84, 218-221 (1978)
VIROLOGY
Temperature-Sensitive Mutants of Human Cytomegalovirus: and Partial Characterization of DNA-Minus Mutants SEIJI IHARA,’ Department
of Molecular
Biology,
KANJI
HIRAI,
Tokai University,
AND
YASUSHI
School of Medicine,
Isolation
WATANABE Bohseidai,
Zsehara, Japan 259-11
Accepted September 14,1977 Fifteen ts mutants of human cytomegalovirus which are unable to form normal plaques at 39” but which do form plaques at 34” have been isolated following mutagenesis by ultraviolet light. Five mutants defective in viral DNA synthesis at 39” were classified into two complementation groups, A and B, normal and defective in the induction of viral DNA polymerase, respectively.
Ultraviolet (uv) light irradiation of human cytomegalovirus (HCMV) results in differential inactivation of early viral functions such as induction of cell rounding, early antigens, viral DNA synthesis, cellular DNA synthesis, virus-specific DNA polymerase, and cellular DNA polymerases (I), suggesting that virus-coded functions expressed after infection are responsible for these early events. To explore the significance of these events in the replication of HCMV, we have undertaken a study of early temperature-sensitive (ts) mutants of the virus. Here we report isolation and partial characterization of such mutants. Wild-type (wt) HCMV (Towne strain) which had been triply plaque-purified and propagated on human embryo lung (HEL) cells was exposed to uv light at 24 ergs/ mm2/sec at the irradiated surface. After irradiation for 6, 8, and 10 min, the exposure times selected for mutagenesis, viral infectivity was reduced exponentially to about 1, 0.5, and 0.025% of the initial level, respectively. Mutants which made plaques at 34 but not at 39” were sought in these three samples of irradiated virus; nine mutants were obtained from 365 plaque isolates (2.5%), six from 163 plaque isolates (3.7%), and none from 24 plaque isolates, respectively. Each isolate was 1 To whom dressed.
requests
for reprints
should
be ad-
further purified by single-plaque isolation and the virus propagated was plaqueassayed on HEL cells (for the procedure see reference 1) at 34, 36, and 39” as summarized in Table 1. The ratio of plating efficiencies (39’134”) of wt was about 2, while those of mutants fell in the range of 5.9 x lO-‘j (ts676) to 1.2 x 10-l (ts526). Among the mutants, ts526 and ts569 formed only tiny aggregates of several rounded cells even after 3 weeks of incubation at 39” and these were scored as plaques in Table 1. One mutant, ts197, did not form plaques at 36” and two others, ts567 and ts589, exhibited some temperature sensitivity at 36”. To determine whether the ts mutation is in an early or late function, 15 mutants were examined for their ability to synthesize viral DNA at 39”. The viral DNA synthesized was determined by hybridization of DNA extracted from the infected cells with a tritiated RNA complementary to HCMV (13HlcRNA) which was prepared in vitro by using Escherichia coli RNA polymerase as described previously (1). A test with wt (at a multiplicity of 5 PFU/ cell) indicated that the amount of viral DNA in infected cells reached a maximum 4 days after infection when incubated at 39” and 5 to 6 days when incubated at 34”. A similar test with ts197 which at 34” caused cytopathic effect more slowly than the other mutants also resulted in maximum accumulation of viral DNA 6 days
218 0042-6822/78/0841-0218$02.00/O Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
SHORT
TABLE Virus
wt ts71 ts197 ts212 ts256 ts380 ts442 ts526 ts567 ts569 ts589 ts614 ts633 ts637 ts664 ts676
219
COMMUNICATIONS 1
PLAQUE FORMATION AND PLATING EFFICIENCIES OF ts MUTANTS” Ratio of plating efPlaque formation (PFU/ml) ficiencies (39”/34”) 36 39” 34 2.6 5.4 4.4 3.4 1.5 1.1 6.0 5.2 1.6 4.4 6.0 1.6 3.0 1.0 2.3 1.7
x x x x x x x x x x x x x x x x
10fi 1OJ 10’ 105 106 105 10” 10’ loj 106 loj lo5 l@ 106 loj 106
3.6 x lo6 7.4 x 10
VIROLOGY
Temperature-Sensitive Mutants of Human Cytomegalovirus: and Partial Characterization of DNA-Minus Mutants SEIJI IHARA,’ Department
of Molecular
Biology,
KANJI
HIRAI,
Tokai University,
AND
YASUSHI
School of Medicine,
Isolation
WATANABE Bohseidai,
Zsehara, Japan 259-11
Accepted September 14,1977 Fifteen ts mutants of human cytomegalovirus which are unable to form normal plaques at 39” but which do form plaques at 34” have been isolated following mutagenesis by ultraviolet light. Five mutants defective in viral DNA synthesis at 39” were classified into two complementation groups, A and B, normal and defective in the induction of viral DNA polymerase, respectively.
