESMER, P., FAN, H., PASKIND, M., and BALTIMORE, D. (1979). Isolation and characterization of a mouse cell line containing a defective Moloney murine leukemia virus genome. .Z. viral 29,1023-1034. BILELLO, J. A., STRAND, M., and AUGUST, J. T. (1977). Expression of viral envelope glycoproteins and transformation genes in cells transformed by a defective Kirsten murine sarcoma virus. VirdoBy 77,233-244. CHESEBRO, B., WEHFUY, K., CLOYD, M., BRIIT, W., PORTIS, J., COLLINS, J., and NISHIO, J. (1981). Characterization of mouse monoclonal antibodies specific for Friend murine leukemia virus-induced erythroleukemia cells: Friend-specific and FMRspecific antigens. virdogy 112, 131-144. COLLINS, J. K., BRI’IT, W. J., and CHESEBRO, B. (1980).
CELLS
129
Cytotoxic T lymphocyte recognition of gp70 on Friend virus-induced erythroleukemia cell clones. J. Immund 125,1318-1324. COLLINS, J. K., and CHESEBRO, B. (1980a). Spontaneous cessation of Friend murine leukemia virus production by leukemia cell line Y57: Overgrowth by nonproducer cells. J. Nat. Cancer Inst. 64,11531159. COLLINS, J. K., and CHESEBRO, B. (1980b). Synthesis, processing and cell surface expression of Friend and xenotropic murine leukemia virus gp70 antigens on Friend virus-induced erythroleukemia cell clones. J. Zmmund 12,1325-1331. COLLINS, J. K., and CHESEBRO, B. (1981). Replicationdefective Friend murine leukemia virus particles containing uncleaved gag polyproteins and decreased levels of envelope glycoprotein. J. viral. 37, 161-170. DELARCO, J., and TODARO, G. J. (1976). Membrane receptors for murine leukemia viruses: Characterization using purified viral envelope glycoprotein, gp71. Cell 8, 365-371. HARTLEY, J. W., and ROWE, W. P. (1975). Cloned cell lines from a feral mouse embryo which lack host range restrictions for murine leukemia viruses. Virology 65,128-134. HARTLEY, J. W., and ROWE, W. P. (1976). Naturally occurring murine leukemia viruses in wild mice: Characterization of a new “amphotropic” class. J. ViroL 19, 19-25. HUNSMANN, G., MOENNING, V., PISTER, L., SEIFERT, E., and SCHAFER, W. (1974). Properties of mouse leukemia viruses. VIII. The major viral glycoprotein of Friend leukemia virus. Seroimmunological, interfering and hemagglutinating capacities. virologp 62,307-318. SARA, P. S., CHEONG, M., HARTLEY, J. W., and HUEBNER, R. J. (1967). A viral interference test for mouse leukemia viruses. Virology 33.180-184. STECK, F. T., and RUBIN, H. (1966a). The mechanism of interference between an avian leukosis virus and Rous sarcoma virus. I. Establishment of interference. Virology 29. 628-641. STECK, F. T., and RUBIN, H. (196613). The mechanism of interference between an avian leukosis virus and Rous sarcoma virus. II. Early steps of infection by RSV of cells under conditions of interference. &rology 29,642-653. VOGT, P. K., and ISHIZAKI, R. (1965). Reciprocal patterns of genetic resistance to avian tumor viruses in two lines of chickens. virology 26, 664-672. VOGT, P. K., and ISHIZAKI, R. (1966). Patterns of viral interference in the avian leukosis and sarcoma complex. Virolog?/ 30, 368-374.
