JOURNAL OF VIROLOGY, Aug. 1975, p. 275-283 Copyright 0 1975 American Society for Microbiology
Vol. 16, No. 2 Printed in U.S.A.
Temperature-Sensitive Mutants of Herpes Simplex Virus Type 1 Defective in Lysis but Not in Transformation R. G. HUGHES, JR.,* AND W. H. MUNYON Department of Medical Viral Oncology, Roswell Park Memorial Institute, Buffalo, New York
14263
Received for publication 19 February 1975
Twelve temperature-sensitive (ts) mutants of herpes simplex virus type 1 (HSV-1), representing seven complementation groups, were isolated subsequent to 5-bromodeoxyuridine mutagenesis. These mutants were identified by their inability to replicate in a line of monkey (CV-1) cells at 39 C. Seven of these mutants, representing six complementation groups, induced thymidine kinase (tk) and transformed Ltk- cells, a line of mouse L cells lacking tk, to a tk+ phenotype at both the permissive (34 C) and nonpermissive (39 C) temperatures. Thus, the defective cistrons in these six complementation groups, although necessary for lysis, have no essential function in this transformation system. Transformation by these 12 mutants was dependent on prior UV irradiation. Infection of cells with unirradiated virus under conditions which did not permit virus replication was not sufficient to allow cell transformation. Five mutants, representing two complementation groups, were tk- and were incapable of causing the tk--to-tk+ transformation at either 34 C or 39 C. The tk defects in these mutants are probably unrelated to the ts defects, since one of these complementation groups contains a tk+ member. Therefore, transformation of Ltk- cells to a tk+ phenotype by HSV-1 requires an active viral tk gene. One complementation group was represented by a single tk - member. The role of this cistron in transformation remains undetermined since the primary block to transformation is presumed to be the tk- phenotype. Mutants representing the seven complementation groups were unable to replicate at 39 C in two lines of HSV-1-transformed cells, indicating that the activities of resident wild-type copies of the defective cistrons, if present, could not be detected by complementation.
Herpes simplex virus (HSV) can transform cultured mammalian cells to new phenotypes. Mouse (16), rat (12), and human (7) cells have been shown to acquire thymidine kinase (tk), hamster cells have been shown to acquire new HSV antigens, altered growth patterns, and malignant potential (9), and human cells have been shown to be morphologically transformed by HSV (6). Stevens and Cook (19) have shown that HSV can establish a latent infection in the dorsal root ganglia of mice and rabbits. Other studies have shown (2, 3) that herpes simplex type 1 (HSV-1) can infect human trigeminal ganglia and remain in a latent state to give rise to recurrent herpetic episodes. Furthermore, it has recently been shown that herpes simplex type 2 (HSV-2) can latently reside in the lumbosacral ganglia of mice after vaginal infection, suggesting a mechanism for recurrent genital herpes lesions in the human (21).
