VIROLOGY
95, 185-196 (1979)
Fusion
Activity DAVID
Department
of Pathology
of Virions
of Murine
A. ZARLING2
Leukemia
AND ILANA
and Immunobiology Research Center, Madison, Wisconsin 53?‘06 Accepted
February
Virus1
KESHET University of Wisconsin,
5, 1979
The fusion activity associated with many strains of murine leukemia virus (MLV) is commonly used to measure MLV infectivity. Other strains of MLV lack fusion activity and some of these can produce variants which have fusion acitivity. The SC-l/uv-XC fusion assay (Rowe et al., 1970) measures the infectivity of MLV grown in SC-l cells by the XC cell fusion activity of the progeny virus. The fusion activity of parental MLV in XC cells can be measured directly within a few hours after infection. No new macromolecular synthesis (early after infection) is required for MLV-induced fusion. Treatment of MLV virions with proteolytic enzymes destroys their ability to fuse XC cells. The fusion activity of MLV, like the infectivity of MLV, is specifically inhibited by anti-MLV gp’70 serum but not by antiserum against other MLV polypeptides. The infectivity of MLV is much more susceptible to uv irradiation than viral fusion activity and noninfectious (uv irradiated) MLV can cause XC cell fusion. When MLV virions are disrupted with NP-40 detergent, the fusion activity can be restored after high-speed centrifugation and dialysis of the detergent. The fusion activity of NP-40 disrupted MLV (after high-speed centrifugation and dialysis) is inhibited by antiserum against MLV gp70. Thus, the fusion activity of MLV is associated with the virion envelope glycoprotein.
duced fusion of XC cells is a virion activity associated with the viral envelope glycoprotein.
INTRODUCTION
Some laboratory and naturally occurring strains of murine leukemia virus (MLV) induce the fusion of established rat cell lines such as XC (fibroblasts transformed by the Prague strain of Rous sarcoma virus), FU-1 (rat myoblasts defective in differentiation), or RFL (normal rat fibroblasts). Mixed cultures containing MLV-infected cells and XC cells cause the XC cells to fuse with one another (Klement et al., 1969) and is commonly used as an assay for measuring the infectivity of these MLV strains in tissue culture (Rowe et al., 1970). Direct infection of XC (Johnson et al., 1971), FU-1 (Wong et al., 1977a, b), or RFL (Koga, 1977) cells with MLV also causes the fusion of these cells. The mechanism of MLV induced cell fusion is not established. In this communication, we present evidence that MLV-in-
MATERIALS
’ Supported by American Cancer Society Grant CA-14520. * Author to whom reprint requests should be addressed. 185
AND METHODS
Cells, media, viruses, and sera. SC-l (Rowe et al., 1970) feral mouse cells and XC (Klement et al., 1969; Svoboda, 1960) Rous sarcoma virus-transformed OsborneMendel rat cells were from R. Risser, McArdle Lab, Madison, Wisconsin, who obtained these lines from J. Hartley and W. Rowe of the National Institute of Allergy and Infectious Diseases. SC-l cells were propagated in modified McCoy-5A medium with 10% heat-inactivated fetal calf serum (FCS). XC cells were propagated in Temin-modified Eagle MEM supplemented with 10% FCS (referred to as complete media). The sources of all the virus strains used are listed in Table 1. All viruses listed in Table 1 were aliquoted and stored frozen at -70”. The following specific MLV antisera were obtained from R. Wilsnack, Huntingdon Research Center, Brooklandville, Marlyand: 0042~6822/79/070185-12$02.00/O Copyright 0 1979 by Academic Press, All rights of reproduction in any form
Inc. reserved.
186
ZARLING
AND KESHET
goat anti-Rauscher-MLV gp70, lot 58-678, RIA titer 800; goat anti-Rauscher-MLV p30, lot 68-492, RIA titer 38,000; goat antiRauscher-MLV ~15, lot 58-716, RIA titer 2000, goat anti-Rauscher-MLV ~12, lot 58-37, RIA titer 18,000; goat anti-AKRMLV ~10, lot 53-465, RIA titer 150; goat anti-Rauscher-MLV DNA polymerase, lot 69-425, reverse transcriptase inhibition titer 500, and goat anti-Moloney-MLV, lot 63-142, fixed cell immunofluorescence titer 80. Rabbit anti-Rauscher-MLV p15(E), lot 21574, was obtained from I. Fleissner, Sloan-Kettering Institute, New York. All antiviral sera were assayed by radioimmunoprecipitation to verify specificity and titer as described by Zarling et al. (1978). Normal goat serum was purchased from GIBCO, Grand Island, New York, and normal rabbit serum was purchased from Cappel, Cochranville, Pennsylvania. AntiSendai virus serum was a gift from K. Sugamura, Department of Microbiology, Kumamoto University Medical School,
Japan. Anti-Sendai virus serum neutralized the infectivity of Sendai virus but did not neutralize the infectivity or fusion activity of murine leukemia virus. All sera and antisera were heat inactivated at 56” for 30 min and stored frozen at -70”. Virus assays. The SC-l/uv-XC cell fusion assay was modified from the method of Rowe et al. (1970). Low passage SC-l cells (2 x lo5 tells/60-mm petri dish) seeded on the previous day were inoculated with 0.2 ml of virus in complete media containing 2 pug/ml Polybrene (Aldrich Chemical, Milwaukee, Wis.). Absorption was allowed for 1 hr at 37” and the cultures were overlayed with 5 ml of complete media. Four days after infection the media was changed (and changed again on Day 5 in some experiments for serial viral passage) and on Day 6 the infected SC-l cells were lethally irradiated with uv light (30 set at 650 pw/cm*, UltraViolet Products Inc., Model 5225, San Gabriel, Calif.) and overlayed with 1 x lo6 low passage XC cells. The media was
TABLE SOURCES
Virus ATCC Rauscher-MLV ATCC Gross Passage A-MLV ATCC rat-adapted Gross Passage A-MLV ATCC Tennant-BR-BcMLV ATCC Moloney-MLV 302 AKR-Ll-MLV MCF AKR-MLV 303 Moloney-MLV JLS-V9 Rauseher-MLV NIH Moloney-MLV NIH AKR-Ll-MLV NIH Gross-MLV
OF
1
MLV STRAINS
Source
Reference
ATCC VR.294-Lot ATCC VR.590-Lot
2D” 3D
Rauscher, 1962 Gross, 1957
ATCC VR.589-Lot
1
Gross, 1963
ATCC VR.596-Lot
1
Tennant, 1966
ATCC VR. 190-Lot 3 JH, AKR-Ll-302-MLVgrown in NIH cells6 JH, AKR-MCF24’7-MLV JH, Moloney-303-MLV grown in NIH cells VOR Lot 623, from JLS-V9 cells” VOR Lot 5003-19-55, grown in NIH cells VOR AKR-Ll-MLV Lots 734-58-4 and 733-40-5, grown in NIH cells VOR XC plaque-purified Gross Passage A-MLV, Lots 500-89-5 and 500-64, grown in NIH cells
Moloney, 1960 Hartley et al., 1970 Hartley et al., 197’7 Hartley et al., 1970 Wright et al., 1967 Benveniste and Scolnick, 1973; Bassin et al., 1975 Rhim et al., 1971 Gross, 1957; Hartley et al., 1970
DAmerican Type Culture Collection. b Dr. Janet Hartley, National Institute of Allergy and Infectious Diseases. c Viral Oncology Resources, National Cancer Institute.