115,
I%-129
(1981)
Expression of Cell Surface Friend Virus gp70 Does Not Block Reinfection by Ecotropic Murine Leukemia Viruses B. CHESEBRO’,
J. K. COLLINS,
K. WEHRLY,
J. NISHIO,
AND M. CLOYD
U. S. Department of Health and Human Services, Public Health Service, National Institutes of Health, National Institute of Allergy and Iv&ctious Diseases, Laboratory of Persisttmt Viral Diseases, Rocky Mountain Laboratories, Hamilton, Montana 598.40 Received Apn’l 7, 1981; accepted June 25, 1981 The effect of cell surface gp70 expression on reinfection by murine leukemia virus was studied in Friend virus (FV)-induced erythroleukemia cell clones. A clone (2C) which released gp70-positive infectious virions and expressed large amounts of cell surface ecotropic FV gp70 was not superinfectable by ecotropic viruses, whereas another clone (7C) which expressed similar amounts of cell surface FV gp70 but released gp70-deficient virions showed no resistance to superinfection. The lack of interference to superinfection of 7C cells suggested that newly budded gp70-positive virions, rather than cell surface gp70 molecules, were responsible for the interference to superinfection seen in 2C cells which released infectious virus. INTRODUCTION
Acute exposure of uninfected cells to one oncornavirus can interfere with subsequent infection by a second related oncornavirus (Steck and Rubin, 1966a). This interference was shown to occur at the level of virus-specific cell surface receptors involved in the infection process (Vogt and Ishizaki, 1965; Steck and Rubin, 1966b). These receptors appear to recognize viral envelope glycoproteins. This was suggested by the observation that oncornaviruses can be divided into interference groups which appear to correlate with relatedness of the viral envelope glycoproteins (Vogt and Ishizaki, 1966). Furthermore, exposure of uninfected cells to large amounts of purified viral envelope glycoprotein, gp70, has been shown to cause interference (Hunsmann et al., 1974). Interference has also been frequently observed in chronically infected cells; such cells cannot be superinfected by viruses from related interference groups. However, the mechanism of this interference ’ To whom reprint requests should be addressed.
has not been adequately addressed. It is possible that since oncornavirus-releasing cells have large amounts of viral envelope glycoprotein on their cell surfaces, production and/or release of this protein could interfere with superinfection by related viruses by receptor blockade similar to that seen in uninfected cells. We have recently studied a Friend virus-induced erythroleukemia cell line which releases gp70-deficient virus particles but expresses amounts of cell surface gp70 similar to productively infected cells (Collins and Chesbro, 1980a, b; Collins et al., 1981). Surprisingly, this cell line can be easily superinfected with homologous ecotropic virus suggesting that cell surface expression of viral envelope glycoproteins per se does not lead to the interference with superinfection seen in chronically infected cells. MATERIALS
AND METHODS
Cells and viruses. Clones 2C, 7C, and 6C derived from the Y57 erythroleukemia line induced by B-tropic FV complex in a (C57BL/lO X A.BY)F1 mouse were de125
0042-6822/81/150125-05$02.00/O Copyright Q 1981 by Academic Press. Inc. All rights of reproduction in any form reserved
126
CHESEBRO
scribed previously (Collins and Chesebro, 1980a). F-MuLV was assayed on the D56 clone of mouse SL cells (Bassin et al., 1971). C243 and 3T3FL cells (Bassin et al., 1970) were kindly provided by Dr. Alan Rein, Frederick Cancer Research Center, Frederick, Maryland. Moloney sarcoma virus (MSV) pseudotypes were made by superinfection of C243 (SL) cells with FMuLV. MSV was assayed on 3T3FL cells. Friend murine leukemia helper virus (FMuLV) was obtained by limiting dilution infection of SC-1 cells (Hartley and Rowe, 1975) with a FV-induced leukemic spleen homogenate from a (BIO.A X A)Fi mouse. Peptide mapping. Cells from the 2C and 7C clones were surface labeled with ‘%I, lysed, precleared, immunoprecipitated, and digested with V3 protease as described (Collins and Chesebro, 1980b). Digests were separated by SDS-PAGE using 10% acrylamide gels, and were analyzed by autoradiography with intensifying screens. Membrane immunofluorescence. Monoclonal antibodies specific for F-MuLV were obtained from culture supernatants of hybridomas. These reagents were used in indirect membrane immunofluorescence tests on live cells with FITCconjugated goat anti-mouse Ig serum. The origin of the hybridomas and the details of the fluorescence assay were described previously (Chesebro et al., 1981). RESULTS
F-MuLV Reirlfection of an F-MuLV Positive Cell Line
gp70-
Previous results indicated that two variant FV-induced erythroleukemia cell clones, 6C and 7C, did not produce infectious MuLV as detected by infectious center assay on mouse S+L cells; however, both of these clones could be reinfected with ecotropic F-MuLV (Table 1) or MuLV derived from a B/T-L virus-induced leukemia cell line (Collins and Chesebro, 1980a). In the case of clone 7C, reinfectability with ecotropic F-MuLV was an unexpected finding because these cells expressed cell surface F-MuLV gp70 in amounts equal to that on a virus-producing clone, 2C (Collins et al., 1980). In contrast, clone 6C was negative for cell sur-
ET AL. TABLE REINFECTION
1
OF ERYTHROLEUKEM~A WITH F-MuLV Infectious
Erythroleukemia clone 6C 7C 2c
CELL CLONES
centers/lo6 cells”
Preinfection
Postinfeetion”
40 40 3.8 x lo4
3.6 x lo4 3.4 x lo4 5.4 x lo4
’ Mouse S+L- assay on D56 cells. * l-2 X lo6 cells were incubated 20 min at 37” with DEAE-dextran (0.005 mg/ml), then cells were washed once with buffer and infected with F-MuLV at a multiplicity of 2 PFU/cell. Cells were cultured overnight and then assayed as infectious centers.