In vitro cell transformation and in vivo latency may be related, the former occurring with nonproductive virus in a permissive cell and the latter with virulent virus in a nonpermissive cell. If cell transformation by HSV, that is, nonlytic infection which changes one or more properties of the cell, occurs in its human host, we presume that specific viral functions and thus specific viral genes are needed to accomplish this transformation. Temperature-sensitive (ts) mutants of HSV have been described by Brown et al. (4) and Schaffer et al. (17, 18). Since in principle it may be possible to isolate ts mutants of any viral gene which is trans acting (11), we have embarked on a study of ts mutants of HSV to determine whether particular mutants of HSV alter the ability of the virus to transform thymidine kinaseless L (Ltk -) cells to a tk-positive (tk+) phenotype. From such studies we hope to determine which HSV genes must function for HSV to accomplish cell trans-
275
276
HUGHES AND MUNYON
formation. This system of transformation has a powerful advantage over nonselective embryo cell transformation systems in that selection can be applied to permit only the growth of the transformed cell. That is, in a medium containing methotrexate, purine nucleosides, glycine, and thymidine, only those cells that have acquired tk will survive. A second objective of this study has been the detection of functional HSV genes in Ltk- cells transformed to a tk+ phenotype by wild-type HSV. If a line of transformed cells contained a functional wild-type copy of the defective cistron of a given ts mutant, its presence could be detected by the formation of progeny virus in infected cells incubated at the nonpermissive temperature. These studies should determine whether different lines of HSV-transformed cells display the same or different collections of HSV functions and thus permit a choice between "random" and "specific" models of in vitro HSV transformation. That is, if specific genes were always found in transformed cells, it would suggest that they were essential for transformation. However, if different HSV genes were present in different lines of HSVtransformed cells, it would suggest that the viral products expressed had no essential function in transformation and that the viral genetic information included in the transformed cell might be random. MATERIALS AND METHODS Media. A modified Eagle medium (10) containing the non-essential amino acids, 0.2 Ag of Fe (NO3)39H2O per ml and 110 vg of sodium pyruvate per ml, and supplemented with 5% calf serum (GIBCO), was the basic medium for all cell culture (EM5C). This medium was further modified for the selection of tk+ cells by the addition of methotrexate, purine nucleosides, and thymidine (MTAGGEM5C) as previously described (7). The overlay medium for the plaque assay was EM5C (without phenol red) made 1% in 4,000 centipoise methylcellulose (MCEM5C). Plaques were visualized by overlaying with the same medium but made 2% in 15 centipoise methylcellulose and containing 100 jAg of neutral red per ml. Viruses and virus assay. HSV-1 strain KOS was obtained from Edmundo Kraiselburd. HSV-1 strain B2006, a tk-negative (tk-) variant, was obtained from and described by Dubbs and Kit (8). All cell and virus culture was carried out in humidified, water-jacketed incubators in an atmosphere of 10% CO2 and 90% air. Viruses were grown in CV-1 cells in EM5C and were assayed by plaque formation on CV-1 monolayers. Adsorption of all viruses was carried out for 1 h at the permissive temperature, and the cultures were then overlaid with MCEM5C. Cells. The origins of Ltk-, a line of mouse L cells
J. VIROL.
lacking tk, and clone 139 cells, a clone of Ltk- cells transformed by HSV-1, have been described (7, 16). CV-1, a line of monkey kidney cells, were obtained from the American Type Culture Collection, Rockville, Md. Clone 11U cells were derived in this laboratory and arose as a single transformed colony after infection of Ltk- cells with unirradiated KOS. This colony is unique in that it is the first and only instance of transformation due to unirradiated HSV which we have ever observed. Isolation of ts mutants. Mutagenization of the virus and isolation of the ts mutants were effected by modifications of the methods of Schaffer et al. (17). Briefly, KOS was plaque purified at 39 C (KOS 1.1) and used to infect CV-1 cells. After virus adsorption, the infected cells were cultured in EM5C containing 5 Ag of 5-bromo-2'-deoxyuridine (BUdR). Cultures were incubated at 31 C for 1 to 4 days with multiplicities of infection (MOI) ranging from 3 to 3 x 10-4 PFU/cell. The mutagenized stocks were plaqued on CV-1 monolayers at