JOURNAL OF VIROLOGY, Mar. 1975, p. 675-678 Copyright 0 1975 American Society for Microbiology
Vol. 15, No. 3
Printed in U.S.A.
Complementation of Human Adenovirus Type 5 ts Mutants by Human Adenovirus Type 12 JIM WILLIAMS,* HAMISH YOUNG,' AND PAUL AUSTIN2
M.R.C. Virology Unit, Institute of Virology, Glasgow, Gll 5JR, Scotland
Received for publication 29 October 1974
Temperature-sensitive mutants of type 5 adenovirus belonging to eight complementation groups were complemented in mixed infection by type 12 adenovirus, whereas mutants of 7 other groups were not enhanced. In some crosses, phenotypic mixing took place. No evidence of recombination between type 5 ts mutants and type 12 was found.
playing abnormal hexon transport (tsl, ts2, ts3, and ts4), and the two mutants tsl8 and ts24, whose defects are not known but which probably reside in viral products made and/or functioning late in the virus growth cycle since they make normal amounts of the major capsid components (9, 10). The complementation index compared favorably with those obtained in crosses between type 5 ts mutants (19). Therefore, in crosses with these particular mutants, type 12 products function effectively to replace defective type 5 functions and enhance the yield of infectious virus. The other class of mutants comprised those complemented poorly 6r not at all by type 12. This class includes the DNA-negative mutants ts36 (15) and ts125 (2), the fiber-deficient mutants ts5, ts9, and ts22, and two mutants tsl9 and ts3l, whose defects are also not known but which are late mutants in that they make normal amounts of major capsid antigens (9, 10). In these crosses, the type 12 products did not substitute for the defective type 5 functions, so that the yields of infectious virus were enhanced less than 10-fold (the cut-off point for positive complementation). All of these noncomplementing mutants are somewhat leaky, and it could be argued that the leakiness might obscure possible complementation. However, since all of these leaky mutants showed positive complementation of up to 1,000-fold in crosses with other type 5 mutants, leakiness is not likely to contribute to the apparent lack of complementation with type 12 in these cases. It should be pointed out that our result with ts125 differs from that of H. S. Ginsberg (personal ' Present address: Department of Microbiology, College of communication), who has found that a type 12 Physicians and Surgeons, Columbia University, New York, ts mutant, ts303, complemented type 5 ts125 in N.Y. 10032. 2Present address: Connaught Laboratories Ltd., Willow- mixed infection at restrictive temperature. We have no good explanation for this difference. dale, Ontario, Canada.
Human adenoviruses of group A are highly oncogenic in rodents and those of group C are nononcogenic, whereas representatives of both groups transform rodent cells in vitro (11). Adenoviruses of these two groups show minimal genetic relatedness in terms of the complementarity of their DNA sequences (3, 4). To assess the degree to which groups A and C are functionally related we carried out complementation tests between type 5 (group C) temperature-sensitive (ts) mutants (18, 19) of 15 different complementation groups, and wild-type adenovirus 12 of strain 1131 (8). In this case, all type 12 functions are wild type, but complementation of the type 5 mutants can be scored because we find that although strain 1131 virus grows in HeLa cells (less well than type 5), it fails to form plaques on monolayers of these cells at either 38.5 or 32.5 C. Thus, in a yield from mixedly infected cells, only type 5 will form plaques, and only at 32.5 C unless considerable reversion of type 5, or recombination, has taken place. Complementation tests were carried out as described in the legend to Table 1 by infecting confluent monolayers either singly at an input multiplicity of 10 PFU/cell, or doubly with mutant and type 12 each at an input of 5 PFU/cell. As shown by the two sets of experimental data given in Table 1, the type 5 mutants fell broadly into two classes, depending on the outcome of their interaction with type 12. One class comprised those mutants enhanced by type 12. This category includes the hexondeficient mutants ts17 and ts2O, mutants dis-
