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Rescue of one phenotype in mixed infections with heat-defective mutants of type 1 poliovirus
26, 431.438 (1965)
VIROLOGY
Rescue
of One
Phenotype Mutants
in Mixed
Infections
of Type
1 Poliovirus
PETER Department
of Microbiology,
John Curtin
with
Heat-Defective
D. COOPER
School of Medical Research, Canberra, Australia
Australian
National
University,
Accepted November 20, 1964 When cells are mixedly infected with two heat-defective (hd) poliovirus mutants at temperatures above those that are optimal for growth of these mutants, the virus yield is 4-14 times the sum of the yields of each mutant when grown separately under identical conditions (“leak” yields). The mixed infection yields are only one thousandth of the yield of wild type (ho!+) under these conditions. The leak and mixed infection yields are 90-997, mutant rather than hdf, but only one of the mutants was detected in the mixed infection yields. Any rescue of the hd mutants by hd+ was too small to be detected. INTRODUCTION
Conditional lethal mutations of bacteriophage T4, which prevent its growth either in certain hosts or at higher temperatures, have been used with great effect (Epstein et al., 1963) to map the T4 genome and to indicate many of its gene functions. The discovery that 5-fluorouracil produces many heat defective (hd) mutants in poliovirus (Cooper, 1964) suggests the possibility of a similar approach to the genetic analysis of a small animal ribovirus. The hd mut,ants that have been isolated are now being examined in several ways. One way is to classify them by means of a complementation test, with the object of defining those groups of mutants which have defects in the same gene. This test showed that mixed infection at high temperature with many but not all pairs of hd mutants gave an enhanced yield. The enhancement was always very small, however. This paper describes the complementation test and reports the results of some experiments in which the interaction between two of the mutants is examined in some detail with the object of defining optimal conditions. It is shown that, for this particular pair at least, the enhancement was
not reciprocal within test.
the sensitivity
of the
METHODS
Temperature control. Well-circulated water baths controlled by Braun (Melsungen, Germany) Thermomix II mercury contact units maintained temperature to fO.05’; the most convenient monitors were clinical thermometers graduated in O.l”C. Tubes were stoppered and were immersed to a depth of 3 cm in open racks; maximum temperature was reached within 3 minutes. Plaque assays of virus at high temperatures were made with a bicarbonate medium (Cooper, 1961) contained in screw-capped bottles immersed inverted in water baths in open baskets, in such a way as not to impede water circulation. In some experiments (indicated in the text) petri plates were enclosed in a sealed box that was immersed in a water bath at a supraoptimal temperature, but this method was discontinued because poor initial temperature control produced a spuriously high plaque count with the hd mutants. Infectivity assays. The agar cell-suspension plaque method (Cooper, 1961) was used, with the human amnion strain U cells
431
432
COOPEIt.
(Pohjanpelto, 1961) grown in medium CSV.6 (Cooper et al., 1959). Virus strains. All virus strains were poliovirus type 1. Strains hd-5 and hd-19 are two of 26 heat-defective and doubly plaquepurified clones isolated from a stock of hd+ that was grown in the presence of 1 mM 5-fluorouracil (Cooper, 1964); hd+ is a clone grown at 40” from a large plaque picked from a 40” assay of strain VS.41, which itself is the Sabin vaccine strain LSc adapted by Dr. A. Lwoff to grow at 41”. Both the leak and reversion rates are low but not zero in both hd-5 and hd-19. Virus stocks. The mutant and hdf stocks used in these experiments were grown in monolayers infected with a multiplicity of 2-3 PFU/cell. After infection the cells were washed, overlaid with Eagle’s medium containing 10 % calf serum and 4 pg neutral red per milliliter, and incubated at 36” for 16 hours in darkness. All manipulations up to irradiation (see below) were