MURINE
LEUKEMIA
VIRUS
FUSION
ACTIVITY
187
changed after another 2 days and the cul- and serially diluted to measure the original tures were stained with methylene blue on viral infectivity (SC-l/uv-XC cell assay) and Day 10 after infection. Positive (Moloneyfusion activity (direct assay). The solution was adjusted to 0.25% with respect to MLV) and negative (mock infected) controls were included in every assay. Viral titers NP-40 and incubation was allowed for 30 with shaking are expressed as XC plaque-forming units min at room temperature (PFUlml). every few minutes. The entire solution was In the direct XC cell fusion assay for centrifuged at 32,000 rpm (140,OOOg) for MLV, cultures of low passage XC cells 60 min at 4” in a Spinco SW60 rotor. The seeded the previous day at 1.25 x lo6 cells/ supernatant was dialyzed in sterile dialysis 60-mm petri dish were infected with 0.2 ml tubing against phosphate-buffered saline of virus in complete media containing 2 lacking calcium and magnesium (PBS-) at wg/ml Polybrene. Absorption was allowed 4”. The absence of detergent (after dialexaminfor 1 hr at 37” and the cultures were over- ysis) was checked by immediately layed with 5 ml of complete media. Giant ing cell cultures innoculated with the dia(fused) cells were scored microscopically lyzed solutions for lysis by residual detergent. (63x) on Day 2 in unstained or methylene In some experiments dialysis against PBS blue-stained cultures. Identical numbers of was performed at 15 to 30 psi of nitrogen plaques are obtained in stained or unstained gas in an Amicon 8MC Micro-UF system cultures. The number of direct XC plaques fitted with a XM50 filter. Pressure dialyzed was proportional to the virus dilution (lo- samples were reduced in volume to approxifold dilutions) and cultures with at least 30 mately 0.6 ml using the Amicon microfiltrato about 300 XC plaques were used to de- tion system. termine the XC plaque-forming titer (PFU). Controls for spontaneous XC cell fusion RESULTS were included in every assay. A background Quantitative Comparison of the Indirect of approximately 4 to 10 spontaneously fused and Direct Assays of MLV by XC Cell XC cells per 60-mm petri dish culture was Fusion subtracted from the data. Viral DNA polymerase activity in cellIn the classical XC cell fusion assay for free culture media of virus-producing cells the infectivity of MLVs developed by Rowe was measured as described by Temin and et al. (1970), SC-1 wild mouse cells are inBaltimore (1972) using NP-40 (Nonidet P40, fected with MLV and on the sixth day after Particle Data Lab, Elmhurst, Ill.) to dis- infection the SC-l cells are uv irradiated rupt virus and activated calf-thymus DNA and overlayed with XC cells. The XC cells is used as an exogenous template-primer. fuse with one another in areas containing Relative polymerase activity is expressed MLV-producing SC-1 cells and plaques are as 3H-counts per minute ([3H]dTTP, New scored 10 days after infection. Comparisons England Nuclear, NET-221X) incorporated between the indirect and direct MLV syninto trichloroacetic acid-insoluble material in cytial plaque assays are shown in Table 2. a standard 1-hr reaction per milliliter of XC cells were directly infected with MLV and the fused cells were scored 2 days after unconcentrated culture fluid. A background of approximately ‘70 cpm is subtracted from infection (Table 2). The XC plaques observed the data. DNA polymerase reactions are in the direct infection of XC cells could be linear with respect to time (up to 2 hr) and easily counted in unstained cultures (as well virus concentration. as methylene blue-stained cultures) as early Solubilixation of MLV. Double density as 1 day after infection. No increase in plaque gradient-purified (Toplin and Sottong, 1972) number was observed at 2 or subsequent MLV virions in 1 ml of 0.01 M Trisdays after infection. The concentration hydrochloride, 0.1 M NaCl, 0.001 M EDTA of 1.25 x lo6 XC cells per 60-mm petri (TNE pH 7.2), was thawed and diluted with dish was optimal for assaying direct XC cell 3.0 ml of TNE. A 0. l-ml sample was removed fusion. Table 2 shows that there was a signif-
133
ZARLING
AND KESHET TABLE
COMPARISON OF THE DIRECT AND INDIRECT
2 ASSAYS
OF
MLV BY XC CELL FUSIONS XC PFU/ml
Virus
mg/ml Protein0
NIH Moloney-MLV JLS-V9 Rauscher-MLV NIH Gross-MLV NIH AKR-MLV
0.64 1.25 0.53 3.52
Indirect assay (infectivity) 3.5 2.5 1.2 1.0
x x x x
107 105 108 10’
Direct assay (fusion activity) 2.2 3.5 1.7 1.5
x x x x
105 103 106 104
n Virus was assayed for XC plaque formation by infecting duplicate cultures of SC-l or XC cells with 0.2 ml of serial lo-fold virus dilutions in complete media containing 2 pg/ml Polybrene. Infected SC-l cell cultures were uv irradiated and overlayed with XC cells 6 days after infection and stained 10 days after infection (indirect assay). Infected XC cell cultures were stained 2 days after infection (direct assay). The number of XC plaque-forming units (PFU) was determined by counting plaques containing fused cells at 63~ using an inverted microscope. * The protein concentration of purified MLV stocks was assayed as described by Lowry et al., 1951.