J. VIROL.
NOTES
676
TABLE 1. Complementation between type 12 wild-type and type 5 ts mutants in HeLa cells at 38.5 Ca Expt 1
Expt 2
Type 5 virus
S
Wild type tsl ts2 ts3 ts4
tsl7 ts18 ts2O ts24 ts5
ts9 tsl9 ts22
ts3l ts36 tsl25
D
2.5 x 109 1.1 x 10s 7.0 x 102 2.1 6.0 3.7 1.1 1.3 2.0
x 104 x 103 x 103 X 104 x 103 x 104
Ratio D/S
S
1.0
1.9 x 109 1.2 x 10s
2.5 x 109 4.5 5.3 8.0 6.0 3.6 2.1 1.1 4.3
x x x x x x
106 106 106 106
106 107
x 106 x 107
4.0 7.6 4.0 1.0 1.0 2.0 8.0 2.1
x x x x x
Ratio D/S
D
1.7 x 109
0.9
x x x x x
3.1 x 103 2.3 x l0 1.1 X 103 1.5 x 103 1.1 x 104 1.4 x 103 1.0 X 104 1.0 X 104
3.7 3.5 3.5 5.6 6.7 1.4 2.9 2.4
106 101
103 103 102 103 103 x 103 x 102 x 103
1.5 3.3 3.6 6.0 1.0 2.7 2.5
0.8 2.4
7.8 x 106 1.6 x 106
6.2 x 10' 6.0 x 106
0.8 3.8
x x x x
103 103
10s
102 x 102 x 103 x 103
101
106 106 x 101 x 107 x 107
1.7 x 106 1.8 x 106
1.3 x 106 4.4 x 106
9.0 x 1.2 x 1.5 x 8.0 x 3.0 x
105
1.4 x 106
1.5
107 107
4.0 x 107 1.1 x 108
3.3 7.3
5.3 x 101 5.5 x 106
5.6 x 106 3.0 x 107
1.0 5.5
106 106
1.6 x 107 3.4 x 106
2.0 1.1
6.5 x 107
2.2 x 107
0.3
a For complementation tests, HeLa cells were either infected singly with each mutant at an input multiplicity of 10 PFU/cell or doubly with the mutant and strain 1131 virus each at 5 PFU/cell. Virus was adsorbed for 1 h at 38.5 C, and then monolayers were washed with Eagle medium, treated for 15 min with a mixture of type 5 and type 12 antisera each diluted to 1:200, and washed again. Cultures were overlaid with Eagle medium plus 2% calf serum and incubated at 38.5 C for 36 to 40 h. Cells were then scraped from the dishes, suspended in Tris-saline, and frozen and thawed three times to release virus; samples were assayed (16) at 32.5 C to measure total yield and at 38.5 C to determine the proportion of wild type in the yield. Complementation was measured by comparing the total virus yields (corrected for wild type if necessary) of the single (S) and the double (D) infections, and is expressed as the ratio D/S-the complementation index. When that ratio exceeded 10, complementation was considered to be positive.
Adenovirus type 5 replicates normally (12, 17) in hamster embryo cells whereas type 12 adenovirus replication is abortive, with no DNA and late capsid antigens being made, although some viral-specific RNA and tumor antigen is made (13). Thus, in these cells, probably only a few early viral functions are expressed, and therefore one would expect complementation of few, if any, type 5 mutants to take place. To determine whether this was the case, hamster embryo cells were infected singly and doubly as described in the legend to Table 1. As expected, no mutants were enhanced in these cells. Table 1 also shows that co-infection of HeLa cells with type 12 and type 5 had little or no effect on the replication of wild-type adenovirus 5 in these cells, and the same was true of type 5 replication in hamster cells. This result was found on many occasions and differs from results reported previously which showed that type 12 infection inhibited type 2 replication (6, 14). Whether this reflects a difference between types 2 and 5 or a strain difference in the type 12 used is not known, because we have tested neither type 2 nor strains of type 12 other than 1131.
The serotypic specificity of types 5 and 12 provides additional markers for analyzing the yields of mixed infections. Antigenic analysis of the yields from the complementing crosses by using sera specific for types 5 and 12 revealed that in some of them the virus produced bore antigenic determinants from both parents. Monospecific antisera against types 5 and 12 each neutralized between 80 and 90% of the virus in the yields (assayed on HeLa cells) of crosses of type 12 with type 5 tsl, ts2, ts3, ts4, and tsl 7, whereas in the case of ts24, virus in the yield was neutralized only by type 5 antiserum (Table 2). The yield from cells co-infected by wild-type adenovirus 5 and strain 1131 was likewise only neutralized by type 5 antiserum and not by type 12 serum. This could stem from the fact that type 5 wild type grows somewhat more rapidly and to 30-to 100-fold higher levels of infectivity in HeLa cells than strain 1131; therefore, type 5 products are likely to be in excess, and as a result phenotypic mixing will probably occur at a low and undetectable level. The ts mutants, on the other hand, grow more slowly or not at all at 38.5 C and make lower levels of certain viral products than wild-type
TABLE 2. Virus neutralization in the yields of type 12 wild type x type 5 ts mutant crosses by antisera to type 5 and type 12 Cross
1131 1131 1131 1131 1131
x tsl x ts2 x ts17 x ts24 x Ad5 wild type
677
NOTES
VOL. 15, 1975
Virus surviving antiserum treatment (%)a Adl2 serumb Ad5 serumb
23
10 16
11 1
Vol. 15, No. 3
Printed in U.S.A.
