JOURNAL OF VIROLOGY, May 1975, P. 1168-1175 Copyright 0 1975 American Society for Microbiology
Vol. 15, No. 5 Printed in U.SA.
Heat-Stable Variant of Human Adenovirus Type 5: Characterization and Use in Three-Factor Crosses C. S. H. YOUNG1 * AND J. F. WILLIAMS Medical Research Council, Virology Unit, Institute of Virology, Glasgow Gll 5JR, Scotland
Received for publication 20 January 1975
A variant of human adenovirus type 5 which is heat stable (hs) in vitro has been isolated following three rounds of heat inactivation at 52 C. The variant is genetically stable, both through vegetative viral passage and through recombination into other genetic backgrounds, which suggests that it arises from a single mutation. Three-factor crosses, using this mutant in conjunction with previously described temperature-sensitive mutants, suggest the hs mutation lies near the left-hand end of the genetic map. The mutant has been used to demonstrate the production of reciprocal recombinants in two-factor crosses. The mutational lesion is unknown, but phenotypic mixing occurs in hs x hs+ infections, which suggests that it lies in a gene specifying a virion structural protein. Other biological parameters examined have shown no differences from the wild-type hs+. A set of temperature-sensitive (ts) mutants of human adenovirus type 5 (Ad5), isolated in this laboratory (16), has been partially characterized by complementation analysis (17) and examined physiologically in terms of the structural antigens (8), polypeptides (9), and viral DNA (14) made in HeLa cells on infection at the restrictive temperature. These and other investigations (3) are beginning to give an estimate of the number of genes in this virus and an indication of their functions. Knowledge of the relative positions of the genes on the genome will be important in understanding how their functions are co-ordinated during viral replication. Accordingly, the ts mutants have been crossed to obtain recombination data from which a genetic map has been constructed (J. F. Williams, C. S. H. Young, and P. E. Austin, Cold Spring Harbor Symp. Quant. Biol., in press). In any set of two-factor crosses, good additivity of recombinant frequencies leads to an unambiguous genetic map, but such additivity is not found in all crosses, especially for markers which are close together, so that the gene order is to some extent uncertain. The uncertainty can be resolved if an additional third marker is used in the cross along with the two markers being mapped. This approach has been used with poliovirus (2) and herpes simplex virus type 1 (1). Theoretically, it could be applied to the adenoviruses, where, in addition to ts mu-
tants, other classes have been described; for example, cytocidal mutants in type 12 (13) and host range mutants in type 5 (12). The observation that two of our Ad5 ts mutants, ts 18 and ts 19, were extremely heat labile (S. Ustacelebi, Ph.D. thesis, University of Glasgow, 1973) suggested the possibility of searching for virion heat-stable mutants which could be used in classical three-factor crosses. This report describes the isolation and partial characterization of one such mutant and illustrates the use of this marker in a set of three-factor crosses. MATERIALS AND METHODS Virus and cells. The wild-type Ad5 and the ts mutants derived from it have been described previously (16), as have the methods for virus propagation and titration by plaque assay in HeLa cells (15). Heat inactivation. The kinetics of heat inactivation of virus at 52 C was measured in the following way. A 0.1-ml aliquot of virus was added to 0.9 ml of prewarmed Tris-hydrochloride buffer (pH 7.4) in a 20-ml cylindrical bottle in a water bath. At intervals, 0.1-ml samples were taken and diluted directly into ice-cold medium. When comparing the heat stability of different plaque isolates, 0.1 ml of each isolate was added to 0.9 ml of prewarmed buffer in 5-ml bottles held in a rack in the water bath, and the inactivation was stopped at a given time by placing all the bottles directly in an ice-water mixture. Inactivations were carried out in a Grant stirring water bath in which the
temperature could be maintained at 52.0 + 0.5 C. Recombination. The details of setting up mixed ' Present address: Department of Microbiology, College of infections and single-parent controls have been dePhysicians and Surgeons of Columbia University, New York, scribed previously (17). The recombinant frequency is N.Y. 10032. expressed as: titer at 38.5 C/titer at 32.5 C x 2 x 1168
HEAT-STABLE VARIANT OF ADENOVIRUS
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from a temperature-sensitive mutant so that it would be possible immediately to analyze threefactor crosses of the type tsx-hs+ x tsy-hs. Mutant tsl, which has much the same heat stability as the wild type (Fig. 1A), was chosen as starting material because it lay near the middle of the two-factor genetic map as it existed at that time. Since the location of an hs mutation was not known a priori, there was a RESULTS good chance that the mutant would not be so Isolation of a heat-stable (hs) mutant. distant from the ts marker as to be of no use in Selection of a heat-stable variant was made three-factor analysis. 100%. In general, cells singly infected yielded negligible titers at 38.5 C compared with doubly infected cells; thus the recombinant frequencies did not have to be corrected for reversion or leakiness in any parent. The factor of 2 in the expression corrects for the production of undetected double ts recombinants which are expected to arise with the same frequency as the ts+ class.
Minutes of inactivation 0
2
4
6
8
10
tsl -hs
12 0
2
4
b \ \ -~
FIG. 1. Heat inactivation of Ad5 wild type and mutants at 52 C. The data are expressed as the logarithm of the surviving fraction.
