0022-538X/78/0025-0187$02. 00/0 JOURNAL OF VIROLOGY, Jan. 1978, p. 187-192 Copyright © 1978 American Society for Microbiology
Vol. 25, No. 1 Printed in U.S.A.
Prelimninary Characterization of a Temperature-Sensitive Mutant of Moloney Murine Leukemia Virus That Produces Particles at the Restrictive Temperature P. K. Y. WONG* AND GARY E. GALLICK Department of Microbiology and School of Basic Medical Sciences, University of Illinois, Urbana, Illinois 61801
Received for publication 27 June 1977
The isolation and preliminary characterization of a new temperature-sensitive mutant of Moloney murine leukemia virus, designated ts7, are reported. The infectivity of ts7, determined by a focus-forming unit assay, was reduced at least 100-fold when the virus was assayed at the nonpermissive temperature (39°C) as compared with assay at the permissive temperature (340C). However, several lines of evidence indicated that the diminution of ts7 titer at 390C is not due to its inability to form virus particles at that temperature. The supernatant from ts7-infected cells grown at 390C showed significant infectivity when assayed at 34°C; only small reductions in reverse transcriptase activity and fusion ability were observed when compared with supernatant from 340C ts7-infected cultures. That particles are produced at the nonpermissive temperature was confirmed by transmission electron microscopy of the supernatant from ts7-infected cells at 390C and by transmission electron microscope observations of mature particles trapped in the intercellular spaces of pelleted thin cell sections. A possible explanation for the productivity at 390C of particles that are infectious at 34°C but not at 390C is that the virus is heat labile at the nonpermissive temperature. Consistent with this hypothesis is the extreme heat lability of virus harvested at 340C. Such virus, when incubated at 390C, has a half-life one-sixth that of identical virus incubated at 340C, or that of wild-type virus at either temperature.
This paper reports the isolation and prelimiUnderstanding of the replicative cycle of many viruses has been greatly facilitated by the nary characterization of a new, spontaneous, isolation and characterization of virus mutants. .temperature-sensitive mutant of the Moloney Temperature-sensitive conditionally lethal mu- strain of murine leukemia virus (Mo-MuLV), tants have proven invaluable in characterizing designated ts7. Infectivity, fu-1 fusion, reversedifferent stages in viral life cycles. However, transcription, electron microscopic, and heat laanimal virus temperature-sensitive mutants bility studies indicate that ts7 is unique among have been slow and tedious to isolate compared the temperature-sensitive mutants of RNA tuwith those of bacterial viruses, and ts mutants mor viruses so far studied. These and other of RNA tumor viruses have proven to be no preliminary studies on ts7 are the subject of this exception. Thus, knowledge of the protein prod- report. ucts encoded by the RNA tumor virus genome MATERIALS AND METHODS has been derived largely by biochemical analyses rather than by characterization of mutants. DeCell cultures. Virus was propagated in mouse TB fining the viral functions biologically by the cells, a thymus bone marrow cell line derived from characterization of temperature-sensitive mu- CFW/D mice (1). Cells used for the assay of virus tants remains extremely important but is prov- production were 15F and fu-1. 15F cells are a nontransleukemia-neging to be difficult. Although considerable effort formed, nonproducer, sarcoma-positive, derived by infecting TB cells with Mohas been devoted to determining the ts defects ative cell line sarcoma virus (2). fu-1 cells were derived loney murine of the relatively few RNA tumor virus mutants, from an La rat myoblast cell line (6) and kindly supas yet none is completely understood on a mo- plied by S. J. Kaufman. All cell lines were grown in lecular basis; furthermore, the mutants isolated Eagle minimal essential medium supplemented with thus far probably are not representative of all 10% fetal calf serum (Grand Island Biological Co., the possible functions and products coded by Grand Island, N.Y.), 50 IU of penicillin per ml, and 50 ug of streptomycin per ml. Cell cultures were seeded the entire viral genome. 187
188
WONG AND GALLICK
