VIROLOGY
63, 176-190 (1975)
Cell Killing tl. Cell Killing
by Vesicular
by Viruses
Stomatitis
Derived
Virus:
a Requirement
Transcription
l
PHILIP I. MARCUS AND MARGARET Microbiology
Section,
U-44, Biological
Sciences Group, University Accepted
September
for Virion-
J. SEKELLICK of Connecticut,
Storrs, Connecticut
06268
3, 1974
Single-cell survival curves generated by infecting the Vero line of green monkey kidney (GMK) cells with Indiana-type vesicular stomatitis virus (VSV) were used to establish the cell killing particle (CKP) activity of active, uv-irradiated and heat-inactivated (50°) virions, as well as that of conditional-lethal mutants tested at permissive (30”) and nonpermissive (40”) temperatures. CKP activity of virions treated in this manner was compared to that of infectivity, PFP, and virion-derived transcriptase activity measured in uiuo. Heat denaturation revealed that all three of these functions were lost at the same rate, the absolute value apparently set by the intrinsic lability of the virion-associated transcriptase. For VSV-HR and mutant ts G114, these rates were K = 0.23 min-’ and K = 1.44 min-I, respectively. Ultraviolet-radiation inactivated infectivity (PFP) at an exponential rate with the 37% survival dose, D” = 52.3 ergs min- 2. Virion-derived transcription as measured in uivo and CKP activity were lost at coincident rates upon irradiation, but were 5 times more resistant to inactivation--D’ = 263 ergs min-*. In all cases, in oivo virionderived transcriptase and CKP activities were inactivated at coincident rates. With mutant ts G114 (group I), the CKP and PFP titers were equivalent at 30”. However, ts G114 did not kill cells at 40”, nor was significant virion-derived transcription observed. In contrast, mutant ts WlO (group IV) produced almost equivalent CKP titers over a temperature range of 30”-40”. Although large amounts of primary transcripts accumulate in the presence of cycloheximide in cells infected with ts G114 at 30”, these transcripts act inefficiently to kill cells upon removal of the drug and a temperature-shift to 40’. Mutant ts G114 requires 18-20 hr at 30” to express maximum CKP activity. The lethal action of 50% of the CKP is extant by 6 hr at 30°, yet less than 10% of newly synthesized virus has been released. Cell killing factor is produced during normal infection and upon exposure of cells to (1) defective CKP, (2) uv-irradiated virions and (3) mutant ts WlO at nonpermissive temperatures. Virion-derived primary transcription is required to produce cell killing by VSV, but accumulation of transcripts per se does not suffice to kill cells. It appears likely that formation of a putative cell killing factor requires at least two reactants: (1) primary transcripts and (2) transcriptase molecules, functioning in a reaction(s) subsequent to primary transcription which could be replicative or transcriptive in nature.
by the single cell survival procedure as described in the first report of this series Studies of cell killing particle activity of demonstrated that (1) the virion per se is vesicular stomatitis virus (VSV) measured not intrinsically toxic, (2) cell killing does INTRODUCTION
‘This research was supported by USPHS Grant AI-09312 from the National Institute of Allergy and Infectious Diseases and benefited from use of the Cell presented at the 74th annual meeting of the American Culture Facility supported by National Cancer Insti- Society for Microbiology, May 17, 1974 (Amer. Sot. tute Grant CA-14733. Some aspects of this paper were Microbial. Abst., p. 266). 176 Copyright 0 1975 by AcademicPress.Inc. All rights of reproductionin any form reserved.
CELL KILLING
not depend upon complete viral replication and (3) an activity not expressed in cells infected solely with defective interfering particles is required for cell killing (Marcus and Sekellick, 1974). Furthermore, we noted that cell killing by VSV is expressed under two different conditions of viral interference where primary transcription is extant: (1) homotypic interference (Huang and Manders, 1972; Perrault and Holland, 1972) and (2) intrinsic interference (Hunt and Marcus, 1974). In contrast, cell killing is prevented under conditions where viriondirected transcription is inhibited significantly by the interferon system (Marcus et al., 1971; Manders et al., 1972). These findings led us to hypothesize that virionassociated transcription was a requisite, though not necessarily sufficient, event for cell killing by VSV. The present communication provides evidence to support this hypothesis by demonstrating that the rate of loss of cell killing particle (CKP) activity and of virion-associated transcription are coincident under three different experimental conditions-inactivation of virions by (1) ultraviolet radiation, (2) heat (50”) and (3) when temperature-sensitive mutants defective in virion-associated transcriptase are used to infect cells at the restrictive temperature. In addition, we describe experiments which indicate that virion-type transcriptase molecules may be required in a post-primary transcriptive reaction essential for cell killing by VSV. Subsequent reports in this series will examine the role of the interferon system and of double-stranded RNA in cell killing by viruses. MATERJALS
AND
Cells and characteristics
METHODS
of cell growth.