the permissive temperature, and wellisolated plaques were picked under a low-power microscope from unstained dishes. Mutants were tested for temperature sensitivity by their inability to form plaques on CV-1 monolayers at the nonpermissive temperature. The mutants that were presumptively ts were plaque purified and retested for temperature sensitivity. tk assay. The tk assay was a modification of the method of Munyon et al. (16). Pellets of about 4 x 106 cells were suspended in 100 ,l of 100 mM sodium maleate, pH 6.5, and frozen at -20 C and thawed once. The disrupted cells were sedimented at 500 x g, and the supernatant was used as the source of enzyme. The reaction mixture consisted of 20 Mmol of sodium maleate, pH 6.5, 1 gmol of ATP, 1 gmol of MgCl2, and 2 nmol of [3H JTdR (specific activity, 200 MCi/Mmol) in a total volume of 200 Ml. The reaction mixture also contained from about 400 to 600 Mg of protein, as determined by the method of Lowry et al. (13). The reaction was extinguished by the addition of 20Mgl of 50% trichloroacetic acid after incubation for 20 min at 37 C. Aliquots of the supernatant were spotted on DEAE-cellulose disks, which were then washed six times with 250-ml aliquots of 1 mM ammonium formate, pH 8.0, and once with 95% ethanol and dried. The disks were extracted in scintillation vials with 1-rnl aliquots of 0.1 N NaOH, after which was added 9 ml of a counting solution made by mixing 1 volume of Triton X-100 with 2 volumes of toluene containing 8.25 g of 2,5-diphenyloxazole and 0.25 g of dimethyl 1,4-bis-(5-phenyloxazolyl)benzene per liter. Counting efficiency was about 30%. Transformation assay. The transformation assay was that of Munyon et al. (16). Virus was diluted in EM5C to achieve the desired MOI and irradiated for 8 min at 280 MW/cm2 on a rotary shaker. All virus adsorptions were carried out for 1 h at the permissive temperature. The day after infection, EM5C was replaced with MTAGGEM5C made 0.1% in human gamma globulin (Hyland). This medium was replaced on days 6, 10, 14, and 18 postinfection, and the colonies were stained with crystal violet on day 21 postinfection.
VOL. 16, 1975
ts MUTANT TRANSFORMATION
RESULTS Characterization of productive infection by ts mutants. HSV-1 strain KOS was plaque purified at 39 C to produce a stock free of pre-existing ts mutants and which would grow well at the nonpermissive temperature. All of the ts mutants were derived from this virus, KOS 1.1. The nonpermissive temperature was defined as 39 C, and the permissive temperature was either 34 or 31 C. Twelve ts mutants were isolated as described in Materials and Methods. The course of maturation of KOS 1.1 and one of the mutants, ts 661, is compared in Fig. 1. Although ts 661 grew at 34 C, it was still clearly different from wild-type virus even at the permissive temperature. Similar observations on other HSV ts mutants have been made by Brown et al. (4) and Schaffer et al. (17). None of the mutants produced infectious virus at the nonpermissive temperature in contrast to KOS 1.1, which grew better at 39 than at 34 C (Table 1). Figure 1 also indicates that, for ts 661, any new virus synthesis at 39 C, if present at all, is insufficient to replace that lost by thermal inactivation, since the virus titer declines throughout the course of infection. After a cycle of growth at 39 C, plaques with wild-type morphology were observed in ts 661 (Table 1) and at low levels in samples grown at 39 C and titrated at 39 C in the complementation analyses (data not shown). Plaques with wild-type morphology could not be found when virus stocks themselves were titrated at 39 C, although wild-type virus occurring at low fre-
277
quency in the ts mutant cell population would not have been seen. No further search for revertants in the stocks was made. One of the mutants, ts 478, produced very small plaques at the nonpermissive temperature in contrast to
0
7
E
oSg
5
_
-
10 20 30 HOURS POST INFECTION FIG. 1. Course of maturation of KOS 1.1 and ts 661. Cultures of infected CV-1 cells were incubated at 34 C or 39 C. Samples were taken at the indicated times, and infectious virus was titrated at 34 C; 0, KOS 1.1 at 34 C; 0, KOS 1.1 at 39 C; 0, ts 661 at 34 C; *, ts 661 at 39 C. 0
TABLE 1. Progeny virus productiona and plaque formation at the permissive and nonpermissiL ?temperatures PFU/ml Virus
EOPb Ea
KOS 1.1 ts84 ts 478 ts656 ts661 ts730 ts 756 ts 606 ts629 ts807 ts 822
ts833 ts901
2.9 7.8 1.0 1.1 3.3 6.9 4.4 6.9 7.1 5.3 8.0 5.1 1.1
x x x x x x x x x x x
39 C
106
105 10"
106 105 10' 105 105 105 10'
10'
x 10' x 106
1.2 x 10" 5.0 x 10' 3.3 x 10' 1.0 x 105 1.8 x 10'c 5.0 x 103 1.1 x 105 1.0 x 104
Vol. 16, No. 2 Printed in U.S.A.