at a density of 2 x 105 to 3 x 105 cells per 75-cm2 flask and subcultured every 3 to 4 days, before reaching confluency. Virus. ts7 was isolated by a selection procedure described elsewhere (16). Preparation of virus stocks was as previously published (14), except that fresh virus stock was obtained by changing medium 24 h before harvest, which occurred after 3 days of incubation at 340C. All supernatant-containing virus was filtered through a 0.45-,um membrane filter (Millipore Corp., Bedford, Mass.), the larger volumes through a 0.45-pm Nalgene filter (Nalge Sybron Corp., Rochester, N.Y.), and was stored at -80°C. Viral assays. The 15F assay for focus-forming units per milliliter as detailed by McCarter (7) was used to determine surviving virus in the heat lability experiment because this assay was relatively more sensitive than the modified XC assay (16). Briefly, 15F cells were removed with trypsin (0.25% in isotonic saline citrate) before the cultures became confluent and were transferred in complete medium to spinner flasks kept at 370C. After 4 h of continuous stirring, the cells were counted, diluted to 106 per ml, and infected in suspension with the supernatant to be assayed for MuLV. Virus was diluted in medium lacking serum and containing 25 pug of DEAE-dextran per ml. Diluted virus (1.0 ml) was mixed with 1.0 ml of cell suspension in plastic tubes and was shaken at 37°C for 30 min. The cell suspension was then diluted, and 2 x 105 infected cells were plated in each of a number of plastic petri dishes (60 mm). Cultures infected with ts mutants were incubated at 340C, the permissive temperature, or 39°C, the nonpermissive temperature. A focus results from both division of initially infected cells and recruitment of neighboring cells by subsequent rounds of viral replication. Maximal focus formation required 5 days at 340C and 4 days at 390C. The fu-1 assay was performed as described by Wong et al. (17). Virus stock to be tested was diluted (1:2) with 25 pg of DEAE-dextran per ml. The diluted virus was inoculated in 0.4-ml aliquots into a subconfluent cell monolayer (2 x 105 cells seeded on each 35-mm plate overnight). After 40 min of absorption at 370C, unabsorbed virus was removed, and fresh complete medium was added. Cells were fixed and stained after 6 h of incubation at 370C. Multinucleate cells containing three or more nuclei were counted in four or five randomly selected fields in each plate. Duplicate plates were counted, and the viral titer was expressed as number of syncytia per square centimeter. Heat lability. Heat lability of a 340C (the permissive temperature) harvest of ts7 and wild-type MoMuLV was determined by infecting TB cells as described above, filtering immediately after harvesting, and dividing the supernatant into two 10-ml aliquots that were placed in 34 and 390C water baths. At fixed times after incubation at these temperatures, 1-ml samples were collected and frozen. The titer of surviving infectious virus was determined by the 15F assay. Reverse-transcriptase assay. Assay for viral-associated reverse transcriptase (3, 11) was as described previously (19). Briefly, tissue culture fluids removed from infected cells were filtered through 0.45-pum Millipore membrane filters and centrifuged at 60,000 x g for 90 min at 40C. The pellets were then suspended
J. VIROL. in 0.1 ml of 0.01 M Tris-0.05 M KCl-0.001 M dithiothreitol (pH 7.4) at 200C and assayed. Portions (30 p1) were assayed in 100-pl incubation mixtures containing 50 mM Tris (pH 8.0), 60 mM KCl, 1 mM MnCl2, 2.5 mM dithiothreitol, 0.05% Nonidet P-40, and 0.04 optical density unit (260 nm) of polyribocytidylic acid* oligodeoxyguanylic acid 12-18 (rC:dG). To start the reaction, 10 id of 0.2 mM [3H]dGTP (10 Ci/mmol) was added. The mixtures were incubated at 370C for 30 min. Acid-precipitable radioactivity, collected on Millipore filters, was estimated in a liquid scintillation counter. Transmission electron microscopy. Procedures for the preparation of thin sections and negative staining of virus particles have been described (20). For quantitation of virus in thin sections, cell sections were chosen at random, and the virions were counted at a magnification of x80,000. Negative staining. Formvar-carbon-coated grids were lowered onto a drop of clarified concentrated virus suspended in Tris-NaCl-EDTA buffer and allowed to adsorb for 1 to 3 min. The excess fluid was then drained off, and the grids were stained in 1% uranyl acetate or 1% ammonium molybdate. Scanning electron microscopy. Details of the scanning electron microscopy procedure used have been previously described (15).