The Vero line, clone M3, of green monkey kidney cells (GMK) was used in all experiments reported here. These cells clone with high efficiency (70-lOO%), even at 40”, and can maintain this plating efficiency after 20-24 hr at 30”-an important attribute for host cells of ts-mutants. Parental VSV replicates to high titer in GMKVero cells over a broad temperature range (30”-40”), and all ts-mutants described
177
BY VIRUSES
herein manifest their characteristic temperature-sensitive lesions in plaquing efficiency and in in vivo virion-associated transcriptase activity when tested at nonpermissive temperatures. This line of Vero is incapable of being stimulated to produce interferon, and is tested periodically for the presence of Mycoplasma. The growth of GMK-Vero cells and optimal conditions for their cloning have been described in the first report of this series (Marcus and Sekellick, 1974). Viruses: source and stock preparations.
The heat-resistant (HR) strain of VSV (Indiana serotype) was graciously provided by Dr. D. V. Cormack and Dr. A. F. Holloway, as were the temperature sensitive (ts) mutants that carry the Winnipeg, W (Holloway et al., 1970), and the Toronto, T (Farmilo and Stanners, 1972), designations. The Glasgow, G, mutants (Pringle, 1970) were obtained through the courtesy of Dr. Alice Huang. Stocks of VSV-HR and ts-mutants were produced from confluent monolayers of primary chick embryo cells grown in attachment solution (Marcus and Carver, 1965) and infected with a m.o.i.ppp = 0.001-0.01. Infected cells were incubated for 36-48 hr at 30”; the medium overlaying the cells was collected and centrifuged for 20 min at 1000 g to remove cell debris, The supernatant fluid constituted a standard virus stock preparation, which usually assayed at about 5-50 x lOa PFP/ml. Revertant titers for mutants ts WlO, T1026, and G41 measured by plaquing at 40.0” were usually around lo’-lo6 PFP/ml, whereas revertants for ts-G114 were never observed, i.e., were
63, 176-190 (1975)
Cell Killing tl. Cell Killing
by Vesicular
by Viruses
Stomatitis
Derived
Virus:
a Requirement
Transcription
l
PHILIP I. MARCUS AND MARGARET Microbiology
Section,
U-44, Biological
Sciences Group, University Accepted
September
for Virion-
J. SEKELLICK of Connecticut,
Storrs, Connecticut
06268
3, 1974
Single-cell survival curves generated by infecting the Vero line of green monkey kidney (GMK) cells with Indiana-type vesicular stomatitis virus (VSV) were used to establish the cell killing particle (CKP) activity of active, uv-irradiated and heat-inactivated (50°) virions, as well as that of conditional-lethal mutants tested at permissive (30”) and nonpermissive (40”) temperatures. CKP activity of virions treated in this manner was compared to that of infectivity, PFP, and virion-derived transcriptase activity measured in uiuo. Heat denaturation revealed that all three of these functions were lost at the same rate, the absolute value apparently set by the intrinsic lability of the virion-associated transcriptase. For VSV-HR and mutant ts G114, these rates were K = 0.23 min-’ and K = 1.44 min-I, respectively. Ultraviolet-radiation inactivated infectivity (PFP) at an exponential rate with the 37% survival dose, D” = 52.3 ergs min- 2. Virion-derived transcription as measured in uivo and CKP activity were lost at coincident rates upon irradiation, but were 5 times more resistant to inactivation--D’ = 263 ergs min-*. In all cases, in oivo virionderived transcriptase and CKP activities were inactivated at coincident rates. With mutant ts G114 (group I), the CKP and PFP titers were equivalent at 30”. However, ts G114 did not kill cells at 40”, nor was significant virion-derived transcription observed. In contrast, mutant ts WlO (group IV) produced almost equivalent CKP titers over a temperature range of 30”-40”. Although large amounts of primary transcripts accumulate in the presence of cycloheximide in cells infected with ts G114 at 30”, these transcripts act inefficiently to kill cells upon removal of the drug and a temperature-shift to 40’. Mutant ts G114 requires 18-20 hr at 30” to express maximum CKP activity. The lethal action of 50% of the CKP is extant by 6 hr at 30°, yet less than 10% of newly synthesized virus has been released. Cell killing factor is produced during normal infection and upon exposure of cells to (1) defective CKP, (2) uv-irradiated virions and (3) mutant ts WlO at nonpermissive temperatures. Virion-derived primary transcription is required to produce cell killing by VSV, but accumulation of transcripts per se does not suffice to kill cells. It appears likely that formation of a putative cell killing factor requires at least two reactants: (1) primary transcripts and (2) transcriptase molecules, functioning in a reaction(s) subsequent to primary transcription which could be replicative or transcriptive in nature.