done under a red safe light (Wilson and Cooper, 1963). Standard complementation test. Barely confluent monolayers of 3 X lo5 U cells were grown on the base of stoppered flatbottomed tubes (20 mm diameter) by overnight incubation in 1 ml medium CSV.6; the tubes were always used standing upright. These cultures provided reproducible replicates. After immersion in ice water and removal of the medium, each tube received 3 drops of virus dilutions from Pasteur pipettes calibrated to deliver drops of 0.02 ml. The tubes were shaken and stood for 90 minutes at 0”; the inocula were then removed, and the tubes were washed once with 2 ml of PBS. One milliliter of cold Eagle’s medium containing 10 % calf serum and 0.03% bicarbonate was added per tube, stoppers were replaced, and the tubes were plunged into a water bath at the appropriate temperature. It is estimated that the cells reached water bath temperature within 1 minute. After 2-3 hours the cultures were irradiated with white light without removing them from the water bath, a treatment which reduced noneclipsed infectivity to < 102 PFU per culture so as not to obscure the low yields obtained. After incubation for a further 4-5 hours the tubes were frozen for
assay of infectivity. Tubes were usually in duplicate, and each experiment included controls of cultures infected with each mutant alone plus medium, to give the same final inoculum dilution as in t’he mixed infections. Measurement of thymidine uptake by infected cells. Replicate tubes containing 3 X lo5 cells were completely infected with each virus stock in duplicate at 0”. The stocks used were clones isolated from a complementation experiment, and stocks of hd-5, hd-19, and hd+; some tubes were left uninfected to serve as controls. The cells were washed, overlaid with 1 ml of medium and duplicate tubes were incubated at 37.0” and at 39.5”. After 3.75 hours’ incubation, 2 equal drops of a solution of thymidine-2-C’* (New England Nuclear Corporation, 0.1 mc/0.96 mg) were added per tube to give about lo4 cpm per milliliter of culture; incubation was continued for a further 1.75 hours, when the cultures were abruptly chilled, the medium was drained off, and the cells were washed thoroughly 3 times with 2 ml PBS. Four ml of cold 10 70 trichloroacetic acid were then added to each tube, and after standing overnight at 4” the tubes were washed again with trichloroacetic acid. The tubes were then heated (20 minutes at 100’) and dried; the contents, dissolved at, 100” in 0.2 ml 0.2 N NaOH, were transferred to planchettes and counted in an end-window counter. Duplicate cultures all agreed within a few counts per minute. Terminology used. The leak refers to the ability of a mutant stock to produce some mutant progeny under the restrictive growth conditions, as distinct from reversion, which refers to the ability of purified mutant stocks to produce progeny that are wild type in respect of the character examined. RESULTS
The Thermal Growth Requirements of hd+, hd-5 and hd-19 Figure 1 compares the effect of temperature on the abilities of hd+, hd-5, and hd-19 to form plaques, and also shows the effect of temperature on the 6-hour yield of hdf in liquid medium. It is seen that plaque production by hd+ decreases above 39,0”,
COMPLEMENTATION
y
, 36’
,
370 380 TEMPERATU FE OF
BETWEEN
\,-., 39O 4o” INCUBATION
FIG. 1. Thermal growth requirements of the poliovirus strains described in this paper. Three curves (0, a, A) show the effect of temperature on plaque formation, plaque assay bottles of hd+, k-5 and h&19, respectively, being immersed for 3 days in water baths at the indicated t.emperatures. The fourth curve (0) shows the &hour yield of hd+ when grown in tube cultures at the indicated temperatures.