face F-MuLV gp70, although the cells did express a xenotropic MuLV gp70 (Collins and Chesebro, 1980b). Reinfectability of this line by F-MuLV was not surprising since ecotropic and xenotropic MuLV belong to different interference subgroups (Besmer and Baltimore, 1977). Comparison 6C
of gp70 frcmz Clones 2C, 7C, and
Since the quantitation of gp70 molecules on clones 2C and 7C was based on a radioimmunoassay, further data were sought to determine if small antigenic or peptide sequence differences were present which could explain the different reinfection properties of the two cell clones. The clones were analyzed by membrane immunofluorescence with a panel of monoclonal antibodies specific for F-MuLV gp70 and p15 (Table 2). Clone 7C and 2C cells reacted with each of the monoclonal antigp70 antibodies, and the patterns of fluorescence appeared indistinguishable. Since each monoclonal anti-gp70 antibody detected a unique antigenic determinant on the gp70 molecules (Chesebro et al., 1981), these data indicated that all gp70 determinants detected by this antibody panel were present on both 2C and 7C cells. In contrast, clone 6C did not react with any anti-F-MuLV gp70 monoclonal antibodies. Anti-p15 monoclonal antibodies
REINFECTION
OF gp’70-POSITIVE
TABLE 2 ANALYSIS OF CELL SURFACE F-MuLV ANTIGENS ON ERYTHROLEUKEMIA CLONES USING MONOCLONAL ANTIBODIES Membrane immunofluorescence on erythroleukemia clones” Hybridoma 48 273 55 350 59 66 47 307 34 257 None
Specificity
2C
7C
6C
Anti-gp’70 Antigp70 Anti-gp70 Anti-gp70 Anti-gp70 Anti-gp70 Antigp70 Anti-gp70
+ + + + + + + +
+ + + + + + + +
-
+ + -
-
+ + -
Anti-p15 Anti-p15 -
127
CELLS
with F-MuLV envelope (F-MuLV pseudotype) was used. Infection by MSV could be measured on 3T3FL cells even in the presence of MuLV infection. When 6C, 7C, and 2C cells were infected with various dilutions of MSV (F-MuLV), the 7C and 6C cells appeared identical and loo-fold more infectious centers were seen with these clones than with the 2C clone infected at the same multiplicity (Fig. 2). Thus, in spite of similar quantitative expression of cell surface F-MuLV gp70 in 7C and 2C cells, these clones differed markedly in reinfectability by MSV (FMuLV). DISCUSSION
The present experiments demonstrated that expression of cell surface F-MuLV 2c 1234
7c 5678
u (+) Greater than 95% of cells positive; (-) less than 1% of cells positive.
reacted with clones 2C and 6C, but failed to react with clone 7C which was shown previously to express reduced amounts of gag cell surface antigens detected by conventional antisera (Collins et al., 1980). In order to confirm the identity of the gp’70 molecules found on 7C and 2C cells, these gp7Os were compared by peptide mapping. Anti-gp70 immunoprecipitates from ‘?-surface-labeled cells were digested with V8 protease for various times and then compared by SDS-PAGE. The results indicated that these gp7Os were indistinguishable from each other (Fig. 1). Previous results with this technique indicated that xenotropic MuLV gp70 from clone 6C differed markedly from that on clone 2C (Collins and Chesebro, 1980b). Infection of SC, XT, and 2C Cells by MSV (F-MuL V) Since 2C cells were already high producers of MuLV detectable on S+L- cells, these cells could not be tested for interference using F-MuLV alone. Therefore, to compare the infectability of 7C and 2C clones, which expressed the same cell surface gp70, Moloney sarcoma virus (MSV)
FIG. 1. V8 protease digestion of ‘?-gp70 from 2C and 7C cells. Tracks 1 and 5, undigested, tracks 2 and 6, digested 46 min; tracks 3 and 7, digested 90 min; tracks 4 and 8, digested 180 min. Analysis by SDSPAGE in 10% acrylamide.