RESULTS Virus production at 39 and 34°C compared by infectivity assay. The infectivity of ts7, when assayed by the focus-forming units assay at 390C, was at least 100-fold less than when the assay was performed at 340C, regardless of whether the viral harvest was obtained from infected cells incubated at 34 or 390C. However, virus harvested from infected cells grown at 390C was only about 10-fold less infectious than that grown at 340C, when the assay was done at 340C. (A fourfold difference in infectivity was obtained when wild-type virus grown at the permissive and nonpermissive temperatures was assayed at 340C.) These data, indicating that virus was produced at the nonpermissive temperature, are confirmed by the following lines of evidence and are summarized in Table 1. Induction of syncytia formation in fu-1 cells. fu-1 cells are a nonfusing myoblast cell line derived from rat L8 cells by Kaufinan (6). These cells form syncytia within 1 h when infected with Mo-MuLV (17). It was further shown that UV-inactivated MuLV still possesses the ability to induce syncytia formation (18), which suggests that infectious particles are not needed to cause syncytia formation. When the viral harvests from ts7-infected cells grown at the nonpermissive and permissive temperature were compared to determine their ability to cause syncytia formation in fu-1 cells, only a twofold difference in the number of syncytia was found, although approximately a 10-fold difference in
VOL. 25, 1978
189
MuLV ts MUTANT PRODUCING DEFECTIVE PARTICLES
TABLE 1. Comparison of some properties of ts7 and wild-type Mo-MuL V at 34 and 390C Infectivity (FFU/ml) as-
Virus
sayed ata:
Growth temp
(OC) ts7 ts7 MuLV MuLV
34 39 34 39
390C
4 2 2 7
X 104 x 104
34°C
3 X 107 3 X 106 6 x 107 1.5 X 107
Reverse-transcriptase
Syncytia formation in fu-1 cells (no./cm2)
7.2 X 3.5 x 1.5 x 1.0 X
102 102
activity (pmol/40 pl)
Virus particles observed per cell section
Intracellular 1.3 1.3
sociated 0.20 0.05
Extracellular 631.3 58.1 353.1
Cell as-
Extracellularb + +
0.8 + 0.22 103 + 0.6 92.3 0.20 X 106 103 aSupematant from infected cells grown at the temperature indicated was assayed for infectivity at both 34 and 39°C by the 15F focus-forming units (FFU) assay described in the text. bAlthough extracellular particles (i.e., particles trapped in the intercellular space) were observed for both ts7 and Mo-MuLV at 34 and 39°C, they occurred too infrequently to quantitatively estimate. x 107
infectivity was obtained. In contrast, ts3, an MoMuLV mutant that does not produce particles at 390C, shows a 100-fold difference in fusion ability between viral harvests from these two temperatures (18). Reverse-transcriptase activity. No significant difference in the amount of intracellular reverse-transcriptase activity was found between ts7-infected cells grown at the nonpermissive and at the permissive temperature. Extracellular reverse transcriptase decreased 10-fold in ts7 grown at 390C; there was also a fourfold decrease in wild-type Mo-MuLV at this temperature.
20
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Vol. 25, No. 1 Printed in U.S.A.
Prelimninary Characterization of a Temperature-Sensitive Mutant of Moloney Murine Leukemia Virus That Produces Particles at the Restrictive Temperature P. K. Y. WONG* AND GARY E. GALLICK Department of Microbiology and School of Basic Medical Sciences, University of Illinois, Urbana, Illinois 61801
Received for publication 27 June 1977
The isolation and preliminary characterization of a new temperature-sensitive mutant of Moloney murine leukemia virus, designated ts7, are reported. The infectivity of ts7, determined by a focus-forming unit assay, was reduced at least 100-fold when the virus was assayed at the nonpermissive temperature (39°C) as compared with assay at the permissive temperature (340C). However, several lines of evidence indicated that the diminution of ts7 titer at 390C is not due to its inability to form virus particles at that temperature. The supernatant from ts7-infected cells grown at 390C showed significant infectivity when assayed at 34°C; only small reductions in reverse transcriptase activity and fusion ability were observed when compared with supernatant from 340C ts7-infected cultures. That particles are produced at the nonpermissive temperature was confirmed by transmission electron microscopy of the supernatant from ts7-infected cells at 390C and by transmission electron microscope observations of mature particles trapped in the intercellular spaces of pelleted thin cell sections. A possible explanation for the productivity at 390C of particles that are infectious at 34°C but not at 390C is that the virus is heat labile at the nonpermissive temperature. Consistent with this hypothesis is the extreme heat lability of virus harvested at 340C. Such virus, when incubated at 390C, has a half-life one-sixth that of identical virus incubated at 340C, or that of wild-type virus at either temperature.