by the single cell survival procedure as described in the first report of this series Studies of cell killing particle activity of demonstrated that (1) the virion per se is vesicular stomatitis virus (VSV) measured not intrinsically toxic, (2) cell killing does INTRODUCTION
‘This research was supported by USPHS Grant AI-09312 from the National Institute of Allergy and Infectious Diseases and benefited from use of the Cell presented at the 74th annual meeting of the American Culture Facility supported by National Cancer Insti- Society for Microbiology, May 17, 1974 (Amer. Sot. tute Grant CA-14733. Some aspects of this paper were Microbial. Abst., p. 266). 176 Copyright 0 1975 by AcademicPress.Inc. All rights of reproductionin any form reserved.
CELL KILLING
not depend upon complete viral replication and (3) an activity not expressed in cells infected solely with defective interfering particles is required for cell killing (Marcus and Sekellick, 1974). Furthermore, we noted that cell killing by VSV is expressed under two different conditions of viral interference where primary transcription is extant: (1) homotypic interference (Huang and Manders, 1972; Perrault and Holland, 1972) and (2) intrinsic interference (Hunt and Marcus, 1974). In contrast, cell killing is prevented under conditions where viriondirected transcription is inhibited significantly by the interferon system (Marcus et al., 1971; Manders et al., 1972). These findings led us to hypothesize that virionassociated transcription was a requisite, though not necessarily sufficient, event for cell killing by VSV. The present communication provides evidence to support this hypothesis by demonstrating that the rate of loss of cell killing particle (CKP) activity and of virion-associated transcription are coincident under three different experimental conditions-inactivation of virions by (1) ultraviolet radiation, (2) heat (50”) and (3) when temperature-sensitive mutants defective in virion-associated transcriptase are used to infect cells at the restrictive temperature. In addition, we describe experiments which indicate that virion-type transcriptase molecules may be required in a post-primary transcriptive reaction essential for cell killing by VSV. Subsequent reports in this series will examine the role of the interferon system and of double-stranded RNA in cell killing by viruses. MATERJALS
AND
Cells and characteristics
METHODS
of cell growth.
The Vero line, clone M3, of green monkey kidney cells (GMK) was used in all experiments reported here. These cells clone with high efficiency (70-lOO%), even at 40”, and can maintain this plating efficiency after 20-24 hr at 30”-an important attribute for host cells of ts-mutants. Parental VSV replicates to high titer in GMKVero cells over a broad temperature range (30”-40”), and all ts-mutants described
177
BY VIRUSES
herein manifest their characteristic temperature-sensitive lesions in plaquing efficiency and in in vivo virion-associated transcriptase activity when tested at nonpermissive temperatures. This line of Vero is incapable of being stimulated to produce interferon, and is tested periodically for the presence of Mycoplasma. The growth of GMK-Vero cells and optimal conditions for their cloning have been described in the first report of this series (Marcus and Sekellick, 1974). Viruses: source and stock preparations.
The heat-resistant (HR) strain of VSV (Indiana serotype) was graciously provided by Dr. D. V. Cormack and Dr. A. F. Holloway, as were the temperature sensitive (ts) mutants that carry the Winnipeg, W (Holloway et al., 1970), and the Toronto, T (Farmilo and Stanners, 1972), designations. The Glasgow, G, mutants (Pringle, 1970) were obtained through the courtesy of Dr. Alice Huang. Stocks of VSV-HR and ts-mutants were produced from confluent monolayers of primary chick embryo cells grown in attachment solution (Marcus and Carver, 1965) and infected with a m.o.i.ppp = 0.001-0.01. Infected cells were incubated for 36-48 hr at 30”; the medium overlaying the cells was collected and centrifuged for 20 min at 1000 g to remove cell debris, The supernatant fluid constituted a standard virus stock preparation, which usually assayed at about 5-50 x lOa PFP/ml. Revertant titers for mutants ts WlO, T1026, and G41 measured by plaquing at 40.0” were usually around lo’-lo6 PFP/ml, whereas revertants for ts-G114 were never observed, i.e., were