Complementation of Human Adenovirus Type 5 ts Mutants by Human Adenovirus Type 12 JIM WILLIAMS,* HAMISH YOUNG,' AND PAUL AUSTIN2
M.R.C. Virology Unit, Institute of Virology, Glasgow, Gll 5JR, Scotland
Received for publication 29 October 1974
Temperature-sensitive mutants of type 5 adenovirus belonging to eight complementation groups were complemented in mixed infection by type 12 adenovirus, whereas mutants of 7 other groups were not enhanced. In some crosses, phenotypic mixing took place. No evidence of recombination between type 5 ts mutants and type 12 was found.
playing abnormal hexon transport (tsl, ts2, ts3, and ts4), and the two mutants tsl8 and ts24, whose defects are not known but which probably reside in viral products made and/or functioning late in the virus growth cycle since they make normal amounts of the major capsid components (9, 10). The complementation index compared favorably with those obtained in crosses between type 5 ts mutants (19). Therefore, in crosses with these particular mutants, type 12 products function effectively to replace defective type 5 functions and enhance the yield of infectious virus. The other class of mutants comprised those complemented poorly 6r not at all by type 12. This class includes the DNA-negative mutants ts36 (15) and ts125 (2), the fiber-deficient mutants ts5, ts9, and ts22, and two mutants tsl9 and ts3l, whose defects are also not known but which are late mutants in that they make normal amounts of major capsid antigens (9, 10). In these crosses, the type 12 products did not substitute for the defective type 5 functions, so that the yields of infectious virus were enhanced less than 10-fold (the cut-off point for positive complementation). All of these noncomplementing mutants are somewhat leaky, and it could be argued that the leakiness might obscure possible complementation. However, since all of these leaky mutants showed positive complementation of up to 1,000-fold in crosses with other type 5 mutants, leakiness is not likely to contribute to the apparent lack of complementation with type 12 in these cases. It should be pointed out that our result with ts125 differs from that of H. S. Ginsberg (personal ' Present address: Department of Microbiology, College of communication), who has found that a type 12 Physicians and Surgeons, Columbia University, New York, ts mutant, ts303, complemented type 5 ts125 in N.Y. 10032. 2Present address: Connaught Laboratories Ltd., Willow- mixed infection at restrictive temperature. We have no good explanation for this difference. dale, Ontario, Canada.
Human adenoviruses of group A are highly oncogenic in rodents and those of group C are nononcogenic, whereas representatives of both groups transform rodent cells in vitro (11). Adenoviruses of these two groups show minimal genetic relatedness in terms of the complementarity of their DNA sequences (3, 4). To assess the degree to which groups A and C are functionally related we carried out complementation tests between type 5 (group C) temperature-sensitive (ts) mutants (18, 19) of 15 different complementation groups, and wild-type adenovirus 12 of strain 1131 (8). In this case, all type 12 functions are wild type, but complementation of the type 5 mutants can be scored because we find that although strain 1131 virus grows in HeLa cells (less well than type 5), it fails to form plaques on monolayers of these cells at either 38.5 or 32.5 C. Thus, in a yield from mixedly infected cells, only type 5 will form plaques, and only at 32.5 C unless considerable reversion of type 5, or recombination, has taken place. Complementation tests were carried out as described in the legend to Table 1 by infecting confluent monolayers either singly at an input multiplicity of 10 PFU/cell, or doubly with mutant and type 12 each at an input of 5 PFU/cell. As shown by the two sets of experimental data given in Table 1, the type 5 mutants fell broadly into two classes, depending on the outcome of their interaction with type 12. One class comprised those mutants enhanced by type 12. This category includes the hexondeficient mutants ts17 and ts2O, mutants dis-