and is only 25 % of the maximum value at 40.0”. The 6-hour yield of hd+ drops more rapidly and is only 4 % of the maximum value at 40.0”. The mutations that gave rise to hd-5 and to hd-19 have had a very large effect on growth at high temperatures: at 38.9”, the plaque formation by both mutant strains is 2 to 3 X 10e3 of the maximum value, while at 39.2” that of hd-5 is 10-3, and that of hd-19 is 2 to 3 X 1OV. The opposite differences in sensitivity of hd-5 and hd-19 above and below 38.9” are reproducible. The plaques produced by both mutants above 39” are small and indistinct; they are clearly distinguishable from those of hd+, which are large and sharp. Mixed Infection with hd-5 and hd-19 at High Temperatures-Effect of Multiplicity of Infection Table 1 shows the marked enhancement in yield that occurs when cells are mixedly infected with hd-5 and hd-19 and are then
POLIOVIRUS
MUTANTS
433
incubated at a temperature above the optimum for growth of these mutants. The replicate assays at 39.2” (in parentheses) show that the enhanced yields are predominantly mutant, not wild type. The yields from each mutant alone are also mainly mutant, and hence represent leak rather than content of wild-type revertant. As is to be expected, enhancement of yield is small where not all cells are infected. However, a phenomenon found in most experiments with many hd mutants is shown by the undiluted hd-19 stock; the yield is often less enhanced at higher multiplicities. The highest mixed infection yield was about 10-S of wild-type yields, and 4-6 times the sum of the leak yields. Table 1 also shows another consistent finding: the increase in multiplicity of infection of the mutant controls above that needed to infect all cells does not increase the leak yield. For this reason, and also because only 1 mutant may predominate in the mixed infection yield (see below), the true control value for comparing with mixed infection yields is probably less than the sum of the individual leak yields. However, since this “true value” cannot be arrived at, the sum will be used. The Proportion of Each Mutant Infection Yield
in the Mixed
In order to see whether the apparent complementation was fully reciprocal, i.e., whether both mutants appeared equally in the yield, a standard complementation test (duplicate tubes, 6 hours at 38.8”) was performed with approximately equal multiplicities of hd-5 and hd-19. The average mixed infection yield was 5.3 X lo* PFU per 3 x lo5 cells, while the single infection yields were 1.1 X lo* (hd-19) and 5 X lo3 (hd-5). Four-inch petri dishes were employed for the assays, and about 50 plaques per dish were present in the assay of the mixed infection yields; after spraying with stain (Cooper, 1964) plaques were picked at random and clones were grown from them. Wild-type poliovirus inhibits the uptake of thymidine by cells. Strain hd-5 is defective in this inhibition at 39.5” but not at 37.0”, while h&19 is not defective in this function
434
COOPER TABLE EFFECT
OF MULTIPLICITY
OF INFECTION hd-5 .\ND
1
ON MIXED GROWTH hd-19 AT 39.2””
OF Po~,~ovraas
STRAINS
hd-5 inoculum (PFU/cell) lzd-19 inoculum (PFU/cell)
20
24 (1)
54 (2)b 136 (5) 89 (7)
20 6 2 0.6 0
2
6
85 72 31 5
68 (2) 7 (3)
25 36 24 2 1
(3) (3) (0) (1)
0.6
(1) (0) (1) (3) (0)
0
18 (0)
10 (1)
21 (0)
12 (0)
13 (1) 0 (0) 0 (0)
5 (2) 1 (1) -
144 (15O)c
hd+ (20 PFU/cell)
a The conditions were those of the standard complementation test (see Methods), in tube cultures at 39.2”. Tubes were irradiated with white light at 2.5 hours, and frozen for infectivity assay at 6.5 hours. b The figures in the table are the plaque counts on plates containing 0.1 ml of 10-l dilution of the 6.5 hour yields and incubated at 37”; the figures in parentheses are plaque counts on replicate petri plates incubated in boxes immersed in a water bath at 39.2’. c Plaques from 0.1 ml of 10-d dilutions. TABLE EFFECT
OF INFECTION
WITH
CLONES
CELLULAR
Controls
Uninfected Uninfected hd-5 hd-5 hd-19 hd-19 hd+ hd+
Thymidine” uptake at 37.0”
39.5”
48 47 11 13 11 8 15 10
57 59 3 -2 29 23 26 24
2
ISOLATED UPTAKE
Clone no.
1 2 3 4 5 6 7 8
FRO&~ A COWLEMENTATION
TEST
ON
OF THYMIDINE~
Thymidineh uptake at 37.0”
39.5”
17 15 13 9 13 17 8 20
21 17 21 20 15 14 20 17
Clone no.
9 10 11 12 13 14 15 16
Thymidineb uptake at 37.0”
39.5”
8 16 9 10 18 12 8 3
18 24 13 20 31 19 15 20
a Procedure described in Methods. b Thymidine Cl4 cpm/3 X lo5 cells taken up in 1.75 hours; uninfected values represent observed uptakes, other values represent the differences between observed uptakes and the mean uninfected values at the respective temperature.