icantly lower (approximately 70- to 6’70-fold lower) virus titer obtained by direct assay in comparison with indirect assay. In other similar experiments, different preparations of all these (concentrated, purified, and frozen) virus strains (Table 2) consistently showed approximately 30- to 500-fold lower direct titers compared to the indirect titers. Similar results were also obtained with either frozen or fresh (not frozen, concentrated, or purified) Moloney-MLV harvested from newly infected SC-l cells (data not shown). It is likely that the direct assay measures the parental MLV or MLV-induced fusion activity whereas the indirect assay measures the infectivity of MLV in mouse SC-l cells by the fusion activity of the mouse cell progeny virus. Kinetics of MLV-Induced Cells
Fusion of XC
XC cells infected with the Moloney, Rauscher, Gross, or AKR strains of MLV were observed to induce the formation of giant cells during the first 3 hr of infection. Figure 1 shows representative results from experiments where the effects of MLV infection on total XC cell numbers were measured. By 3 hr after infection with NIH Gross-MLV there was a significant reduction in the total number of single XC cells per infected culture (Fig. 1) depending on
the multiplicity of infection (m.0.i.). During counting of these trypsinized cultures (3 hr after infection) XC cells in the process of fusing were observed. The decrease in the number of single cells per infected culture relative to parallel uninfected cultures reached a maximum at 6 to 24 hr after infection (Fig. 1). There was no significant increase in the number of dead cells (stained with eosin) during these times (Fig. 1). Thus, MLV-induced fusion of XC cells occurs early after infection and causes a reduction in the number of single cells during this period (Fig. 1). Effects of Metabolic Inhibitors Fusion of XC Cells by MLV
on the
The ability of MLV to fuse XC cells could be virally induced in XC cells. Therefore, the effects of metabolic inhibitors on the direct fusion of XC cells by MLV was tested. MLV-infected XC cell cultures were treated with cycloheximide, cytosine arabinoside (Ara-C), actinomycin D, or no inhibitor for 3 hr (Table 3). Table 3 shows that neither the cyclohexamide (10 pg/ml) nor Ara-C (10 pg/ml) treatments significantly inhibited XC plaque formation by Rauscher-MLV. Actinomycin D treatment (either 0.25 or 0.5 tLglm1, Sigma Lot 126C-0219) was extremely cytotoxic to XC cells and thus its effects on XC plaque formation were not determined.
MURINE
LEUKEMIA
189
VIRUS FUSION ACTIVITY
A
MOI
q
0.002
MOI = 0.02
8
25 -
k z 0’
01
’ 3
1 24
6 TIME
AFTER
INFECTION
(HRS.1
FIG. 1. Cultures of XC cells (1.25 x 10” cells per 60-mm dish seeded the previous day) were mock infected or infected with NIH Gross-MLV at a multiplicity of either 0.02 or 0.002 XC PFU (direct assay) per cell. At each time after infection the total number of single cells per culture was determined. Giant cells-were scored as 1 cell and at least 200 cells were counted to determine the total number of single cells per culture. At each time 295% of the cells in mock-infected or infected cultures were viable (excluding eosin dye). The percentage of the control cell number at each time was calculated from the formula: 100 x
number of single cells per infected culture number of single cells per mock-infected culture ’
Control experiments showed that cycloheximide and Ara-C treatment at these concentrations significantly inhibited protein (>99% inhibition) and DNA (>89% inhiTABLE
3
EFFECT OF METABOLIC INHIBITORS ON THE FUSION OF XC CELLS BY MLV” Inhibitor
Fusion activity (XC PFU/ml)
Cycloheximide Ara-C None
2.7 x 104 1.4 x 104 3.8 x lo4
D Replicate cultures of XC cells were inoculated with serial lo-fold dilutions of JLS-V9 Rauscher-MLV and following 45 min virus absorption the cultures were overlayed with complete medium containing either cycloheximide (10 pg/ml), Ara-C (10 pg/ml), or no inhibitor. After a 3-hr exposure, the cultures were washed twice and overlayed with fresh complete media. The number of XC plaques was scored 2 days after infection. Cycloheximide and Ara-C at these concentrations inhibited the respective incorporation of [3H]serine or [3H]thymidine into trichloroacetic acid-precipitable material by 99% (cycloheximide) and 89% (Ara-C) compared to mock-treated control cultures.