Temperature-Sensitive Mutants of Herpes Simplex Virus Type 1 Defective in Lysis but Not in Transformation R. G. HUGHES, JR.,* AND W. H. MUNYON Department of Medical Viral Oncology, Roswell Park Memorial Institute, Buffalo, New York
14263
Received for publication 19 February 1975
Twelve temperature-sensitive (ts) mutants of herpes simplex virus type 1 (HSV-1), representing seven complementation groups, were isolated subsequent to 5-bromodeoxyuridine mutagenesis. These mutants were identified by their inability to replicate in a line of monkey (CV-1) cells at 39 C. Seven of these mutants, representing six complementation groups, induced thymidine kinase (tk) and transformed Ltk- cells, a line of mouse L cells lacking tk, to a tk+ phenotype at both the permissive (34 C) and nonpermissive (39 C) temperatures. Thus, the defective cistrons in these six complementation groups, although necessary for lysis, have no essential function in this transformation system. Transformation by these 12 mutants was dependent on prior UV irradiation. Infection of cells with unirradiated virus under conditions which did not permit virus replication was not sufficient to allow cell transformation. Five mutants, representing two complementation groups, were tk- and were incapable of causing the tk--to-tk+ transformation at either 34 C or 39 C. The tk defects in these mutants are probably unrelated to the ts defects, since one of these complementation groups contains a tk+ member. Therefore, transformation of Ltk- cells to a tk+ phenotype by HSV-1 requires an active viral tk gene. One complementation group was represented by a single tk - member. The role of this cistron in transformation remains undetermined since the primary block to transformation is presumed to be the tk- phenotype. Mutants representing the seven complementation groups were unable to replicate at 39 C in two lines of HSV-1-transformed cells, indicating that the activities of resident wild-type copies of the defective cistrons, if present, could not be detected by complementation.
Herpes simplex virus (HSV) can transform cultured mammalian cells to new phenotypes. Mouse (16), rat (12), and human (7) cells have been shown to acquire thymidine kinase (tk), hamster cells have been shown to acquire new HSV antigens, altered growth patterns, and malignant potential (9), and human cells have been shown to be morphologically transformed by HSV (6). Stevens and Cook (19) have shown that HSV can establish a latent infection in the dorsal root ganglia of mice and rabbits. Other studies have shown (2, 3) that herpes simplex type 1 (HSV-1) can infect human trigeminal ganglia and remain in a latent state to give rise to recurrent herpetic episodes. Furthermore, it has recently been shown that herpes simplex type 2 (HSV-2) can latently reside in the lumbosacral ganglia of mice after vaginal infection, suggesting a mechanism for recurrent genital herpes lesions in the human (21).
In vitro cell transformation and in vivo latency may be related, the former occurring with nonproductive virus in a permissive cell and the latter with virulent virus in a nonpermissive cell. If cell transformation by HSV, that is, nonlytic infection which changes one or more properties of the cell, occurs in its human host, we presume that specific viral functions and thus specific viral genes are needed to accomplish this transformation. Temperature-sensitive (ts) mutants of HSV have been described by Brown et al. (4) and Schaffer et al. (17, 18). Since in principle it may be possible to isolate ts mutants of any viral gene which is trans acting (11), we have embarked on a study of ts mutants of HSV to determine whether particular mutants of HSV alter the ability of the virus to transform thymidine kinaseless L (Ltk -) cells to a tk-positive (tk+) phenotype. From such studies we hope to determine which HSV genes must function for HSV to accomplish cell trans-
275
276
HUGHES AND MUNYON
formation. This system of transformation has a powerful advantage over nonselective embryo cell transformation systems in that selection can be applied to permit only the growth of the transformed cell. That is, in a medium containing methotrexate, purine nucleosides, glycine, and thymidine, only those cells that have acquired tk will survive. A second objective of this study has been the detection of functional HSV genes in Ltk- cells transformed to a tk+ phenotype by wild-type HSV. If a line of transformed cells contained a functional wild-type copy of the defective cistron of a given ts mutant, its presence could be detected by the formation of progeny virus in infected cells incubated at the nonpermissive temperature. These studies should determine whether different lines of HSV-transformed cells display the same or different collections of HSV functions and thus permit a choice between "random" and "specific" models of in