128
CHESEBRO
UNDIL.
l/ID
VIRUS DILUTION l/100
1/1000
FIG. 2. Infection of 2C, ?C, and 6C cells with MSV (F-MuLV pseudotype) at various dilutions was followed by analysis of cells as MSV infectious centers on 3T3FL cells.
gp’70 on 7C erythroleukemia cells did not lead to interference with reinfection of these cells by F-MuLV or the F-MuLV pseudotype of MSV. This lack of interference was not due to a quantitative difference in gp70 expression on 7C cells since the amount of gp70 on 7C cells was similar to the gp70 on the 2C erythroleukemia clone, which did show interference to superinfection. Furthermore, the gp70 molecules on these two cell lines had identical peptide maps and demonstrated identical reactivity with monoclonal antibodies detecting different F-MuLV gp70-specific antigens. Therefore, any differences in these two gp7Os would most likely be quite minor. It would be surprising if differences so difficult to detect could give rise to an altered interference pattern because even obvious differences in gp70 antigenicity or peptide maps do not eliminate the cross-interference seen between AKR and FMR serogroups of ecotropic viruses (Sarma et al., 1967; Hartley and Rowe, 1976). However, these results do not completely exclude the possibility that the gp70 on 7C cells had some alteration or defect in a small portion of the molecule critical for interference. If so, then more detailed biochemical comparison of the gp7Os on 2C and 7C cells might provide an
ET AL.
indication as to which portion of the gp70 molecule is responsible for interference, and presumably also receptor specificity. Although 7C cells do not produce infectious MuLV, these cells do release noninfectious viral particles which incorporate [‘Hluridine into 70 S RNA and band at the usual MuLV buoyant density (Collins and Chesebro, 1981). The lack of infectivity in these particles is not surprising as they contain no active reverse transcriptase. Furthermore, they do not process the gag core polyprotein pr65 into its smaller components. Possibly as a result of this core defect, virions from 7C cells incorporate markedly reduced amounts of gp70 and p15E into their envelopes in spite of the abundance of gp70 on the cell surface (Collins and Chesebro, 1981). The reduced amounts of gp70 in 7C virions might account for the lack of interference to superinfection of 7C cells by nondefective viruses. This would be expected if interference in virus producer cells was medicated by blockade of cell surface receptors by virus particles just released from the same cell surface. Due to lack of multivalent binding, free soluble gp70 molecules or virus particles with reduced amounts of gp70 might have a decreased receptor binding affinity compared to virions with normal amounts of gp70. This mechanism of interference could also account for the decreased number of cell surface gp70 binding receptors reported on infected producer cells (DeLarco and Todaro, 1976) since many receptors might be blocked by released virions. Newly budded virions would have an advantage in reaching such receptors as they would be ‘in close proximity and in high concentations near the cell surface, in contrast to other extrinsically added virions. The present results do not easily account for the resistance to superinfection previously noted in some gp70-positive virus nonproducer cells (Bile110 et al., 1977; Besmer et al., 1979). In some cases virus release was measured by infectivity (Bilello et al., 1977), and the possibility remains that noninfectious envelope-positive particles were released and caused the interference. Alternatively, if free gp70 molecules were released in large enough
REINFECTION
OF gp70-POSITIVE
amounts, they might reach very high concentrations in the immediate vicinity of the cell surface. This could result in significant receptor occupancy with subsequent interference. The lack of interference in gp70-positive 7C cells indicated that 7C cells probably did not release free gp70 in high enough concentrations to cause interference by this mechanism. This would suggest that the quantity of released gp70 is not necessarily proportional to the amount on the cell surface, and that the former may be more important than the latter in resistance to reinfection. ACKNOWLEDGMENT The paring
authors thank the manuscript.
Mrs.
Helen
Blahnik
for
pre-
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