bition) synthesis (Table 3). Similar results to those in Table 3 were obtained in two other experiments (data not shown). Furthermore, similar results were obtained by Johnson et al. (1971) with either actinomycin D (0.5 pg/ml), puromycin (20 pug/ml), cycloheximide (10 pg/ml), rifamycin (120 pg/ml), or rat serum interferon (60 units in 0.1 ml rat serum) added at the time of infection of XC cells with Moloney-MLV and included in the media for 6 hr. Thus, fusion of XC cells by MLV does not appear to require de novo protein or DNA synthesis (during the first 3 hr of infection) which can be inhibited by cycloheximide or Ara-C (Table 3). The fusion of XC cells by MLV could either be an activity of the MLV virion or preexisting in the XC cell and activated by MLV infection. Effects of Treatment of MLV with Proteolytic Enzymes, Antiviral Serum, or uv Irradiation on XC Cell Fusion
The effects of Pronase or trypsin treatment of MLV on the fusion of XC cells by MLV are shown in Table 4. Treatment of NIH AKR-MLV with 20 pg/ml of either
190
ZARLING
AND KESHET
Pronase or trypsin inhibited the fusion of XC cells by approximately 99% (Pronase) and 96% (trypsin) (Table 4) and significantly reduced viral infectivity in the SC-l/uv-XC cell assay (data not shown). As a control, DFP-inactivated trypsin (20 pg/ml, Worthington Biochemical Corp., Freehold, N. J.) did not inhibit AKR-MLV fusion activity (Table 4) or the infectivity of MLV in the SC-lluv-XC cell assay (not shown). Similar results to those in Table 4 were obtained with the Moloney strain of MLV. Thus, treatment of MLV virions with Pronase or trypsin prevents fusion of XC cells (Table 4). The envelope glycoprotein of MLV (gp70) is known to be cleaved by trypsin or Pronase treatment and is marked by losses in viral infectivity, in the ability of virus to interfere with infection by other MLVs, to hemagglutinate erythrocytes, or in the ability of the virus to absorb neutralizing antibody to the viral envelope glycoprotein, gp70 (Schafer and Bolognesi, 1977). Goat antiserum against purified MoloneyMLV virions or normal goat serum was incubated with NIH Moloney-MLV for 45 min TABLE
4
EFFECT OF PROTEOLYTIC ENZYMES ON XC CELL FUSION BY MLV” Enzyme treatment Pronase DPCC trypsin* DFP-inactivated None
trypsin’
Fusion activity (total XC PFU) 4.0 2.0 7.2 5.2
x x x x
IO’ 102 103 103
” NIH AKR-MLV was incubated with 20 pg/ml of either Pronase, DPCC-treated trypsin, DFP-inactivated trypsin, or with enzyme buffer alone for 30 min at 37”. Following incubation an equal volume of soybean trypsin inhibitor (20 pg/ml) was added and the samples were serially (lo-fold) diluted in complete media containing 2 kg/ml Polybrene. The virus was then assayed (direct assay) for plaque formation (PFU) in duplicate cultures of XC cells. b Treatment of trypsin with diphenyl carbamyl chloride (DPCC) inactivates chymotrypsin activity but does not affect trypsin activity. c Trypsin inactivated with diisopropyl fluorophosphate (DFP).
at room temperature. Relative to normal goat serum, anti-Moloney-MLV serum caused approximately a 325-fold reduction (299.6% virus neutralization) in MoloneyMLV fusion activity in XC cell cultures and a similar reduction in viral infectivity as measured by the SC-l/uv-XC cell assay. In another different experiment, the effects on MLV infectivity and fusion activity were measured after incubating Moloney-MLV with normal sera or antisera prepared against purified MLV polypeptides (Table 5). Table 5 shows that in comparison to normal goat serum, goat antiserum against Moloney-MLV (whole virus) or anti-RauscherMLV gp70 serum significantly neutralized (299.4% neutralization) both the infectivity and fusion activity of the virus. In contrast, high-titered goat antiserum prepared against Rauscher-MLV ~30, ~15, ~12, or against AKR-MLV p10 did not significantly neutralize either the infectivity or the fusion activity of the virus (Table 5). Similarly, in comparison to normal rabbit serum, specific rabbit antiserum against RauscherMLV p15(E) did not significantly neutralize MLV infectivity or fusion activity (Table 5). These experiments (Table 5) show that the fusion activity or infectivity of MoloneyMLV is strongly neutralized only with antiserum prepared against the MLV envelope glycoprotein, gp70. Thus, MLV fusion activity appears to be associated- with viral infectivity and is inhibited by proteolytic enzymes which cleave the MLV envelope glycoprotein and by neturalizing antiserum directed against antigenic determinants of the viral envelope glycoprotein (Schafer and Bolognesi, 1977). The effects of irradiation with uv light for various times on the fusion activity (direct assay) and the infectivity (indirect assay) of Moloney-MLV were measured (Fig. 2). Figure 2 shows that uv irradiation of MoloneyMLV inactivated viral infectivity at a very rapid rate whereas the fusion activity of the virus was relatively resistant to uv irradiation. After 15 min of uv irradiation the infectivity of Moloney-MLV was reduced by over 5 logs of activity, in contrast the viral fusion activity was reduced by less than 1 log of activity (Fig. 2). In this and other
MURINE
LEUKEMIA
TABLE EFFECT
OF ANTISERUM
AGAINST
MLV
191
VIRUS FUSION ACTIVITY 5
POLYPEPTIDES
ON XC CELL
FUSION
BY
MLV”
XC PFU/ml Serum or antiserum Goat, normal Goat anti-Moloney-MLV Goat anti-Rauscher-MLV gp70 Goat anti-Rauscher-MLV p30 Goat anti-Rauscher-MLV ~15 Goat anti-Rauscher-MLV p12 Goat anti-AKR-MLV ~10 Goat anti-Rauscher-MLV DNA polymerase Rabbit, normal Rabbit anti-Rauscher-MLV p15(E)
Infectivity 4.6 1.0 2.5 6.2 4.3 5.6 6.0 6.0 7.5 5.7
x x x x x x x x x x
Fusion activity
10” 104 lo4 10” 10” 10” 10” 10” 106 106
8.8 x lo4 4.0 x 10’
95, 185-196 (1979)
Fusion
Activity DAVID
Department
of Pathology
of Virions
of Murine
A. ZARLING2
Leukemia
AND ILANA
and Immunobiology Research Center, Madison, Wisconsin 53?‘06 Accepted
February
Virus1
KESHET University of Wisconsin,
5, 1979
The fusion activity associated with many strains of murine leukemia virus (MLV) is commonly used to measure MLV infectivity. Other strains of MLV lack fusion activity and some of these can produce variants which have fusion acitivity. The SC-l/uv-XC fusion assay (Rowe et al., 1970) measures the infectivity of MLV grown in SC-l cells by the XC cell fusion activity of the progeny virus. The fusion activity of parental MLV in XC cells can be measured directly within a few hours after infection. No new macromolecular synthesis (early after infection) is required for MLV-induced fusion. Treatment of MLV virions with proteolytic enzymes destroys their ability to fuse XC cells. The fusion activity of MLV, like the infectivity of MLV, is specifically inhibited by anti-MLV gp’70 serum but not by antiserum against other MLV polypeptides. The infectivity of MLV is much more susceptible to uv irradiation than viral fusion activity and noninfectious (uv irradiated) MLV can cause XC cell fusion. When MLV virions are disrupted with NP-40 detergent, the fusion activity can be restored after high-speed centrifugation and dialysis of the detergent. The fusion activity of NP-40 disrupted MLV (after high-speed centrifugation and dialysis) is inhibited by antiserum against MLV gp70. Thus, the fusion activity of MLV is associated with the virion envelope glycoprotein.