1170
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YOUNG AND WILLIAMS
The mutant was isolated in the following way. A sample of a fourth-passage stock of mutant ts 1 was inactivated at 52 C to give a surviving fraction of about 2.5%. A 0.1-ml aliquot of this fraction was seeded on HeLa cells to obtain a high-titer stock. A sample of this stock was inactivated to about 5%, and a second high-titer stock was obtained. Finally, an aliquot from this second stock was inactivated, and samples were taken after 5, 10 and 15 min of inactivation, giving 0.4, 0.03, and 0.007% survival, respectively. Ten plaques were isolated from each sample, and aliquots were tested for heat stability by heating for 10 min at 52 C. Several plaque isolates showed an enhanced heat stability compared with a tsl control, and one from the 0.04% surviving fraction was plaque purified and grown in HeLa cells to give a high-titer stock. This putative heat-stable mutant (tslhs) was tested by heating at 52 C and compared to tsl and Ad5 wild type. Figure 1A shows heat inactivation curves for tsl-hs, tsl, wild type, and tsl8; clearly tsl-hs is heat stable, whereas tsl8 is heat labile. The heat stability of tsl-hs remained unchanged through numerous subsequent vegetative virus passages so that we can conclude that the hs mutation is genetically stable. An important question is whether the heatstable phenotype is caused by a single mutation or by several mutations with more or less additive phenotypic effects. The way in which tsl-hs was selected, which was designed to enhance the frequency of any pre-existing hs mutant, could favour the enrichment of "multiple" mutants. Repeated back-crossing of the mutant to its parent would be expected to reveal its multiple nature, but this is extremely time consuming in adenovirus. So, we proceeded on the assumption that the hs phenotype results from a single mutation, and that if it does not, the fact would be revealed in subsequent two-factor and three-factor crosses. Construction of derivative hs strains by genetic recombination. For complete genetic analysis by three-factor crosses, it is necessary to have the third unselected marker in all the ts mutants of the set to be tested. Accordingly, the hs marker was transferred to some of our ts mutants, though not all, since no rapid method was available to facilitate the transfer. To obtain ts-hs strains, the hs marker was transferred to a wild-type background from which it could be transferred to other ts markers. Transfer to wild type was first made in the cross tslhs x tsl7-hs+ in which several of the ts+ progeny proved to be hs. One of these plaques was purified, grown to high titer, and tested
again for heat stability. The new derivative was found to be as heat stable as the original tsl-hs parent (Fig. 1B). This isolate was used as the hs parent in crosses designed to transfer the marker to various ts mutants. In such crosses the ts+-hs parent was normally in excess of the ts-hs+ parent. The infected cells were incubated at 32.5 C for 4 days, and plaques were isolated from the yield titrated at the same temperature. Each plaque was checked for its temperature sensitivity and, if ts, was checked for heat stability. The data are given in Table 1, which shows clearly that it is possible to transfer hs into a variety of ts backgrounds and that it is not closely linked to any of the ts markers used. Some of these ts-hs plaques from the different crosses were purified, grown to high titer, and tested for heat stability. Inactivation curves for two of the derivatives, ts5-hs and tsl7-hs, are shown in Fig. 1C and D. Although they appear to be slightly less heat stable than ts 1-hs, the difference is considered to be within the limits of experimental variation. The extent of this variation can be seen when the inactivation curves for ts+-hs in Fig. 1B and E are compared. No segregants displaying intermediate levels of heat stability were observed among the progeny from any of the crosses, so we conclude that the hs marker may be transferred into a number of genetic backgrounds without loss of heat stability. These observations do not support the view that the hs phenotype results from a number of additive mutations but suggest rather that it arises from a single mutational lesion. Use of the hs marker in three-factor cross analysis. The most ambiguous region of the current two-factor genetic map (Fig. 2) is near tsl, where five complementation groups are closely linked and where also there are a large number of individual mutants in complementation group A (Williams et al., Cold Spring Harbor Symp. Quant. Biol., in press). Thus it was of considerable interest to determine if the hs marker could be used to order these mutants. Crosses were set up in the usual way, but parents were added at a number of different input multiplicities of infection, each parental stock being titrated at the time of infection. TABLrE 1. Analysis of ts progeny from two-factor crosses involving ts-hs+ x ts+-hs viruses No. of ts progeny tested
No. of ts which were hs
ts3-hs+ ts5-hs+ tsl3-hs+
13 12
2 3
10
1
ts17-hs+
18
1
Cross
ts+-hs x ts+-hs x ts+-hs x ts+-hs x
%
15 25 10 5.6
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and output ratios of hs/hs+ (data not shown). In the other crosses in Table 2, mutants spanning the two-factor genetic map were used. In all cases except one, there is an hs to hs+ ratio among the ts+ recombinants of approximately 1.0. The exception, cross tsl-hs x ts49-hs+ (line 8), which has a ratio which deviates significantly from 1.0 (P = 0.000014), involves
Only those crosses which had an approximate equality of input were analyzed fully. Wellisolated ts+ plaques were picked from assay plates incubated at 38.5 C, and the heat stability was compared with that of ts+ plaques obtained from the isogenic cross in which neither parent contained the hs marker. In many cases they were also compared with plaques from a standard ts+-hs strain. A representative set of inactivation data is shown in Fig. 3, where it can be seen that the plaque isolates fall into two clearly distinguishable categories, hs and hs+. There are a few exceptions which, on further analysis, prove to be mixed plaques. This will be discussed below. The results of crosses between ts mutants which are closely linked are shown in the top six lines of Table 2, from which it can be seen that the ratio of hs to hs+ phenotypes among ts+ recombinants is not significantly different from 1.0. This is true for mutants in the same or in different complementation groups (line 1 and lines 2 to 5, respectively) and for a pair of reciprocal crosses (lines 4 and 5). Since the hs marker cannot lie between all the ts markers in all possible pairwise combinations, the results strongly suggest that it lies a long distance away from any of the ts markers involved. It is important to note that the high frequency of both hs and hs+ phenotypes among the recombinants cannot be accounted for by (i) a high rate of mutation from hs to hs+, since the cross tsl-hs x tsl7-hs (line 6) only yields ts+-hs recombinants, nor by (ii) the reverse from hs+ to hs, since the accumulated data for ts-hs+ x ts-hs+ crosses (line 13) reveal only ts+-hs+ offspring. Furthermore, the results cannot be explained by selective enrichment for hs or hs+ progeny in the yields, since in two-factor crosses of both ts+-hs x ts-hs+ and ts+-hs+ x ts-hs, there is an approximate equality of the input
or0
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*
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Batch No FiG. 3. Data from heat inactivation of ts+ plaques from the crosses ts3-hs+ x ts17-hs and ts3-hs+ x ts 17-hs+. Plaques were analyzed in batches on different days. Inactivation was for 4 min at 52 C. Symbols: 0, Plaques from ts3-hs+ x ts17-hs; *, plaques from ts 3-hs+ x ts17-hs+; 0, plaques of intermediate heat stability which were progeny tested (Table 3). 30
7 1
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FIG. 2. Summary of the current two-factor map. Distances are 2 x
ts+ frequency expressed as percentages.
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YOUNG AND WILLIAMS
mutant ts49, which lies at the left-hand end of the two-factor genetic map. This result is consistent with the view that hs itself lies at the left-hand end, close to but separable from ts49. The location of hs would explain the approximately random distribution of hs among the ts+ recombinants from crosses involving mutants in the right-hand end of the map. Two points are illustrated by the cross ts5-hs x tsl7-hs (line 12). (i) The rate of reversion from hs to hs+ is not high enough to confuse the three-factor cross results, since all 32 ts+ recombiants are also hs. This extends the observation made in the cross tsl-hs x tsl7-hs (line 6). (ii) Although the two parents are second-generation derivatives from the original isolate, no ts+ recombinants of intermediate heat-stable phenotype segregated in the cross. If the tsl-hs phenotype resulted from the additive effects of a number of randomly located mutations, two independently constructed derivatives, such as ts5-hs and tsl7-hs, would be expected to contain different sets of mutations. Hence in recombination between these ts-hs derivatives, either intermediate or wild type, heat-labile phenotypes might arise by recombination, depending on the numbers of mutations and their linkage relationships. This expectation is not borne out, nor is there any segregation of true-breeding intermediates in any of the three-factor crosses. These observations lend support to the view that the hs phenotype is not caused by multiple mutation. Mixed plaques. As mentioned earlier, a few ts+ plaques from three-factor crosses showed