vitro HSV transformation. That is, if specific genes were always found in transformed cells, it would suggest that they were essential for transformation. However, if different HSV genes were present in different lines of HSVtransformed cells, it would suggest that the viral products expressed had no essential function in transformation and that the viral genetic information included in the transformed cell might be random. MATERIALS AND METHODS Media. A modified Eagle medium (10) containing the non-essential amino acids, 0.2 Ag of Fe (NO3)39H2O per ml and 110 vg of sodium pyruvate per ml, and supplemented with 5% calf serum (GIBCO), was the basic medium for all cell culture (EM5C). This medium was further modified for the selection of tk+ cells by the addition of methotrexate, purine nucleosides, and thymidine (MTAGGEM5C) as previously described (7). The overlay medium for the plaque assay was EM5C (without phenol red) made 1% in 4,000 centipoise methylcellulose (MCEM5C). Plaques were visualized by overlaying with the same medium but made 2% in 15 centipoise methylcellulose and containing 100 jAg of neutral red per ml. Viruses and virus assay. HSV-1 strain KOS was obtained from Edmundo Kraiselburd. HSV-1 strain B2006, a tk-negative (tk-) variant, was obtained from and described by Dubbs and Kit (8). All cell and virus culture was carried out in humidified, water-jacketed incubators in an atmosphere of 10% CO2 and 90% air. Viruses were grown in CV-1 cells in EM5C and were assayed by plaque formation on CV-1 monolayers. Adsorption of all viruses was carried out for 1 h at the permissive temperature, and the cultures were then overlaid with MCEM5C. Cells. The origins of Ltk-, a line of mouse L cells
J. VIROL.
lacking tk, and clone 139 cells, a clone of Ltk- cells transformed by HSV-1, have been described (7, 16). CV-1, a line of monkey kidney cells, were obtained from the American Type Culture Collection, Rockville, Md. Clone 11U cells were derived in this laboratory and arose as a single transformed colony after infection of Ltk- cells with unirradiated KOS. This colony is unique in that it is the first and only instance of transformation due to unirradiated HSV which we have ever observed. Isolation of ts mutants. Mutagenization of the virus and isolation of the ts mutants were effected by modifications of the methods of Schaffer et al. (17). Briefly, KOS was plaque purified at 39 C (KOS 1.1) and used to infect CV-1 cells. After virus adsorption, the infected cells were cultured in EM5C containing 5 Ag of 5-bromo-2'-deoxyuridine (BUdR). Cultures were incubated at 31 C for 1 to 4 days with multiplicities of infection (MOI) ranging from 3 to 3 x 10-4 PFU/cell. The mutagenized stocks were plaqued on CV-1 monolayers at the permissive temperature, and wellisolated plaques were picked under a low-power microscope from unstained dishes. Mutants were tested for temperature sensitivity by their inability to form plaques on CV-1 monolayers at the nonpermissive temperature. The mutants that were presumptively ts were plaque purified and retested for temperature sensitivity. tk assay. The tk assay was a modification of the method of Munyon et al. (16). Pellets of about 4 x 106 cells were suspended in 100 ,l of 100 mM sodium maleate, pH 6.5, and frozen at -20 C and thawed once. The disrupted cells were sedimented at 500 x g, and the supernatant was used as the source of enzyme. The reaction mixture consisted of 20 Mmol of sodium maleate, pH 6.5, 1 gmol of ATP, 1 gmol of MgCl2, and 2 nmol of [3H JTdR (specific activity, 200 MCi/Mmol) in a total volume of 200 Ml. The reaction mixture also contained from about 400 to 600 Mg of protein, as determined by the method of Lowry et al. (13). The reaction was extinguished by the addition of 20Mgl of 50% trichloroacetic acid after incubation for 20 min at 37 C. Aliquots of the supernatant were spotted on DEAE-cellulose disks, which were then washed six times with 250-ml aliquots of 1 mM ammonium formate, pH 8.0, and once with 95% ethanol and dried. The disks were extracted in scintillation vials with 1-rnl aliquots of 0.1 N NaOH, after which was added 9 ml of a counting solution made by mixing 1 volume of Triton X-100 with 2 volumes of toluene containing 8.25 g of 2,5-diphenyloxazole and 0.25 g of dimethyl 1,4-bis-(5-phenyloxazolyl)benzene per liter. Counting efficiency was about 30%. Transformation assay. The transformation assay was that of Munyon et al. (16). Virus was diluted in EM5C to achieve the desired MOI and irradiated for 8 min at 280 MW/cm2 on a rotary shaker. All virus adsorptions were carried out for 1 h at the permissive temperature. The day after infection, EM5C was replaced with MTAGGEM5C made 0.1% in human gamma globulin (Hyland). This medium was replaced on days 6, 10, 14, and 18 postinfection, and the colonies were stained with crystal violet on day 21 postinfection.