duced fusion of XC cells is a virion activity associated with the viral envelope glycoprotein.
INTRODUCTION
Some laboratory and naturally occurring strains of murine leukemia virus (MLV) induce the fusion of established rat cell lines such as XC (fibroblasts transformed by the Prague strain of Rous sarcoma virus), FU-1 (rat myoblasts defective in differentiation), or RFL (normal rat fibroblasts). Mixed cultures containing MLV-infected cells and XC cells cause the XC cells to fuse with one another (Klement et al., 1969) and is commonly used as an assay for measuring the infectivity of these MLV strains in tissue culture (Rowe et al., 1970). Direct infection of XC (Johnson et al., 1971), FU-1 (Wong et al., 1977a, b), or RFL (Koga, 1977) cells with MLV also causes the fusion of these cells. The mechanism of MLV induced cell fusion is not established. In this communication, we present evidence that MLV-in-
MATERIALS
’ Supported by American Cancer Society Grant CA-14520. * Author to whom reprint requests should be addressed. 185
AND METHODS
Cells, media, viruses, and sera. SC-l (Rowe et al., 1970) feral mouse cells and XC (Klement et al., 1969; Svoboda, 1960) Rous sarcoma virus-transformed OsborneMendel rat cells were from R. Risser, McArdle Lab, Madison, Wisconsin, who obtained these lines from J. Hartley and W. Rowe of the National Institute of Allergy and Infectious Diseases. SC-l cells were propagated in modified McCoy-5A medium with 10% heat-inactivated fetal calf serum (FCS). XC cells were propagated in Temin-modified Eagle MEM supplemented with 10% FCS (referred to as complete media). The sources of all the virus strains used are listed in Table 1. All viruses listed in Table 1 were aliquoted and stored frozen at -70”. The following specific MLV antisera were obtained from R. Wilsnack, Huntingdon Research Center, Brooklandville, Marlyand: 0042~6822/79/070185-12$02.00/O Copyright 0 1979 by Academic Press, All rights of reproduction in any form
Inc. reserved.
186
ZARLING
AND KESHET
goat anti-Rauscher-MLV gp70, lot 58-678, RIA titer 800; goat anti-Rauscher-MLV p30, lot 68-492, RIA titer 38,000; goat antiRauscher-MLV ~15, lot 58-716, RIA titer 2000, goat anti-Rauscher-MLV ~12, lot 58-37, RIA titer 18,000; goat anti-AKRMLV ~10, lot 53-465, RIA titer 150; goat anti-Rauscher-MLV DNA polymerase, lot 69-425, reverse transcriptase inhibition titer 500, and goat anti-Moloney-MLV, lot 63-142, fixed cell immunofluorescence titer 80. Rabbit anti-Rauscher-MLV p15(E), lot 21574, was obtained from I. Fleissner, Sloan-Kettering Institute, New York. All antiviral sera were assayed by radioimmunoprecipitation to verify specificity and titer as described by Zarling et al. (1978). Normal goat serum was purchased from GIBCO, Grand Island, New York, and normal rabbit serum was purchased from Cappel, Cochranville, Pennsylvania. AntiSendai virus serum was a gift from K. Sugamura, Department of Microbiology, Kumamoto University Medical School,
Japan. Anti-Sendai virus serum neutralized the infectivity of Sendai virus but did not neutralize the infectivity or fusion activity of murine leukemia virus. All sera and antisera were heat inactivated at 56” for 30 min and stored frozen at -70”. Virus assays. The SC-l/uv-XC cell fusion assay was modified from the method of Rowe et al. (1970). Low passage SC-l cells (2 x lo5 tells/60-mm petri dish) seeded on the previous day were inoculated with 0.2 ml of virus in complete media containing 2 pug/ml Polybrene (Aldrich Chemical, Milwaukee, Wis.). Absorption was allowed for 1 hr at 37” and the cultures were overlayed with 5 ml of complete media. Four days after infection the media was changed (and changed again on Day 5 in some experiments for serial viral passage) and on Day 6 the infected SC-l cells were lethally irradiated with uv light (30 set at 650 pw/cm*, UltraViolet Products Inc., Model 5225, San Gabriel, Calif.) and overlayed with 1 x lo6 low passage XC cells. The media was
TABLE SOURCES
Virus ATCC Rauscher-MLV ATCC Gross Passage A-MLV ATCC rat-adapted Gross Passage A-MLV ATCC Tennant-BR-BcMLV ATCC Moloney-MLV 302 AKR-Ll-MLV MCF AKR-MLV 303 Moloney-MLV JLS-V9 Rauseher-MLV NIH Moloney-MLV NIH AKR-Ll-MLV NIH Gross-MLV
OF
1
MLV STRAINS
Source
Reference
ATCC VR.294-Lot ATCC VR.590-Lot
2D” 3D
Rauscher, 1962 Gross, 1957
ATCC VR.589-Lot
1
Gross, 1963
ATCC VR.596-Lot
1
Tennant, 1966
ATCC VR. 190-Lot 3 JH, AKR-Ll-302-MLVgrown in NIH cells6 JH, AKR-MCF24’7-MLV JH, Moloney-303-MLV grown in NIH cells VOR Lot 623, from JLS-V9 cells” VOR Lot 5003-19-55, grown in NIH cells VOR AKR-Ll-MLV Lots 734-58-4 and 733-40-5, grown in NIH cells VOR XC plaque-purified Gross Passage A-MLV, Lots 500-89-5 and 500-64, grown in NIH cells
Moloney, 1960 Hartley et al., 1970 Hartley et al., 197’7 Hartley et al., 1970 Wright et al., 1967 Benveniste and Scolnick, 1973; Bassin et al., 1975 Rhim et al., 1971 Gross, 1957; Hartley et al., 1970
DAmerican Type Culture Collection. b Dr. Janet Hartley, National Institute of Allergy and Infectious Diseases. c Viral Oncology Resources, National Cancer Institute.