J. VIROL.
intermediate hs phenotypes, such as those shown in the data taken from the cross ts3-hs+ x tsl7-hs (Fig. 3). The progeny of the three intermediate plaques from this cross were tested. All were found to be mixed in that the intermediate phenotype did not breed true but segregated in two cases into hs and hs+ and in one case into hs+ and a further intermediate class which was presumed to be mixed also (Table 3). In most three-factor crosses, a few plaque isolates were found to be slightly less heat stable than the standard ts+-hs or slightly more heat stable than the standard ts+-hs+ used (examples of the latter can be seen in Fig. 3, batch 3). When tested, the progeny of these plaques always proved to be either ts+-hs or ts+-hs+, suggesting that the slight initial deviations from the standard values were due to experimental error. In no case did any isolate with intermediate hs phenotype breed true; plaques were either mixed or completely hs or TABLE 3. Analysis of progeny from plaques of
intermediate heat stability from the cross ts3-hs+ x tsl 7-hs No. of progeny No. of progeny No. of progeny plaques plaques plaques Plaque no. which were which were which were ts+-hs ts+-hs+ intermediate
21 27 34
5 2 3
5 0 1
0 2
0
a These two plaques are assumed to be mixed also since other two progeny plaques were genuinely hs+.
TABLE 2. Analysis of ts+ progeny from three-factor crosses of the type ts-hs+ x ts-hs and from some isogenic hs x hseandhs+ x hs+ crosses Cross R.F. (%)a
ts-hs parent
ts-hs+ parent
1 1 17 1 17
7 3 3 17 1
Both tsl and tsl7: hs 1 9 1 49 17 9 14 17 22 5 Both ts5 and ts17: hs Sum of all ts-hs+ x ts-hs+ crosses
0.41 + 0.50 + 1.3 + 1.3 ± 1.4 0.89 ±
15.5± 24.5 ± 9.9 8.0 ± 0.88 9.4 ±
0.02 0.01 0.10 0.10 0.10 0.21 1.5 1.5 0.20 1.0 0.10 0.80
No.
No.
Test of significance of deviation from 1:1 ratio (P)5
21 13 14 12 7 0 16 1 12 11 30 0 39
10 13 22 12 15 8
0.064-0.090 0.22 0.12 0.23
ts.-hs+ of ts+-hs of ts-hs of ts-hs of
0.12
20
0.17
31 18 15 18 32 0
0.000014 0.15 0.19 0.077
a R.F., Recombinant frequency: 2 x frequency of ts+ in the yield (see Materials and Methods). The values are means of two duplicates performed at the same time ± the experimental range. The probability is calculated from Fisher's exact test since the numbers of observations are small. The probability is for the null hypothesis that hs is not linked to either ts parent.
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HEAT-STABLE VARIANT OF ADENOVIRUS
hs+. This is further experimental evidence that hs is not caused by multiple mutations with additive phenotypic effects. The nature of the mixed hs/hs+ plaques is not known, but several explanations are possible. The plaques might arise from clumps of hs and hs+ virus particles, but more interesting possibilities are that they represent segregants from virions containing more than one complete genome or from partially heterozygous structures. The latter situation is found in phage (4) and has been proposed for herpes simplex type 1 (1). At present, we cannot choose between these possibilities. Reciprocal recombinants. For mapping purposes, it is usual to assume that where only one class of recombinant from a two-factor cross can be scored easily, as, for example, ts+ from ts x ts crosses, the other class is present at equal frequency. Thus to obtain the recombinant frequency from such a cross, the ts+ frequency is doubled. However, the hs marker can be used to check this assumption since crosses of the type ts-hs x ts+-hs+ (or the reciprocals) will yield recombinants ts+-hs and ts-hs+, both of which can be detected. Accordingly, test crosses were set up between ts5-hs and ts+-hs+, and plaques were isolated from the yield titrated at 32 C and tested for heat stability. Of the 160 plaques tested, four were ts+-hs and three were ts-hs+. Thus there would appear to be no gross deviation from reciprocality, at least in this cross. One of the ts+-hs recombinant plaque isolates was purified and grown to high titer, and its heat stability was compared with that of the original ts+-hs strain. No difference in the heat stabilities was found (Fig. 1E), despite the fact that the hs marker had been transferred from the original ts+-hs strain to ts5 and then back to tsW. In addition, no isolates of intermediate phenotype segregated from the ts5-hs x ts+-hs+ crosses. These observations are yet further evidence that hs is caused by a single mutation. Phenotypic mixing. At present we do not know the physiological basis for the heat stability of the hs marker. Heat-stable mutants of X and the T-odd bacteriophages are known to be DNA deletion mutants (5-7). In the case of T4, on the other hand, heat stability arises from an alteration to a virion protein (11). As a biological approach to this problem we have tested whether or not phenotypic mixing occurs in mixed infections of hs and hs+ strains. Should phenotypic mixing occur, and particles of intermediate sensitivity arise, this would be strong evidence that heat stability results from a change in a virus structural component. Accordingly, cells were co-infected at a low multi-
plicity with ts+-hs and at increasing multiplicities with tsl-hs+. As a control, cells were infected with ts+-hs and at increasing multiplicities with tsl-hs. Both sets of infections were incubated at 32.5 C for 4 days, and the yields were titrated at 38.5 C to score for tsW. Aliquots from the yields were heated for 10 min at 52 C and titrated at 38.5 C to score for surviving ts+ progeny. As shown in Fig. 4, the effect of including virus of wild-type heat stability is to lower the proportion of phenotypically ts+-hs virus particles emerging from the mixed infection. To determine that this was not due to the production of large numbers of ts+-hs+ recombinants, 10 ts+ plaques were isolated from the yield of the mixed infection in which tsl-hs+ was in greatest excess and tested for heat stability. Nine of these plaques were ts+-hs; thus the lowering of the proportion of ts+ heat-stable particles emerging from the cross must result from the encoating of ts+-hs DNA with hs+ and hs+lhs protein. This evidence for phenotypic mixing strongly suggests that the underlying cause of the heat stability in the hs mutant is an alteration in a virion protein. Other phenotypic properties. The following phenotypic characteristics were found to be identical in hs and hs+ viruses: (i) ability to transform rat embryo cells; (ii) ability to induce interferon on chick embryo fibroblasts; (iii) frequency of ts+ recombinants in crosses of the general types tsx x tsy, tsx-hs x tsy, tsx x tsy-hs, and tsx-hs x tsy-hs; (iv) inactivation by neutralizing antiserum to Ad5; (v) stability on storage at -20 and -70 C; (vi) ability to form U
td5-hs alone tsl-hs alone
i -2
I._
U
* cm
0
0
tst-he alone
c
:F
-
10
20
30
40
50
Ratio of input multiplicities FIG. 4. Heat inactivation of the yields from the mixed infections ts+-hs x ts1-hs+ and ts+-hs x tslhs. Yields were assayed at 38.5 C, both before and after 10-min inactivation at 52 C. ts+-hs was added at a multiplicity of 0.1 PFUper cell in all cases, whereas tsl-hs+ and tsl-hs were added at increasing multiplicities. Symbols: 0, ts+-hs x tsl-hs+; *, ts+-hs x tsl-hs.