VOL. 16, 1975
ts MUTANT TRANSFORMATION
RESULTS Characterization of productive infection by ts mutants. HSV-1 strain KOS was plaque purified at 39 C to produce a stock free of pre-existing ts mutants and which would grow well at the nonpermissive temperature. All of the ts mutants were derived from this virus, KOS 1.1. The nonpermissive temperature was defined as 39 C, and the permissive temperature was either 34 or 31 C. Twelve ts mutants were isolated as described in Materials and Methods. The course of maturation of KOS 1.1 and one of the mutants, ts 661, is compared in Fig. 1. Although ts 661 grew at 34 C, it was still clearly different from wild-type virus even at the permissive temperature. Similar observations on other HSV ts mutants have been made by Brown et al. (4) and Schaffer et al. (17). None of the mutants produced infectious virus at the nonpermissive temperature in contrast to KOS 1.1, which grew better at 39 than at 34 C (Table 1). Figure 1 also indicates that, for ts 661, any new virus synthesis at 39 C, if present at all, is insufficient to replace that lost by thermal inactivation, since the virus titer declines throughout the course of infection. After a cycle of growth at 39 C, plaques with wild-type morphology were observed in ts 661 (Table 1) and at low levels in samples grown at 39 C and titrated at 39 C in the complementation analyses (data not shown). Plaques with wild-type morphology could not be found when virus stocks themselves were titrated at 39 C, although wild-type virus occurring at low fre-
277
quency in the ts mutant cell population would not have been seen. No further search for revertants in the stocks was made. One of the mutants, ts 478, produced very small plaques at the nonpermissive temperature in contrast to
0
7
E
oSg
5
_
-
10 20 30 HOURS POST INFECTION FIG. 1. Course of maturation of KOS 1.1 and ts 661. Cultures of infected CV-1 cells were incubated at 34 C or 39 C. Samples were taken at the indicated times, and infectious virus was titrated at 34 C; 0, KOS 1.1 at 34 C; 0, KOS 1.1 at 39 C; 0, ts 661 at 34 C; *, ts 661 at 39 C. 0
TABLE 1. Progeny virus productiona and plaque formation at the permissive and nonpermissiL ?temperatures PFU/ml Virus
EOPb Ea
KOS 1.1 ts84 ts 478 ts656 ts661 ts730 ts 756 ts 606 ts629 ts807 ts 822
ts833 ts901
2.9 7.8 1.0 1.1 3.3 6.9 4.4 6.9 7.1 5.3 8.0 5.1 1.1
x x x x x x x x x x x
39 C
106
105 10"
106 105 10' 105 105 105 10'
10'
x 10' x 106
1.2 x 10" 5.0 x 10' 3.3 x 10' 1.0 x 105 1.8 x 10'c 5.0 x 103 1.1 x 105 1.0 x 104