MURINE
LEUKEMIA
VIRUS
FUSION
ACTIVITY
187
changed after another 2 days and the cul- and serially diluted to measure the original tures were stained with methylene blue on viral infectivity (SC-l/uv-XC cell assay) and Day 10 after infection. Positive (Moloneyfusion activity (direct assay). The solution was adjusted to 0.25% with respect to MLV) and negative (mock infected) controls were included in every assay. Viral titers NP-40 and incubation was allowed for 30 with shaking are expressed as XC plaque-forming units min at room temperature (PFUlml). every few minutes. The entire solution was In the direct XC cell fusion assay for centrifuged at 32,000 rpm (140,OOOg) for MLV, cultures of low passage XC cells 60 min at 4” in a Spinco SW60 rotor. The seeded the previous day at 1.25 x lo6 cells/ supernatant was dialyzed in sterile dialysis 60-mm petri dish were infected with 0.2 ml tubing against phosphate-buffered saline of virus in complete media containing 2 lacking calcium and magnesium (PBS-) at wg/ml Polybrene. Absorption was allowed 4”. The absence of detergent (after dialexaminfor 1 hr at 37” and the cultures were over- ysis) was checked by immediately layed with 5 ml of complete media. Giant ing cell cultures innoculated with the dia(fused) cells were scored microscopically lyzed solutions for lysis by residual detergent. (63x) on Day 2 in unstained or methylene In some experiments dialysis against PBS blue-stained cultures. Identical numbers of was performed at 15 to 30 psi of nitrogen plaques are obtained in stained or unstained gas in an Amicon 8MC Micro-UF system cultures. The number of direct XC plaques fitted with a XM50 filter. Pressure dialyzed was proportional to the virus dilution (lo- samples were reduced in volume to approxifold dilutions) and cultures with at least 30 mately 0.6 ml using the Amicon microfiltrato about 300 XC plaques were used to de- tion system. termine the XC plaque-forming titer (PFU). Controls for spontaneous XC cell fusion RESULTS were included in every assay. A background Quantitative Comparison of the Indirect of approximately 4 to 10 spontaneously fused and Direct Assays of MLV by XC Cell XC cells per 60-mm petri dish culture was Fusion subtracted from the data. Viral DNA polymerase activity in cellIn the classical XC cell fusion assay for free culture media of virus-producing cells the infectivity of MLVs developed by Rowe was measured as described by Temin and et al. (1970), SC-1 wild mouse cells are inBaltimore (1972) using NP-40 (Nonidet P40, fected with MLV and on the sixth day after Particle Data Lab, Elmhurst, Ill.) to dis- infection the SC-l cells are uv irradiated rupt virus and activated calf-thymus DNA and overlayed with XC cells. The XC cells is used as an exogenous template-primer. fuse with one another in areas containing Relative polymerase activity is expressed MLV-producing SC-1 cells and plaques are as 3H-counts per minute ([3H]dTTP, New scored 10 days after infection. Comparisons England Nuclear, NET-221X) incorporated between the indirect and direct MLV syninto trichloroacetic acid-insoluble material in cytial plaque assays are shown in Table 2. a standard 1-hr reaction per milliliter of XC cells were directly infected with MLV and the fused cells were scored 2 days after unconcentrated culture fluid. A background of approximately ‘70 cpm is subtracted from infection (Table 2). The XC plaques observed the data. DNA polymerase reactions are in the direct infection of XC cells could be linear with respect to time (up to 2 hr) and easily counted in unstained cultures (as well virus concentration. as methylene blue-stained cultures) as early Solubilixation of MLV. Double density as 1 day after infection. No increase in plaque gradient-purified (Toplin and Sottong, 1972) number was observed at 2 or subsequent MLV virions in 1 ml of 0.01 M Trisdays after infection. The concentration hydrochloride, 0.1 M NaCl, 0.001 M EDTA of 1.25 x lo6 XC cells per 60-mm petri (TNE pH 7.2), was thawed and diluted with dish was optimal for assaying direct XC cell 3.0 ml of TNE. A 0. l-ml sample was removed fusion. Table 2 shows that there was a signif-
133
ZARLING
AND KESHET TABLE
COMPARISON OF THE DIRECT AND INDIRECT
2 ASSAYS
OF
MLV BY XC CELL FUSIONS XC PFU/ml
Virus
mg/ml Protein0
NIH Moloney-MLV JLS-V9 Rauscher-MLV NIH Gross-MLV NIH AKR-MLV
0.64 1.25 0.53 3.52
Indirect assay (infectivity) 3.5 2.5 1.2 1.0
x x x x
107 105 108 10’
Direct assay (fusion activity) 2.2 3.5 1.7 1.5
x x x x
105 103 106 104
n Virus was assayed for XC plaque formation by infecting duplicate cultures of SC-l or XC cells with 0.2 ml of serial lo-fold virus dilutions in complete media containing 2 pg/ml Polybrene. Infected SC-l cell cultures were uv irradiated and overlayed with XC cells 6 days after infection and stained 10 days after infection (indirect assay). Infected XC cell cultures were stained 2 days after infection (direct assay). The number of XC plaque-forming units (PFU) was determined by counting plaques containing fused cells at 63~ using an inverted microscope. * The protein concentration of purified MLV stocks was assayed as described by Lowry et al., 1951.