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YOUNG AND WILLIAMS
infectious centers; (vii) particle to PFU ratios (20 10); and (viii) yields of virus at 32 C (and, where applicable, 37 and 38 C) (100 to 1,000 PFU per cell). There is no loss of, or alteration in, any of the wild-type functions examined.
J. VIROL.
a virion protein. It is known that penton base is the first protein to be expelled from the virion when it is heated at 56 C (10), and it is tempting to speculate that the hs mutation lies in the appropriate structural gene. The successful isolation of a new type of Ad5 mutant which is stable, can be transferred to other genetic backgrounds, and can be used in three-factor crosses encourages us in our search for classes of mutants which map in other areas of the genome. Recently, T. Harrison, in our laboratory, has isolated a host-range mutant which we hope will be useful in three-factor crosses. Eventually we hope to obtain other markers which would enable all areas of the genome to be mapped reliably.
DISCUSSION The aim of this work was to develop a third marker suitable for inclusion in ts x ts crosses so that unambiguous gene orders could be deduced. This marker must fulfil two essential criteria; it should be genetically stable and should result from a single mutation. The phenotype of the hs mutant isolated here is stable through many vegetative passages, and thus the first criterion is fulfilled. The marker can be transferred to a number of different ACKNOWLEDGMENTS genetic backgrounds, and genetically stable We should like to thank Lesley Fraser for excellent intermediate phenotypes do not segregate from technical assistance, J. H. Subak-Sharpe for his interest and either two- or three-factor crosses. From these criticism, and P. E. Austin and E. A. C. Follett for performing cell-transformation assay and virus particle counts, results we conclude that the second criterion is the respectively. also upheld. Other criteria, desirable but not absolutely essential, for a third marker are that the phenotype is easily scored and that the locus LITERATURE CITED is not too distant from the markers to be D. A. Ritchie, and J. H. Subak-Sharpe. 1. S. Brown, M., ordered. These latter criteria allow us to esti1973. Genetic studies with herpes simplex virus type 1. mate, rapidly and with statistical reliability, The isolation of temperature-sensitive mutants, their the distribution of the third unselected marker arrangement into complementation groups and recombination analysis leading to a linkage map. J. Gen. among the selected recombinants. With respect Virol. 18:329-346. to these two criteria, the hs marker is less 2. Cooper, P. D. 1968. A genetic map of poliovirus temperauseful. (i) Because of phenotypic mixing, the ture-sensitive mutants. Virology 35:584-596. yield of a three-factor cross cannot be assayed 3. Ensinger, M. J., and H. S. Ginsberg. 1972. Selection and preliminary characterization of temperature-sensitive directly by heat inactivation to measure the mutants of type 5 adenovirus. J. Virol. 10:328-339. frequency of ts+-hs among the ts+ progeny. 4. Levinthal, C. 1954. Recombination in phage T2: its Instead, the analysis depends on the time-conrelationship to heterozygosis and growth. Genetics suming process of picking ts+ plaques and 39:169-184. testing their heat stability. (ii) hs maps close to 5. Parkinson, J. S., and R. J. Huskey. 1971. Deletion mutants of bacteriophage lambda. I. Isolation and ts49 at the left-hand end of our current two-facinitial characterization. J. Mol. Biol. 56:369-384. tor genetic map and is thus a considerable 6. Ritchie, D. A., and F. E. Malcolm. 1970. Heat-stable and distance from those markers towards the rightdensity mutants of phages Ti, T3 and T7. J. Gen. Virol. 9:35-43. hand end whose order is most uncertain. ConseI. 1968. Heat-stable mutants of T5 phage. I. quently, we need to analyze a large number of 7. Rubenstein, The physical properties of the phage and their DNA plaques from any one cross to obtain a reliable molecules. Virology 36:356-376. estimate of the positions of the ts markers 8. Russell, W. C., C. Newman, and J. F. Williams. 1972. Characterization of temperature-sensitive mutants of relative to hs. adenovirus type 5-serology. J. Gen. Virol. 17:265-279. The hs marker has been used to examine the 9. Russell, W. C., J. J. Skehel, and J. F. Williams. 1974. reciprocality of recombinant frequencies in twoCharacterization of temperature-sensitive mutants of factor crosses and, although the data are limadenovirus type 5: synthesis of polypeptides in infected cells. J. Gen. Virol. 24:247-259. ited, it indicates that there is no gross deviation W. C., R. C. Valentine, and H. G. Pereira. 1967. from reciprocality. Thus we feel justified in 10. Russell, The effect of heat on the anatomy of the adenovirus. J. doubling the ts+ frequency in ts x ts crosses to Gen. Virol. 1:509-522. obtain the recombinant frequency. 11. Streisinger, G. 1956. Phenotypic mixing of host range and serological specificities in bacteriophages T2 and T4. The nature of the mutation to heat stability is Virology 2:388-398. unknown, but the observation that phenotypic 12. Takahashi, M. 1972. Isolation of conditional lethal mumixing occurs in hs x hs+ crosses suggests that tants (temperature sensitive and host-dependent muthe lesion resulting from the hs mutation lies in tants) of adenovirus type 5. Virology 49:815-817.
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HEAT-STABLE VARIANT OF ADENOVIRUS
13. Takemori, N., J. L. Riggs, and C. Aldrich. 1968. Genetic studies with tumorigenic adenoviruses. I. Isolation of cytocidal (cyt) mutants of adenovirus type 12. Virology 36:575-586. 14. Wilkie, N. M., S. Ustacelebi, and J. F. Williams. 1973. Characterization of temperature-sensitive mutants of adenovirus type 5: nucleic acid synthesis. Virology 51:499-503. 15. Williams, J. F. 1970. Enhancement of adenovirus plaque
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formation on HeLa cells by magnesium chloride. J. Gen. Virol. 9:251-255. 16. Williams, J. F., M. Gharpure, S. Ustacelebi, and S. McDonald. 1971. Isolation of temperature-sensitive mutants of adenovirus type 5. J. Gen. Virol. 11:95-101. 17. Williams, J. F., and S. Ustacelebi. 1971. Complementation and recombination with temperature-sensitive mutants of adenovirus type 5. J. Gen. Virol. 13:345-348.
Vol. 15, No. 5 Printed in U.SA.