This paper reports the isolation and prelimiUnderstanding of the replicative cycle of many viruses has been greatly facilitated by the nary characterization of a new, spontaneous, isolation and characterization of virus mutants. .temperature-sensitive mutant of the Moloney Temperature-sensitive conditionally lethal mu- strain of murine leukemia virus (Mo-MuLV), tants have proven invaluable in characterizing designated ts7. Infectivity, fu-1 fusion, reversedifferent stages in viral life cycles. However, transcription, electron microscopic, and heat laanimal virus temperature-sensitive mutants bility studies indicate that ts7 is unique among have been slow and tedious to isolate compared the temperature-sensitive mutants of RNA tuwith those of bacterial viruses, and ts mutants mor viruses so far studied. These and other of RNA tumor viruses have proven to be no preliminary studies on ts7 are the subject of this exception. Thus, knowledge of the protein prod- report. ucts encoded by the RNA tumor virus genome MATERIALS AND METHODS has been derived largely by biochemical analyses rather than by characterization of mutants. DeCell cultures. Virus was propagated in mouse TB fining the viral functions biologically by the cells, a thymus bone marrow cell line derived from characterization of temperature-sensitive mu- CFW/D mice (1). Cells used for the assay of virus tants remains extremely important but is prov- production were 15F and fu-1. 15F cells are a nontransleukemia-neging to be difficult. Although considerable effort formed, nonproducer, sarcoma-positive, derived by infecting TB cells with Mohas been devoted to determining the ts defects ative cell line sarcoma virus (2). fu-1 cells were derived loney murine of the relatively few RNA tumor virus mutants, from an La rat myoblast cell line (6) and kindly supas yet none is completely understood on a mo- plied by S. J. Kaufman. All cell lines were grown in lecular basis; furthermore, the mutants isolated Eagle minimal essential medium supplemented with thus far probably are not representative of all 10% fetal calf serum (Grand Island Biological Co., the possible functions and products coded by Grand Island, N.Y.), 50 IU of penicillin per ml, and 50 ug of streptomycin per ml. Cell cultures were seeded the entire viral genome. 187
188
WONG AND GALLICK
at a density of 2 x 105 to 3 x 105 cells per 75-cm2 flask and subcultured every 3 to 4 days, before reaching confluency. Virus. ts7 was isolated by a selection procedure described elsewhere (16). Preparation of virus stocks was as previously published (14), except that fresh virus stock was obtained by changing medium 24 h before harvest, which occurred after 3 days of incubation at 340C. All supernatant-containing virus was filtered through a 0.45-,um membrane filter (Millipore Corp., Bedford, Mass.), the larger volumes through a 0.45-pm Nalgene filter (Nalge Sybron Corp., Rochester, N.Y.), and was stored at -80°C. Viral assays. The 15F assay for focus-forming units per milliliter as detailed by McCarter (7) was used to determine surviving virus in the heat lability experiment because this assay was relatively more sensitive than the modified XC assay (16). Briefly, 15F cells were removed with trypsin (0.25% in isotonic saline citrate) before the cultures became confluent and were transferred in complete medium to spinner flasks kept at 370C. After 4 h of continuous stirring, the cells were counted, diluted to 106 per ml, and infected in suspension with the supernatant to be assayed for MuLV. Virus was diluted in medium lacking serum and containing 25 pug of DEAE-dextran per ml. Diluted virus (1.0 ml) was mixed with 1.0 ml of cell suspension in plastic tubes and was shaken at 37°C for 30 min. The cell suspension was then diluted, and 2 x 105 infected cells were plated in each of a number of plastic petri dishes (60 mm). Cultures infected with ts mutants were incubated at 340C, the permissive temperature, or 39°C, the nonpermissive temperature. A focus results from both division of initially infected cells and recruitment of neighboring cells by subsequent rounds of viral replication. Maximal focus formation required 5 days at 340C and 4 days at 390C. The fu-1 assay was performed as described by Wong et al. (17). Virus stock to be tested was diluted (1:2) with 25 pg of DEAE-dextran per ml. The diluted virus was inoculated in 0.4-ml aliquots into a subconfluent cell monolayer (2 x 105 cells seeded on each 35-mm plate overnight). After 40 min of absorption at 370C, unabsorbed virus was removed, and fresh complete medium was added. Cells were fixed and stained after 6 h of incubation at 370C. Multinucleate cells containing three or more nuclei were counted in four or five randomly selected fields in each plate. Duplicate plates were counted, and the viral titer was expressed as number of syncytia per square centimeter. Heat lability. Heat lability of a 340C (the permissive temperature) harvest of ts7 and wild-type MoMuLV was determined by infecting TB cells as described above, filtering immediately after harvesting, and dividing the supernatant into two 10-ml aliquots that were placed in 34 and 390C water baths. At fixed times after incubation at these temperatures, 1-ml samples were collected and frozen. The titer of surviving infectious virus was determined by the 15F assay. Reverse-transcriptase assay. Assay for viral-associated reverse transcriptase (3, 11) was as described previously (19). Briefly, tissue culture fluids removed from infected cells were filtered through 0.45-pum Millipore membrane filters and centrifuged at 60,000 x g for 90 min at 40C. The pellets were then suspended