icantly lower (approximately 70- to 6’70-fold lower) virus titer obtained by direct assay in comparison with indirect assay. In other similar experiments, different preparations of all these (concentrated, purified, and frozen) virus strains (Table 2) consistently showed approximately 30- to 500-fold lower direct titers compared to the indirect titers. Similar results were also obtained with either frozen or fresh (not frozen, concentrated, or purified) Moloney-MLV harvested from newly infected SC-l cells (data not shown). It is likely that the direct assay measures the parental MLV or MLV-induced fusion activity whereas the indirect assay measures the infectivity of MLV in mouse SC-l cells by the fusion activity of the mouse cell progeny virus. Kinetics of MLV-Induced Cells
Fusion of XC
XC cells infected with the Moloney, Rauscher, Gross, or AKR strains of MLV were observed to induce the formation of giant cells during the first 3 hr of infection. Figure 1 shows representative results from experiments where the effects of MLV infection on total XC cell numbers were measured. By 3 hr after infection with NIH Gross-MLV there was a significant reduction in the total number of single XC cells per infected culture (Fig. 1) depending on
the multiplicity of infection (m.0.i.). During counting of these trypsinized cultures (3 hr after infection) XC cells in the process of fusing were observed. The decrease in the number of single cells per infected culture relative to parallel uninfected cultures reached a maximum at 6 to 24 hr after infection (Fig. 1). There was no significant increase in the number of dead cells (stained with eosin) during these times (Fig. 1). Thus, MLV-induced fusion of XC cells occurs early after infection and causes a reduction in the number of single cells during this period (Fig. 1). Effects of Metabolic Inhibitors Fusion of XC Cells by MLV
on the
The ability of MLV to fuse XC cells could be virally induced in XC cells. Therefore, the effects of metabolic inhibitors on the direct fusion of XC cells by MLV was tested. MLV-infected XC cell cultures were treated with cycloheximide, cytosine arabinoside (Ara-C), actinomycin D, or no inhibitor for 3 hr (Table 3). Table 3 shows that neither the cyclohexamide (10 pg/ml) nor Ara-C (10 pg/ml) treatments significantly inhibited XC plaque formation by Rauscher-MLV. Actinomycin D treatment (either 0.25 or 0.5 tLglm1, Sigma Lot 126C-0219) was extremely cytotoxic to XC cells and thus its effects on XC plaque formation were not determined.
MURINE
LEUKEMIA
189
VIRUS FUSION ACTIVITY
A
MOI
q
0.002
MOI = 0.02
8
25 -
k z 0’
01
’ 3
1 24
6 TIME
AFTER
INFECTION
(HRS.1
FIG. 1. Cultures of XC cells (1.25 x 10” cells per 60-mm dish seeded the previous day) were mock infected or infected with NIH Gross-MLV at a multiplicity of either 0.02 or 0.002 XC PFU (direct assay) per cell. At each time after infection the total number of single cells per culture was determined. Giant cells-were scored as 1 cell and at least 200 cells were counted to determine the total number of single cells per culture. At each time 295% of the cells in mock-infected or infected cultures were viable (excluding eosin dye). The percentage of the control cell number at each time was calculated from the formula: 100 x
number of single cells per infected culture number of single cells per mock-infected culture ’
Control experiments showed that cycloheximide and Ara-C treatment at these concentrations significantly inhibited protein (>99% inhibition) and DNA (>89% inhiTABLE
3
EFFECT OF METABOLIC INHIBITORS ON THE FUSION OF XC CELLS BY MLV” Inhibitor
Fusion activity (XC PFU/ml)
Cycloheximide Ara-C None
2.7 x 104 1.4 x 104 3.8 x lo4
D Replicate cultures of XC cells were inoculated with serial lo-fold dilutions of JLS-V9 Rauscher-MLV and following 45 min virus absorption the cultures were overlayed with complete medium containing either cycloheximide (10 pg/ml), Ara-C (10 pg/ml), or no inhibitor. After a 3-hr exposure, the cultures were washed twice and overlayed with fresh complete media. The number of XC plaques was scored 2 days after infection. Cycloheximide and Ara-C at these concentrations inhibited the respective incorporation of [3H]serine or [3H]thymidine into trichloroacetic acid-precipitable material by 99% (cycloheximide) and 89% (Ara-C) compared to mock-treated control cultures.