Heat-Stable Variant of Human Adenovirus Type 5: Characterization and Use in Three-Factor Crosses C. S. H. YOUNG1 * AND J. F. WILLIAMS Medical Research Council, Virology Unit, Institute of Virology, Glasgow Gll 5JR, Scotland
Received for publication 20 January 1975
A variant of human adenovirus type 5 which is heat stable (hs) in vitro has been isolated following three rounds of heat inactivation at 52 C. The variant is genetically stable, both through vegetative viral passage and through recombination into other genetic backgrounds, which suggests that it arises from a single mutation. Three-factor crosses, using this mutant in conjunction with previously described temperature-sensitive mutants, suggest the hs mutation lies near the left-hand end of the genetic map. The mutant has been used to demonstrate the production of reciprocal recombinants in two-factor crosses. The mutational lesion is unknown, but phenotypic mixing occurs in hs x hs+ infections, which suggests that it lies in a gene specifying a virion structural protein. Other biological parameters examined have shown no differences from the wild-type hs+. A set of temperature-sensitive (ts) mutants of human adenovirus type 5 (Ad5), isolated in this laboratory (16), has been partially characterized by complementation analysis (17) and examined physiologically in terms of the structural antigens (8), polypeptides (9), and viral DNA (14) made in HeLa cells on infection at the restrictive temperature. These and other investigations (3) are beginning to give an estimate of the number of genes in this virus and an indication of their functions. Knowledge of the relative positions of the genes on the genome will be important in understanding how their functions are co-ordinated during viral replication. Accordingly, the ts mutants have been crossed to obtain recombination data from which a genetic map has been constructed (J. F. Williams, C. S. H. Young, and P. E. Austin, Cold Spring Harbor Symp. Quant. Biol., in press). In any set of two-factor crosses, good additivity of recombinant frequencies leads to an unambiguous genetic map, but such additivity is not found in all crosses, especially for markers which are close together, so that the gene order is to some extent uncertain. The uncertainty can be resolved if an additional third marker is used in the cross along with the two markers being mapped. This approach has been used with poliovirus (2) and herpes simplex virus type 1 (1). Theoretically, it could be applied to the adenoviruses, where, in addition to ts mu-
tants, other classes have been described; for example, cytocidal mutants in type 12 (13) and host range mutants in type 5 (12). The observation that two of our Ad5 ts mutants, ts 18 and ts 19, were extremely heat labile (S. Ustacelebi, Ph.D. thesis, University of Glasgow, 1973) suggested the possibility of searching for virion heat-stable mutants which could be used in classical three-factor crosses. This report describes the isolation and partial characterization of one such mutant and illustrates the use of this marker in a set of three-factor crosses. MATERIALS AND METHODS Virus and cells. The wild-type Ad5 and the ts mutants derived from it have been described previously (16), as have the methods for virus propagation and titration by plaque assay in HeLa cells (15). Heat inactivation. The kinetics of heat inactivation of virus at 52 C was measured in the following way. A 0.1-ml aliquot of virus was added to 0.9 ml of prewarmed Tris-hydrochloride buffer (pH 7.4) in a 20-ml cylindrical bottle in a water bath. At intervals, 0.1-ml samples were taken and diluted directly into ice-cold medium. When comparing the heat stability of different plaque isolates, 0.1 ml of each isolate was added to 0.9 ml of prewarmed buffer in 5-ml bottles held in a rack in the water bath, and the inactivation was stopped at a given time by placing all the bottles directly in an ice-water mixture. Inactivations were carried out in a Grant stirring water bath in which the
temperature could be maintained at 52.0 + 0.5 C. Recombination. The details of setting up mixed ' Present address: Department of Microbiology, College of infections and single-parent controls have been dePhysicians and Surgeons of Columbia University, New York, scribed previously (17). The recombinant frequency is N.Y. 10032. expressed as: titer at 38.5 C/titer at 32.5 C x 2 x 1168
HEAT-STABLE VARIANT OF ADENOVIRUS
VOL. 15, 1975
1169
from a temperature-sensitive mutant so that it would be possible immediately to analyze threefactor crosses of the type tsx-hs+ x tsy-hs. Mutant tsl, which has much the same heat stability as the wild type (Fig. 1A), was chosen as starting material because it lay near the middle of the two-factor genetic map as it existed at that time. Since the location of an hs mutation was not known a priori, there was a RESULTS good chance that the mutant would not be so Isolation of a heat-stable (hs) mutant. distant from the ts marker as to be of no use in Selection of a heat-stable variant was made three-factor analysis. 100%. In general, cells singly infected yielded negligible titers at 38.5 C compared with doubly infected cells; thus the recombinant frequencies did not have to be corrected for reversion or leakiness in any parent. The factor of 2 in the expression corrects for the production of undetected double ts recombinants which are expected to arise with the same frequency as the ts+ class.
Minutes of inactivation 0
2
4
6
8
10
tsl -hs
12 0
2
4
b \ \ -~
FIG. 1. Heat inactivation of Ad5 wild type and mutants at 52 C. The data are expressed as the logarithm of the surviving fraction.