at either temperature (fuller details of these findings will be presented elsewhere). A test for the ability to inhibit thymidine uptake at 39.5” should therefore distinguish hd-5 and hd-19 genotypes in the mixed infection yields. Accordingly, 16 of the clones described above were tested for their effect upon thymidine uptake at 37.0” and 39.5”, as described in the Methods. The results are shown in Table 2, which gives the thymidine uptake by uninfected controls and expresses the inhibition of up-
take caused by hd+, hd-5, hd-19, and the clones as the difference between the uptakes by the infected cultures and the average uptake by uninfected cultures. It can be seen that the ratio of the inhibition of uptake at 39.5” to the inhibition of uptake at 37” is similar for M-19 (2.7), hd+ (2.0), and the clones (mean ratio = 1.88). Strain hd-5 gives a normal inhibition at 37” but is virtually completely defective in thymidine uptake inhibition at 39.5”; none of the clones is at all similar to hd-5. Independent assays
COMPLEMENTATION
BETWEEN
of the efficiencies of plating at 39.3” of these clones gave values of 1 to 6 X lop5 (with two exceptions of 10h4, which contained some wild-type plaques). Thus although the clones were wild type in thymidine uptake inhibition, they were nevertheless highly heat defective, having the plating efficiency of h&19 (2 X 10h5) rather than that of hd-5 (10e3). Furthermore, Ad-5 forms small plaques at 37.0” whereas hd-19 plaques are wild type, and few of the plaques in the mixed infection yield could be regarded as typical of hd-5. It is therefore concluded that the apparent complementation observed was not as effective for one mutant as for the other and that hd-19 predominated in the yield. The number of clones tested was insufficient to indicate that hd-5 was entirely excluded from the yield. More extensive testing for this point is likely to be inconclusive because of the leakage of hd-5.
2
3
4
POLIOVIRUS
MUTANTS
Effect of Time of Incubation hancement
435 on Yield En-
Figure 2 compares the one-step growth at 39.2” of hd-5 and hd-19, alone and in mixed infection, with that of hd+. The conditions were those of the standard complementation test described in the Methods, except that tubes were removed and frozen at intervals for assay. The maximum enhancement in yield in mixed infections of hd-5 and hd-19 occurred between 6 and 7 hours after infection, being 3 to 4 times the sum of the individual mutant yields and 0.5 X 1O-3 of hdf yields. The growth of the mixed yield followed roughly the same time course as the leak of both mutants (e.g., the 50% maximum yields all occurred at the same time). However, growth of all three mutants was delayed by almost one hour when compared with that of hd+ under the same conditions. All plaques in the 39.2”
5
6
HOURS
AT
7
8
9
39.2’
FIG. 2. One-step growth at 39.2” of poliovirus strains hd+ (A), k-5 alone (o), h&19 alone (A), and hd-5 plus ho!-19 in mixed infection (0) : the sum of the mutant yields when grown on their own is given by 0. The ha!+ curve is related to the left-hand ordinate, the others to the right. These five curves were obtained by assay at 37”. The sixth curve (0) shows the assay at 39.2” of the hat-5 plus k-19 mixed infection cultures (for this assay, petri plates in sealed boxes were immersed in a water bath at 39.2” for 3 days). The growth conditions were those of the complementation test (see Methods); replicate tubes were removed at intervals and frozen for assay.
436
COOPER
assay of the mixed yield (lowest curve) were small and indistinct, characteristic of mutant rather than hd+; similar assays at 39.2” of the growth cycles of each mutant alone gave plaques of similar number and character. Hence again (a) these growth curves of h,d-5 and M-19 by themselves represent leak at 39.2” and not growth of contaminant wild type, and (b) less than 1OP of the enhanced yield is wild type.
easier to detect at the higher temperatures. Compared with wild-type yields, however, the enhanced yields (calculated as excess over combined leaks) decreased from 2 X 1OV to 3 X 10e4 of hdf yield with increase in temperature from 38.4” to 39.6”. At temperatures above 39” it is evident that the drop with increased temperature in leak, and presumably also in mixed infection yields, reflects some defect in the parental hd+ or in the host cell as much as the defect peculiar to the mutant.
E$ect of Temperature on Yield Enhancement Several standard 6-hour complementation tests were performed concurrently in water baths at several different temperatures. Figure 3 shows that the excess of the mixed infection yields over the combined leak increased with the temperature over the range studied. At 38.4’ the mixed infection yield was less than twice the sum of the individual mutant yields, whereas at 39.6” the mixed infection was 14 times the combined leaks. Hence enhancement of yield is
ii s IO2
I
38O TEMPERATURE
E$ect of pH on Yield Enhancement Four concurrent complementation tests done at pH values of 6.9, 7.1, 7.3, and 7.5, respectively, showed that the degree of enhancement was no greater at other pH values that covered the range optimal for growth of poliovirus. The mixed infection yields were 3.9, 4.3, 4.2, and 3.4 times the sum of individual mutant yields, and about
,
A
39O OF INCUBATION
I 4o”
FIG. 3. The effect of temperature on mixed infection of poliovirus strains hd-5 and hd-19. The procedure was that of the standard complementation test (see Methods); sets of tubes were incubated for 6 hours at the indicated temperatures. The points give the average yield of 4 replicate cultures of hd-5 alone (0) and h&19 alone (A), and 8 replicate cultures of hd-5 plus h&19 in mixed infection (a); 0 equals the sum of the mutant yields on their own, while k equals hd+ yield (right-hand ordinate).