Ultraviolet (uv) light irradiation of human cytomegalovirus (HCMV) results in differential inactivation of early viral functions such as induction of cell rounding, early antigens, viral DNA synthesis, cellular DNA synthesis, virus-specific DNA polymerase, and cellular DNA polymerases (I), suggesting that virus-coded functions expressed after infection are responsible for these early events. To explore the significance of these events in the replication of HCMV, we have undertaken a study of early temperature-sensitive (ts) mutants of the virus. Here we report isolation and partial characterization of such mutants. Wild-type (wt) HCMV (Towne strain) which had been triply plaque-purified and propagated on human embryo lung (HEL) cells was exposed to uv light at 24 ergs/ mm2/sec at the irradiated surface. After irradiation for 6, 8, and 10 min, the exposure times selected for mutagenesis, viral infectivity was reduced exponentially to about 1, 0.5, and 0.025% of the initial level, respectively. Mutants which made plaques at 34 but not at 39” were sought in these three samples of irradiated virus; nine mutants were obtained from 365 plaque isolates (2.5%), six from 163 plaque isolates (3.7%), and none from 24 plaque isolates, respectively. Each isolate was 1 To whom dressed.
requests
for reprints
should
be ad-
further purified by single-plaque isolation and the virus propagated was plaqueassayed on HEL cells (for the procedure see reference 1) at 34, 36, and 39” as summarized in Table 1. The ratio of plating efficiencies (39’134”) of wt was about 2, while those of mutants fell in the range of 5.9 x lO-‘j (ts676) to 1.2 x 10-l (ts526). Among the mutants, ts526 and ts569 formed only tiny aggregates of several rounded cells even after 3 weeks of incubation at 39” and these were scored as plaques in Table 1. One mutant, ts197, did not form plaques at 36” and two others, ts567 and ts589, exhibited some temperature sensitivity at 36”. To determine whether the ts mutation is in an early or late function, 15 mutants were examined for their ability to synthesize viral DNA at 39”. The viral DNA synthesized was determined by hybridization of DNA extracted from the infected cells with a tritiated RNA complementary to HCMV (13HlcRNA) which was prepared in vitro by using Escherichia coli RNA polymerase as described previously (1). A test with wt (at a multiplicity of 5 PFU/ cell) indicated that the amount of viral DNA in infected cells reached a maximum 4 days after infection when incubated at 39” and 5 to 6 days when incubated at 34”. A similar test with ts197 which at 34” caused cytopathic effect more slowly than the other mutants also resulted in maximum accumulation of viral DNA 6 days
218 0042-6822/78/0841-0218$02.00/O Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
SHORT
TABLE Virus
wt ts71 ts197 ts212 ts256 ts380 ts442 ts526 ts567 ts569 ts589 ts614 ts633 ts637 ts664 ts676
219
COMMUNICATIONS 1
PLAQUE FORMATION AND PLATING EFFICIENCIES OF ts MUTANTS” Ratio of plating efPlaque formation (PFU/ml) ficiencies (39”/34”) 36 39” 34 2.6 5.4 4.4 3.4 1.5 1.1 6.0 5.2 1.6 4.4 6.0 1.6 3.0 1.0 2.3 1.7
x x x x x x x x x x x x x x x x
10fi 1OJ 10’ 105 106 105 10” 10’ loj 106 loj lo5 l@ 106 loj 106
3.6 x lo6 7.4 x 10