1170
J. VIROL.
YOUNG AND WILLIAMS
The mutant was isolated in the following way. A sample of a fourth-passage stock of mutant ts 1 was inactivated at 52 C to give a surviving fraction of about 2.5%. A 0.1-ml aliquot of this fraction was seeded on HeLa cells to obtain a high-titer stock. A sample of this stock was inactivated to about 5%, and a second high-titer stock was obtained. Finally, an aliquot from this second stock was inactivated, and samples were taken after 5, 10 and 15 min of inactivation, giving 0.4, 0.03, and 0.007% survival, respectively. Ten plaques were isolated from each sample, and aliquots were tested for heat stability by heating for 10 min at 52 C. Several plaque isolates showed an enhanced heat stability compared with a tsl control, and one from the 0.04% surviving fraction was plaque purified and grown in HeLa cells to give a high-titer stock. This putative heat-stable mutant (tslhs) was tested by heating at 52 C and compared to tsl and Ad5 wild type. Figure 1A shows heat inactivation curves for tsl-hs, tsl, wild type, and tsl8; clearly tsl-hs is heat stable, whereas tsl8 is heat labile. The heat stability of tsl-hs remained unchanged through numerous subsequent vegetative virus passages so that we can conclude that the hs mutation is genetically stable. An important question is whether the heatstable phenotype is caused by a single mutation or by several mutations with more or less additive phenotypic effects. The way in which tsl-hs was selected, which was designed to enhance the frequency of any pre-existing hs mutant, could favour the enrichment of "multiple" mutants. Repeated back-crossing of the mutant to its parent would be expected to reveal its multiple nature, but this is extremely time consuming in adenovirus. So, we proceeded on the assumption that the hs phenotype results from a single mutation, and that if it does not, the fact would be revealed in subsequent two-factor and three-factor crosses. Construction of derivative hs strains by genetic recombination. For complete genetic analysis by three-factor crosses, it is necessary to have the third unselected marker in all the ts mutants of the set to be tested. Accordingly, the hs marker was transferred to some of our ts mutants, though not all, since no rapid method was available to facilitate the transfer. To obtain ts-hs strains, the hs marker was transferred to a wild-type background from which it could be transferred to other ts markers. Transfer to wild type was first made in the cross tslhs x tsl7-hs+ in which several of the ts+ progeny proved to be hs. One of these plaques was purified, grown to high titer, and tested
again for heat stability. The new derivative was found to be as heat stable as the original tsl-hs parent (Fig. 1B). This isolate was used as the hs parent in crosses designed to transfer the marker to various ts mutants. In such crosses the ts+-hs parent was normally in excess of the ts-hs+ parent. The infected cells were incubated at 32.5 C for 4 days, and plaques were isolated from the yield titrated at the same temperature. Each plaque was checked for its temperature sensitivity and, if ts, was checked for heat stability. The data are given in Table 1, which shows clearly that it is possible to transfer hs into a variety of ts backgrounds and that it is not closely linked to any of the ts markers used. Some of these ts-hs plaques from the different crosses were purified, grown to high titer, and tested for heat stability. Inactivation curves for two of the derivatives, ts5-hs and tsl7-hs, are shown in Fig. 1C and D. Although they appear to be slightly less heat stable than ts 1-hs, the difference is considered to be within the limits of experimental variation. The extent of this variation can be seen when the inactivation curves for ts+-hs in Fig. 1B and E are compared. No segregants displaying intermediate levels of heat stability were observed among the progeny from any of the crosses, so we conclude that the hs marker may be transferred into a number of genetic backgrounds without loss of heat stability. These observations do not support the view that the hs phenotype results from a number of additive mutations but suggest rather that it arises from a single mutational lesion. Use of the hs marker in three-factor cross analysis. The most ambiguous region of the current two-factor genetic map (Fig. 2) is near tsl, where five complementation groups are closely linked and where also there are a large number of individual mutants in complementation group A (Williams et al., Cold Spring Harbor Symp. Quant. Biol., in press). Thus it was of considerable interest to determine if the hs marker could be used to order these mutants. Crosses were set up in the usual way, but parents were added at a number of different input multiplicities of infection, each parental stock being titrated at the time of infection. TABLrE 1. Analysis of ts progeny from two-factor crosses involving ts-hs+ x ts+-hs viruses No. of ts progeny tested
No. of ts which were hs
ts3-hs+ ts5-hs+ tsl3-hs+
13 12
2 3
10
1
ts17-hs+
18
1
Cross
ts+-hs x ts+-hs x ts+-hs x ts+-hs x
%
15 25 10 5.6
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HEAT-STABLE VARIANT OF ADENOVIRUS
VOL. 15, 1975
and output ratios of hs/hs+ (data not shown). In the other crosses in Table 2, mutants spanning the two-factor genetic map were used. In all cases except one, there is an hs to hs+ ratio among the ts+ recombinants of approximately 1.0. The exception, cross tsl-hs x ts49-hs+ (line 8), which has a ratio which deviates significantly from 1.0 (P = 0.000014), involves
Only those crosses which had an approximate equality of input were analyzed fully. Wellisolated ts+ plaques were picked from assay plates incubated at 38.5 C, and the heat stability was compared with that of ts+ plaques obtained from the isogenic cross in which neither parent contained the hs marker. In many cases they were also compared with plaques from a standard ts+-hs strain. A representative set of inactivation data is shown in Fig. 3, where it can be seen that the plaque isolates fall into two clearly distinguishable categories, hs and hs+. There are a few exceptions which, on further analysis, prove to be mixed plaques. This will be discussed below. The results of crosses between ts mutants which are closely linked are shown in the top six lines of Table 2, from which it can be seen that the ratio of hs to hs+ phenotypes among ts+ recombinants is not significantly different from 1.0. This is true for mutants in the same or in different complementation groups (line 1 and lines 2 to 5, respectively) and for a pair of reciprocal crosses (lines 4 and 5). Since the hs marker cannot lie between all the ts markers in all possible pairwise combinations, the results strongly suggest that it lies a long distance away from any of the ts markers involved. It is important to note that the high frequency of both hs and hs+ phenotypes among the recombinants cannot be accounted for by (i) a high rate of mutation from hs to hs+, since the cross tsl-hs x tsl7-hs (line 6) only yields ts+-hs recombinants, nor by (ii) the reverse from hs+ to hs, since the accumulated data for ts-hs+ x ts-hs+ crosses (line 13) reveal only ts+-hs+ offspring. Furthermore, the results cannot be explained by selective enrichment for hs or hs+ progeny in the yields, since in two-factor crosses of both ts+-hs x ts-hs+ and ts+-hs+ x ts-hs, there is an approximate equality of the input
or0
~~~~0@
y
*
A
-I
c
0
o .2
0 *
0 -2
c .5 o
0
*
-
S
*
*
0
*
-3
a :
*
U
3
2
Batch No FiG. 3. Data from heat inactivation of ts+ plaques from the crosses ts3-hs+ x ts17-hs and ts3-hs+ x ts 17-hs+. Plaques were analyzed in batches on different days. Inactivation was for 4 min at 52 C. Symbols: 0, Plaques from ts3-hs+ x ts17-hs; *, plaques from ts 3-hs+ x ts17-hs+; 0, plaques of intermediate heat stability which were progeny tested (Table 3). 30
7 1
1l171
21°0,
0, %,
*
0-76
0-6to
4-
N
,
I,-
49
3,6
3.1
11-9
"'
14 10
8,
30 \
7,
'
13 9 2.2,5
12 4'. 1-I 54
6-5 10- 10
FIG. 2. Summary of the current two-factor map. Distances are 2 x
ts+ frequency expressed as percentages.
1172
YOUNG AND WILLIAMS
mutant ts49, which lies at the left-hand end of the two-factor genetic map. This result is consistent with the view that hs itself lies at the left-hand end, close to but separable from ts49. The location of hs would explain the approximately random distribution of hs among the ts+ recombinants from crosses involving mutants in the right-hand end of the map. Two points are illustrated by the cross ts5-hs x tsl7-hs (line 12). (i) The rate of reversion from hs to hs+ is not high enough to confuse the three-factor cross results, since all 32 ts+ recombiants are also hs. This extends the observation made in the cross tsl-hs x tsl7-hs (line 6). (ii) Although the two parents are second-generation derivatives from the original isolate, no ts+ recombinants of intermediate heat-stable phenotype segregated in the cross. If the tsl-hs phenotype resulted from the additive effects of a number of randomly located mutations, two independently constructed derivatives, such as ts5-hs and tsl7-hs, would be expected to contain different sets of mutations. Hence in recombination between these ts-hs derivatives, either intermediate or wild type, heat-labile phenotypes might arise by recombination, depending on the numbers of mutations and their linkage relationships. This expectation is not borne out, nor is there any segregation of true-breeding intermediates in any of the three-factor crosses. These observations lend support to the view that the hs phenotype is not caused by multiple mutation. Mixed plaques. As mentioned earlier, a few ts+ plaques from three-factor crosses showed