COMPLEMENTATION
BETWEEN
10d3 of that expected for hd+. The leak yields were also not much affected by pH. Attempted Rescue of hd-5 and hd-19 by hd+ Previous sections of this paper indicate that the hd-5 and hd-19 stocks used contained few wild-type revertants. Accordingly, if wild type enhances mixed infection yields with as low efficiency as the mutants, then the enhancement described above between hd-5 and hd-19 is most unlikely to result from some interaction between the mutants and traces of wild-type virus contained in the mutant st,ocks. The efficiency of enhancement with wild type was tested for by mixedly infecting cells with several high multiplicities of infection (4 to 20 PFU/cell) of either hd-5 or hd-19, together with hd+ in multiplicities from 10 PFU/cell to 1 PFU per lo4 cells. At the lowest multiplicities the yield of hd+ when grown on its own was about the same as the mutant leak rates. The experimental procedure was that of the standard complementation t,est performed at 39.0”, and the 6.25 hour yields were assayed at 37” and 39.3”. The mutant hd-19 interfered markedly with hd+; investigations of this interference will be described elsewhere (Pohjanpelto and Cooper, 1965). However, in all cases any excess of the titre obtained by the 37” assay over that of the 39.3” assay (an excess presumably equal to the content of’ mutant virus) was no greater than the leak of mutant alone (2 X lo3 PFU per 2 X lo5 cells). It is therefore concluded that a.ny rescue of mutant by wildtype virus is too small to be detected and hence cannot play a significant part in the reaction between hd-5 and hd-19 described above. DISCUSSION
These data show that hd-5 and hd-19 interact during growth at high temperature so as partially t’o overcome the heat defect of at least one of them. In evaluating this complementation of hd-19 by hd-5, two main aspects must be considered. The first aspect is that complementation in other systems is usually reciprocal; i.e., both mutants appear in the enhanced yield. Only hd-19 was detected in the interaction
POLIOVIRUS
MUTANTS
437
between hd-5 and hd-19 under the conditions studied. The reasons for this are being investigated; one very likely reason is the st,rong interference manifest by hd-19 at high temperatures, for which a number of models can be constructed (Pohjanpelto and Cooper, 1965). However, in other systems the yields of complementing genotypes may be grossly unequal (Pittenger et al., 1955; Valentine et al., 1964), and in our case there may have been a small undetected enhancement of hd-5. Whether or not hd-5 and h&19 interact reciprocally, some kind of cooperative gene function is likely to be the cause of the enhanced yields. Such effects are found frequently in many kinds of living things. Among animal viruses, guanidine-sensitive and guanidine-dependent polioviruses can cooperate in growth (Cords and Holland, 1964; Wecker and Lederhilger, 1964; Ago1 and Shirman, 1965) ; other reports of rescue or yield enhancement of animal viruses exist (Kumagai et al., 1961; Hermodsson, 1963; Hanafusa et al., 1963; Rabson et al., 1964). Among plant viruses, a defective virus with a very small genome is found with, and depends upon the growth of, the unreIated tobacco necrosis virus (Kassanis, 1962). The second aspect is that presented by the very low efficiency of the rescue process. Several factors might account for this. First, the mutants may contain overlapping deletions or alterations which reduce or eliminate the possibility of complementation. However, the reversion to wild type of both hd-5 and hd-19 is small but detectable, and this together with the type of mutagen used to induce them (5-fluorouracil) and the type of defect (in which competent gene products are made at 37”) indicate that they contain point mutations rather than extensive changes. Second, hd-5 and hd-19 may contain defects in the same gene, and interallelic complementation is usually of low efficiency. However, these mutants are defective in different physiological functions (for example, the inhibition of thymidine uptake described above) ; hence at least one of the mutants would have to contain two mutations, each leading to a gross defect at