bition) synthesis (Table 3). Similar results to those in Table 3 were obtained in two other experiments (data not shown). Furthermore, similar results were obtained by Johnson et al. (1971) with either actinomycin D (0.5 pg/ml), puromycin (20 pug/ml), cycloheximide (10 pg/ml), rifamycin (120 pg/ml), or rat serum interferon (60 units in 0.1 ml rat serum) added at the time of infection of XC cells with Moloney-MLV and included in the media for 6 hr. Thus, fusion of XC cells by MLV does not appear to require de novo protein or DNA synthesis (during the first 3 hr of infection) which can be inhibited by cycloheximide or Ara-C (Table 3). The fusion of XC cells by MLV could either be an activity of the MLV virion or preexisting in the XC cell and activated by MLV infection. Effects of Treatment of MLV with Proteolytic Enzymes, Antiviral Serum, or uv Irradiation on XC Cell Fusion
The effects of Pronase or trypsin treatment of MLV on the fusion of XC cells by MLV are shown in Table 4. Treatment of NIH AKR-MLV with 20 pg/ml of either
190
ZARLING
AND KESHET
Pronase or trypsin inhibited the fusion of XC cells by approximately 99% (Pronase) and 96% (trypsin) (Table 4) and significantly reduced viral infectivity in the SC-l/uv-XC cell assay (data not shown). As a control, DFP-inactivated trypsin (20 pg/ml, Worthington Biochemical Corp., Freehold, N. J.) did not inhibit AKR-MLV fusion activity (Table 4) or the infectivity of MLV in the SC-lluv-XC cell assay (not shown). Similar results to those in Table 4 were obtained with the Moloney strain of MLV. Thus, treatment of MLV virions with Pronase or trypsin prevents fusion of XC cells (Table 4). The envelope glycoprotein of MLV (gp70) is known to be cleaved by trypsin or Pronase treatment and is marked by losses in viral infectivity, in the ability of virus to interfere with infection by other MLVs, to hemagglutinate erythrocytes, or in the ability of the virus to absorb neutralizing antibody to the viral envelope glycoprotein, gp70 (Schafer and Bolognesi, 1977). Goat antiserum against purified MoloneyMLV virions or normal goat serum was incubated with NIH Moloney-MLV for 45 min TABLE
4
EFFECT OF PROTEOLYTIC ENZYMES ON XC CELL FUSION BY MLV” Enzyme treatment Pronase DPCC trypsin* DFP-inactivated None
trypsin’
Fusion activity (total XC PFU) 4.0 2.0 7.2 5.2
x x x x
IO’ 102 103 103
” NIH AKR-MLV was incubated with 20 pg/ml of either Pronase, DPCC-treated trypsin, DFP-inactivated trypsin, or with enzyme buffer alone for 30 min at 37”. Following incubation an equal volume of soybean trypsin inhibitor (20 pg/ml) was added and the samples were serially (lo-fold) diluted in complete media containing 2 kg/ml Polybrene. The virus was then assayed (direct assay) for plaque formation (PFU) in duplicate cultures of XC cells. b Treatment of trypsin with diphenyl carbamyl chloride (DPCC) inactivates chymotrypsin activity but does not affect trypsin activity. c Trypsin inactivated with diisopropyl fluorophosphate (DFP).
at room temperature. Relative to normal goat serum, anti-Moloney-MLV serum caused approximately a 325-fold reduction (299.6% virus neutralization) in MoloneyMLV fusion activity in XC cell cultures and a similar reduction in viral infectivity as measured by the SC-l/uv-XC cell assay. In another different experiment, the effects on MLV infectivity and fusion activity were measured after incubating Moloney-MLV with normal sera or antisera prepared against purified MLV polypeptides (Table 5). Table 5 shows that in comparison to normal goat serum, goat antiserum against Moloney-MLV (whole virus) or anti-RauscherMLV gp70 serum significantly neutralized (299.4% neutralization) both the infectivity and fusion activity of the virus. In contrast, high-titered goat antiserum prepared against Rauscher-MLV ~30, ~15, ~12, or against AKR-MLV p10 did not significantly neutralize either the infectivity or the fusion activity of the virus (Table 5). Similarly, in comparison to normal rabbit serum, specific rabbit antiserum against RauscherMLV p15(E) did not significantly neutralize MLV infectivity or fusion activity (Table 5). These experiments (Table 5) show that the fusion activity or infectivity of MoloneyMLV is strongly neutralized only with antiserum prepared against the MLV envelope glycoprotein, gp70. Thus, MLV fusion activity appears to be associated- with viral infectivity and is inhibited by proteolytic enzymes which cleave the MLV envelope glycoprotein and by neturalizing antiserum directed against antigenic determinants of the viral envelope glycoprotein (Schafer and Bolognesi, 1977). The effects of irradiation with uv light for various times on the fusion activity (direct assay) and the infectivity (indirect assay) of Moloney-MLV were measured (Fig. 2). Figure 2 shows that uv irradiation of MoloneyMLV inactivated viral infectivity at a very rapid rate whereas the fusion activity of the virus was relatively resistant to uv irradiation. After 15 min of uv irradiation the infectivity of Moloney-MLV was reduced by over 5 logs of activity, in contrast the viral fusion activity was reduced by less than 1 log of activity (Fig. 2). In this and other
MURINE
LEUKEMIA
TABLE EFFECT
OF ANTISERUM
AGAINST
MLV
191
VIRUS FUSION ACTIVITY 5
POLYPEPTIDES
ON XC CELL
FUSION
BY
MLV”
XC PFU/ml Serum or antiserum Goat, normal Goat anti-Moloney-MLV Goat anti-Rauscher-MLV gp70 Goat anti-Rauscher-MLV p30 Goat anti-Rauscher-MLV ~15 Goat anti-Rauscher-MLV p12 Goat anti-AKR-MLV ~10 Goat anti-Rauscher-MLV DNA polymerase Rabbit, normal Rabbit anti-Rauscher-MLV p15(E)
Infectivity 4.6 1.0 2.5 6.2 4.3 5.6 6.0 6.0 7.5 5.7
x x x x x x x x x x
Fusion activity
10” 104 lo4 10” 10” 10” 10” 10” 106 106
8.8 x lo4 4.0 x 10’