J. VIROL. in 0.1 ml of 0.01 M Tris-0.05 M KCl-0.001 M dithiothreitol (pH 7.4) at 200C and assayed. Portions (30 p1) were assayed in 100-pl incubation mixtures containing 50 mM Tris (pH 8.0), 60 mM KCl, 1 mM MnCl2, 2.5 mM dithiothreitol, 0.05% Nonidet P-40, and 0.04 optical density unit (260 nm) of polyribocytidylic acid* oligodeoxyguanylic acid 12-18 (rC:dG). To start the reaction, 10 id of 0.2 mM [3H]dGTP (10 Ci/mmol) was added. The mixtures were incubated at 370C for 30 min. Acid-precipitable radioactivity, collected on Millipore filters, was estimated in a liquid scintillation counter. Transmission electron microscopy. Procedures for the preparation of thin sections and negative staining of virus particles have been described (20). For quantitation of virus in thin sections, cell sections were chosen at random, and the virions were counted at a magnification of x80,000. Negative staining. Formvar-carbon-coated grids were lowered onto a drop of clarified concentrated virus suspended in Tris-NaCl-EDTA buffer and allowed to adsorb for 1 to 3 min. The excess fluid was then drained off, and the grids were stained in 1% uranyl acetate or 1% ammonium molybdate. Scanning electron microscopy. Details of the scanning electron microscopy procedure used have been previously described (15).
RESULTS Virus production at 39 and 34°C compared by infectivity assay. The infectivity of ts7, when assayed by the focus-forming units assay at 390C, was at least 100-fold less than when the assay was performed at 340C, regardless of whether the viral harvest was obtained from infected cells incubated at 34 or 390C. However, virus harvested from infected cells grown at 390C was only about 10-fold less infectious than that grown at 340C, when the assay was done at 340C. (A fourfold difference in infectivity was obtained when wild-type virus grown at the permissive and nonpermissive temperatures was assayed at 340C.) These data, indicating that virus was produced at the nonpermissive temperature, are confirmed by the following lines of evidence and are summarized in Table 1. Induction of syncytia formation in fu-1 cells. fu-1 cells are a nonfusing myoblast cell line derived from rat L8 cells by Kaufinan (6). These cells form syncytia within 1 h when infected with Mo-MuLV (17). It was further shown that UV-inactivated MuLV still possesses the ability to induce syncytia formation (18), which suggests that infectious particles are not needed to cause syncytia formation. When the viral harvests from ts7-infected cells grown at the nonpermissive and permissive temperature were compared to determine their ability to cause syncytia formation in fu-1 cells, only a twofold difference in the number of syncytia was found, although approximately a 10-fold difference in
VOL. 25, 1978
189
MuLV ts MUTANT PRODUCING DEFECTIVE PARTICLES
TABLE 1. Comparison of some properties of ts7 and wild-type Mo-MuL V at 34 and 390C Infectivity (FFU/ml) as-
Virus
sayed ata:
Growth temp
(OC) ts7 ts7 MuLV MuLV
34 39 34 39
390C
4 2 2 7
X 104 x 104
34°C
3 X 107 3 X 106 6 x 107 1.5 X 107
Reverse-transcriptase
Syncytia formation in fu-1 cells (no./cm2)
7.2 X 3.5 x 1.5 x 1.0 X
102 102
activity (pmol/40 pl)
Virus particles observed per cell section
Intracellular 1.3 1.3
sociated 0.20 0.05
Extracellular 631.3 58.1 353.1
Cell as-
Extracellularb + +
0.8 + 0.22 103 + 0.6 92.3 0.20 X 106 103 aSupematant from infected cells grown at the temperature indicated was assayed for infectivity at both 34 and 39°C by the 15F focus-forming units (FFU) assay described in the text. bAlthough extracellular particles (i.e., particles trapped in the intercellular space) were observed for both ts7 and Mo-MuLV at 34 and 39°C, they occurred too infrequently to quantitatively estimate. x 107
infectivity was obtained. In contrast, ts3, an MoMuLV mutant that does not produce particles at 390C, shows a 100-fold difference in fusion ability between viral harvests from these two temperatures (18). Reverse-transcriptase activity. No significant difference in the amount of intracellular reverse-transcriptase activity was found between ts7-infected cells grown at the nonpermissive and at the permissive temperature. Extracellular reverse transcriptase decreased 10-fold in ts7 grown at 390C; there was also a fourfold decrease in wild-type Mo-MuLV at this temperature.
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