J. VIROL.
intermediate hs phenotypes, such as those shown in the data taken from the cross ts3-hs+ x tsl7-hs (Fig. 3). The progeny of the three intermediate plaques from this cross were tested. All were found to be mixed in that the intermediate phenotype did not breed true but segregated in two cases into hs and hs+ and in one case into hs+ and a further intermediate class which was presumed to be mixed also (Table 3). In most three-factor crosses, a few plaque isolates were found to be slightly less heat stable than the standard ts+-hs or slightly more heat stable than the standard ts+-hs+ used (examples of the latter can be seen in Fig. 3, batch 3). When tested, the progeny of these plaques always proved to be either ts+-hs or ts+-hs+, suggesting that the slight initial deviations from the standard values were due to experimental error. In no case did any isolate with intermediate hs phenotype breed true; plaques were either mixed or completely hs or TABLE 3. Analysis of progeny from plaques of
intermediate heat stability from the cross ts3-hs+ x tsl 7-hs No. of progeny No. of progeny No. of progeny plaques plaques plaques Plaque no. which were which were which were ts+-hs ts+-hs+ intermediate
21 27 34
5 2 3
5 0 1
0 2
0
a These two plaques are assumed to be mixed also since other two progeny plaques were genuinely hs+.
TABLE 2. Analysis of ts+ progeny from three-factor crosses of the type ts-hs+ x ts-hs and from some isogenic hs x hseandhs+ x hs+ crosses Cross R.F. (%)a
ts-hs parent
ts-hs+ parent
1 1 17 1 17
7 3 3 17 1
Both tsl and tsl7: hs 1 9 1 49 17 9 14 17 22 5 Both ts5 and ts17: hs Sum of all ts-hs+ x ts-hs+ crosses
0.41 + 0.50 + 1.3 + 1.3 ± 1.4 0.89 ±
15.5± 24.5 ± 9.9 8.0 ± 0.88 9.4 ±
0.02 0.01 0.10 0.10 0.10 0.21 1.5 1.5 0.20 1.0 0.10 0.80
No.
No.
Test of significance of deviation from 1:1 ratio (P)5
21 13 14 12 7 0 16 1 12 11 30 0 39
10 13 22 12 15 8
0.064-0.090 0.22 0.12 0.23
ts.-hs+ of ts+-hs of ts-hs of ts-hs of
0.12
20
0.17
31 18 15 18 32 0
0.000014 0.15 0.19 0.077
a R.F., Recombinant frequency: 2 x frequency of ts+ in the yield (see Materials and Methods). The values are means of two duplicates performed at the same time ± the experimental range. The probability is calculated from Fisher's exact test since the numbers of observations are small. The probability is for the null hypothesis that hs is not linked to either ts parent.
VOL. 15, 1975
1173
HEAT-STABLE VARIANT OF ADENOVIRUS
hs+. This is further experimental evidence that hs is not caused by multiple mutations with additive phenotypic effects. The nature of the mixed hs/hs+ plaques is not known, but several explanations are possible. The plaques might arise from clumps of hs and hs+ virus particles, but more interesting possibilities are that they represent segregants from virions containing more than one complete genome or from partially heterozygous structures. The latter situation is found in phage (4) and has been proposed for herpes simplex type 1 (1). At present, we cannot choose between these possibilities. Reciprocal recombinants. For mapping purposes, it is usual to assume that where only one class of recombinant from a two-factor cross can be scored easily, as, for example, ts+ from ts x ts crosses, the other class is present at equal frequency. Thus to obtain the recombinant frequency from such a cross, the ts+ frequency is doubled. However, the hs marker can be used to check this assumption since crosses of the type ts-hs x ts+-hs+ (or the reciprocals) will yield recombinants ts+-hs and ts-hs+, both of which can be detected. Accordingly, test crosses were set up between ts5-hs and ts+-hs+, and plaques were isolated from the yield titrated at 32 C and tested for heat stability. Of the 160 plaques tested, four were ts+-hs and three were ts-hs+. Thus there would appear to be no gross deviation from reciprocality, at least in this cross. One of the ts+-hs recombinant plaque isolates was purified and grown to high titer, and its heat stability was compared with that of the original ts+-hs strain. No difference in the heat stabilities was found (Fig. 1E), despite the fact that the hs marker had been transferred from the original ts+-hs strain to ts5 and then back to tsW. In addition, no isolates of intermediate phenotype segregated from the ts5-hs x ts+-hs+ crosses. These observations are yet further evidence that hs is caused by a single mutation. Phenotypic mixing. At present we do not know the physiological basis for the heat stability of the hs marker. Heat-stable mutants of X and the T-odd bacteriophages are known to be DNA deletion mutants (5-7). In the case of T4, on the other hand, heat stability arises from an alteration to a virion protein (11). As a biological approach to this problem we have tested whether or not phenotypic mixing occurs in mixed infections of hs and hs+ strains. Should phenotypic mixing occur, and particles of intermediate sensitivity arise, this would be strong evidence that heat stability results from a change in a virus structural component. Accordingly, cells were co-infected at a low multi-
plicity with ts+-hs and at increasing multiplicities with tsl-hs+. As a control, cells were infected with ts+-hs and at increasing multiplicities with tsl-hs. Both sets of infections were incubated at 32.5 C for 4 days, and the yields were titrated at 38.5 C to score for tsW. Aliquots from the yields were heated for 10 min at 52 C and titrated at 38.5 C to score for surviving ts+ progeny. As shown in Fig. 4, the effect of including virus of wild-type heat stability is to lower the proportion of phenotypically ts+-hs virus particles emerging from the mixed infection. To determine that this was not due to the production of large numbers of ts+-hs+ recombinants, 10 ts+ plaques were isolated from the yield of the mixed infection in which tsl-hs+ was in greatest excess and tested for heat stability. Nine of these plaques were ts+-hs; thus the lowering of the proportion of ts+ heat-stable particles emerging from the cross must result from the encoating of ts+-hs DNA with hs+ and hs+lhs protein. This evidence for phenotypic mixing strongly suggests that the underlying cause of the heat stability in the hs mutant is an alteration in a virion protein. Other phenotypic properties. The following phenotypic characteristics were found to be identical in hs and hs+ viruses: (i) ability to transform rat embryo cells; (ii) ability to induce interferon on chick embryo fibroblasts; (iii) frequency of ts+ recombinants in crosses of the general types tsx x tsy, tsx-hs x tsy, tsx x tsy-hs, and tsx-hs x tsy-hs; (iv) inactivation by neutralizing antiserum to Ad5; (v) stability on storage at -20 and -70 C; (vi) ability to form U
td5-hs alone tsl-hs alone
i -2
I._
U
* cm
0
0
tst-he alone
c
:F
-
10
20
30
40
50
Ratio of input multiplicities FIG. 4. Heat inactivation of the yields from the mixed infections ts+-hs x ts1-hs+ and ts+-hs x tslhs. Yields were assayed at 38.5 C, both before and after 10-min inactivation at 52 C. ts+-hs was added at a multiplicity of 0.1 PFUper cell in all cases, whereas tsl-hs+ and tsl-hs were added at increasing multiplicities. Symbols: 0, ts+-hs x tsl-hs+; *, ts+-hs x tsl-hs.