J. VIROL.
NOTES
676
TABLE 1. Complementation between type 12 wild-type and type 5 ts mutants in HeLa cells at 38.5 Ca Expt 1
Expt 2
Type 5 virus
S
Wild type tsl ts2 ts3 ts4
tsl7 ts18 ts2O ts24 ts5
ts9 tsl9 ts22
ts3l ts36 tsl25
D
2.5 x 109 1.1 x 10s 7.0 x 102 2.1 6.0 3.7 1.1 1.3 2.0
x 104 x 103 x 103 X 104 x 103 x 104
Ratio D/S
S
1.0
1.9 x 109 1.2 x 10s
2.5 x 109 4.5 5.3 8.0 6.0 3.6 2.1 1.1 4.3
x x x x x x
106 106 106 106
106 107
x 106 x 107
4.0 7.6 4.0 1.0 1.0 2.0 8.0 2.1
x x x x x
Ratio D/S
D
1.7 x 109
0.9
x x x x x
3.1 x 103 2.3 x l0 1.1 X 103 1.5 x 103 1.1 x 104 1.4 x 103 1.0 X 104 1.0 X 104
3.7 3.5 3.5 5.6 6.7 1.4 2.9 2.4
106 101
103 103 102 103 103 x 103 x 102 x 103
1.5 3.3 3.6 6.0 1.0 2.7 2.5
0.8 2.4
7.8 x 106 1.6 x 106
6.2 x 10' 6.0 x 106
0.8 3.8
x x x x
103 103
10s
102 x 102 x 103 x 103
101
106 106 x 101 x 107 x 107
1.7 x 106 1.8 x 106
1.3 x 106 4.4 x 106
9.0 x 1.2 x 1.5 x 8.0 x 3.0 x
105
1.4 x 106
1.5
107 107
4.0 x 107 1.1 x 108
3.3 7.3
5.3 x 101 5.5 x 106
5.6 x 106 3.0 x 107
1.0 5.5
106 106
1.6 x 107 3.4 x 106
2.0 1.1
6.5 x 107
2.2 x 107
0.3
a For complementation tests, HeLa cells were either infected singly with each mutant at an input multiplicity of 10 PFU/cell or doubly with the mutant and strain 1131 virus each at 5 PFU/cell. Virus was adsorbed for 1 h at 38.5 C, and then monolayers were washed with Eagle medium, treated for 15 min with a mixture of type 5 and type 12 antisera each diluted to 1:200, and washed again. Cultures were overlaid with Eagle medium plus 2% calf serum and incubated at 38.5 C for 36 to 40 h. Cells were then scraped from the dishes, suspended in Tris-saline, and frozen and thawed three times to release virus; samples were assayed (16) at 32.5 C to measure total yield and at 38.5 C to determine the proportion of wild type in the yield. Complementation was measured by comparing the total virus yields (corrected for wild type if necessary) of the single (S) and the double (D) infections, and is expressed as the ratio D/S-the complementation index. When that ratio exceeded 10, complementation was considered to be positive.
Adenovirus type 5 replicates normally (12, 17) in hamster embryo cells whereas type 12 adenovirus replication is abortive, with no DNA and late capsid antigens being made, although some viral-specific RNA and tumor antigen is made (13). Thus, in these cells, probably only a few early viral functions are expressed, and therefore one would expect complementation of few, if any, type 5 mutants to take place. To determine whether this was the case, hamster embryo cells were infected singly and doubly as described in the legend to Table 1. As expected, no mutants were enhanced in these cells. Table 1 also shows that co-infection of HeLa cells with type 12 and type 5 had little or no effect on the replication of wild-type adenovirus 5 in these cells, and the same was true of type 5 replication in hamster cells. This result was found on many occasions and differs from results reported previously which showed that type 12 infection inhibited type 2 replication (6, 14). Whether this reflects a difference between types 2 and 5 or a strain difference in the type 12 used is not known, because we have tested neither type 2 nor strains of type 12 other than 1131.
The serotypic specificity of types 5 and 12 provides additional markers for analyzing the yields of mixed infections. Antigenic analysis of the yields from the complementing crosses by using sera specific for types 5 and 12 revealed that in some of them the virus produced bore antigenic determinants from both parents. Monospecific antisera against types 5 and 12 each neutralized between 80 and 90% of the virus in the yields (assayed on HeLa cells) of crosses of type 12 with type 5 tsl, ts2, ts3, ts4, and tsl 7, whereas in the case of ts24, virus in the yield was neutralized only by type 5 antiserum (Table 2). The yield from cells co-infected by wild-type adenovirus 5 and strain 1131 was likewise only neutralized by type 5 antiserum and not by type 12 serum. This could stem from the fact that type 5 wild type grows somewhat more rapidly and to 30-to 100-fold higher levels of infectivity in HeLa cells than strain 1131; therefore, type 5 products are likely to be in excess, and as a result phenotypic mixing will probably occur at a low and undetectable level. The ts mutants, on the other hand, grow more slowly or not at all at 38.5 C and make lower levels of certain viral products than wild-type
TABLE 2. Virus neutralization in the yields of type 12 wild type x type 5 ts mutant crosses by antisera to type 5 and type 12 Cross
1131 1131 1131 1131 1131
x tsl x ts2 x ts17 x ts24 x Ad5 wild type
677
NOTES
VOL. 15, 1975
Virus surviving antiserum treatment (%)a Adl2 serumb Ad5 serumb
23
10 16
11 1