1174
YOUNG AND WILLIAMS
infectious centers; (vii) particle to PFU ratios (20 10); and (viii) yields of virus at 32 C (and, where applicable, 37 and 38 C) (100 to 1,000 PFU per cell). There is no loss of, or alteration in, any of the wild-type functions examined.
J. VIROL.
a virion protein. It is known that penton base is the first protein to be expelled from the virion when it is heated at 56 C (10), and it is tempting to speculate that the hs mutation lies in the appropriate structural gene. The successful isolation of a new type of Ad5 mutant which is stable, can be transferred to other genetic backgrounds, and can be used in three-factor crosses encourages us in our search for classes of mutants which map in other areas of the genome. Recently, T. Harrison, in our laboratory, has isolated a host-range mutant which we hope will be useful in three-factor crosses. Eventually we hope to obtain other markers which would enable all areas of the genome to be mapped reliably.
DISCUSSION The aim of this work was to develop a third marker suitable for inclusion in ts x ts crosses so that unambiguous gene orders could be deduced. This marker must fulfil two essential criteria; it should be genetically stable and should result from a single mutation. The phenotype of the hs mutant isolated here is stable through many vegetative passages, and thus the first criterion is fulfilled. The marker can be transferred to a number of different ACKNOWLEDGMENTS genetic backgrounds, and genetically stable We should like to thank Lesley Fraser for excellent intermediate phenotypes do not segregate from technical assistance, J. H. Subak-Sharpe for his interest and either two- or three-factor crosses. From these criticism, and P. E. Austin and E. A. C. Follett for performing cell-transformation assay and virus particle counts, results we conclude that the second criterion is the respectively. also upheld. Other criteria, desirable but not absolutely essential, for a third marker are that the phenotype is easily scored and that the locus LITERATURE CITED is not too distant from the markers to be D. A. Ritchie, and J. H. Subak-Sharpe. 1. S. Brown, M., ordered. These latter criteria allow us to esti1973. Genetic studies with herpes simplex virus type 1. mate, rapidly and with statistical reliability, The isolation of temperature-sensitive mutants, their the distribution of the third unselected marker arrangement into complementation groups and recombination analysis leading to a linkage map. J. Gen. among the selected recombinants. With respect Virol. 18:329-346. to these two criteria, the hs marker is less 2. Cooper, P. D. 1968. A genetic map of poliovirus temperauseful. (i) Because of phenotypic mixing, the ture-sensitive mutants. Virology 35:584-596. yield of a three-factor cross cannot be assayed 3. Ensinger, M. J., and H. S. Ginsberg. 1972. Selection and preliminary characterization of temperature-sensitive directly by heat inactivation to measure the mutants of type 5 adenovirus. J. Virol. 10:328-339. frequency of ts+-hs among the ts+ progeny. 4. Levinthal, C. 1954. Recombination in phage T2: its Instead, the analysis depends on the time-conrelationship to heterozygosis and growth. Genetics suming process of picking ts+ plaques and 39:169-184. testing their heat stability. (ii) hs maps close to 5. Parkinson, J. S., and R. J. Huskey. 1971. Deletion mutants of bacteriophage lambda. I. Isolation and ts49 at the left-hand end of our current two-facinitial characterization. J. Mol. Biol. 56:369-384. tor genetic map and is thus a considerable 6. Ritchie, D. A., and F. E. Malcolm. 1970. Heat-stable and distance from those markers towards the rightdensity mutants of phages Ti, T3 and T7. J. Gen. Virol. 9:35-43. hand end whose order is most uncertain. ConseI. 1968. Heat-stable mutants of T5 phage. I. quently, we need to analyze a large number of 7. Rubenstein, The physical properties of the phage and their DNA plaques from any one cross to obtain a reliable molecules. Virology 36:356-376. estimate of the positions of the ts markers 8. Russell, W. C., C. Newman, and J. F. Williams. 1972. Characterization of temperature-sensitive mutants of relative to hs. adenovirus type 5-serology. J. Gen. Virol. 17:265-279. The hs marker has been used to examine the 9. Russell, W. C., J. J. Skehel, and J. F. Williams. 1974. reciprocality of recombinant frequencies in twoCharacterization of temperature-sensitive mutants of factor crosses and, although the data are limadenovirus type 5: synthesis of polypeptides in infected cells. J. Gen. Virol. 24:247-259. ited, it indicates that there is no gross deviation W. C., R. C. Valentine, and H. G. Pereira. 1967. from reciprocality. Thus we feel justified in 10. Russell, The effect of heat on the anatomy of the adenovirus. J. doubling the ts+ frequency in ts x ts crosses to Gen. Virol. 1:509-522. obtain the recombinant frequency. 11. Streisinger, G. 1956. Phenotypic mixing of host range and serological specificities in bacteriophages T2 and T4. The nature of the mutation to heat stability is Virology 2:388-398. unknown, but the observation that phenotypic 12. Takahashi, M. 1972. Isolation of conditional lethal mumixing occurs in hs x hs+ crosses suggests that tants (temperature sensitive and host-dependent muthe lesion resulting from the hs mutation lies in tants) of adenovirus type 5. Virology 49:815-817.
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HEAT-STABLE VARIANT OF ADENOVIRUS
13. Takemori, N., J. L. Riggs, and C. Aldrich. 1968. Genetic studies with tumorigenic adenoviruses. I. Isolation of cytocidal (cyt) mutants of adenovirus type 12. Virology 36:575-586. 14. Wilkie, N. M., S. Ustacelebi, and J. F. Williams. 1973. Characterization of temperature-sensitive mutants of adenovirus type 5: nucleic acid synthesis. Virology 51:499-503. 15. Williams, J. F. 1970. Enhancement of adenovirus plaque
1175
formation on HeLa cells by magnesium chloride. J. Gen. Virol. 9:251-255. 16. Williams, J. F., M. Gharpure, S. Ustacelebi, and S. McDonald. 1971. Isolation of temperature-sensitive mutants of adenovirus type 5. J. Gen. Virol. 11:95-101. 17. Williams, J. F., and S. Ustacelebi. 1971. Complementation and recombination with temperature-sensitive mutants of adenovirus type 5. J. Gen. Virol. 13:345-348.