438
COOPER
39.2”, and one defect would have to be common to both mutants. Since crosses between hd-5 and hd-19 at 37” reproducibly yield enhanced yields of infective units that’ are negligibly defective at 39.2” (Cooper, unpublished), this possibility is also unlikely. Stronger evidence against interallelic complementation as the cause of the inefficient interaction between the hd mutants is that in no case was the enhancement of yield more efficient among over 100 “complementing” (i.e., yield-enhancing) pairs of hd mutants now studied (Cooper, unpublished) ; indeed the pair h,d-5 and hd-19 was selected for further work for its relatively good performance. This study involved 31 different mutants that were isolated independently at a mutation rate of 10 % (Cooper, 1964) or 2-3 % and in which different physiological defects are often demonstrable (Cooper, Garwes, and Johnson, unpublished). Third, the low efficiency, like the inequality in the yields, may reflect the extensive interference which exists between hd+ and many of the hd mutants at high temperatures (Cooper, unpublished). Strain hd-19 happens to be a strong interfering agent, and the interference by hd-19 results from a hindrance to some event occurring after uncoating of the challenge virus and before its RNA synthesis (Pohjanpelto and Cooper, 1965). Fourth, possible effects of sequential gene function, coupled with sequestration of gene products or their dispersion through a large cell, and/or possible needs for some synchrony of gene function, might well reduce complementation efficiencies. The inefficient rescue of hd-5 by hd+ (a cis control of a kind) would imply some such case [hd-5 happens not to interfere with hd+ at high temperatures (Cooper, unpublished)]. REFERENCES AGOL, V. I., and SHIRMAN, G. A. (1964). Interaction of guanidine-sensitive and guanidine dependent variants of poliovirus in mixedly infected cells. Biochem. Biophys. Res. Commun. 17, 28-33. COOPER, P. D. (1961). An improved agar cellsuspension plaque assay for poliovirus: some factors affecting efficiency of plating. Virology 13, 153-157.
COOPER, P. I>. (1964). The mutation of poliovirus by 5.fluorouracil. Virology 22, 186-192. COOPER, P. D., WILSON, J. N., and BURT, A. M. (1959). The bulk growth of animal cells in continuous suspension culture. J. Gen. ~~~crobiol. 21, 702-720. CORDS, C. E., and HOLLAND, J. J. (1964). Replication of poliovirus RNA induced by heterologous virus. Proc. Natl. Acad. Sci. U. S. 51, 1080-1082. EPSTEIN, R. H., BOLLE, A., STEINBERG, C. M., KELLENBERGER, E., BOY DE LA TOCR, E., CHEVALLY, R., EDGAR, R. S., SUSMAN, M., DENHARDT, G. H., and LIELAUSIS, A. (1963). Physiological studies of conditional lethal mutants of bacteriophage T4D. Cold Spring Harbor Symp. Quant. Biol. 28, 375-394. HANAFUSA, H., HANAFUSA, I., and RUBIN, H. (1963). The defectiveness of Rous sarcoma virus. Proc. Natl. Acad. Sci. U.S. 49, 572-580. HERMODSSON, S. (1963). Inhibition of interferon by an infection with parainfluenza virus type 3 (PIV-3). Virology 20, 333-343. KASSANIS, B. (1962). Properties and behaviour of a virus depending for its multiplication on another. J. Gen. Microbial. 27, 477-488. KUMAGAI, T., SHIMIZU, T., IKEDA, S., and MATUMOTO, M. (1961). A new in vitro method (END) for detection and measurement of hog cholera virus and its antibody by means of effect of HC virus on Newcastle disease virus in swine tissue culture. J. Immunol. 87, 245-256. PITTENGER, T. H., KIMBALL, A. W., and ATTWOOD, K. C. (1955). Control of nuclear ratios in Neurospora heterokaryons. Am. J. Botany 42, 954958. POHJANPELTO, P. (1961). Response of enteroviruses to cystine. Vz%oZogy 15, 225-230. POHJANPELTO, P., and COOPER, P. D. (1965). Interference between polioviruses induced by strains that cannot multiply. Virology, 25, 350357, this issue. RABSON, A. S., O’CONNOR, G. T., BEREZESKY, I. K., and PAUL, F. J. (1964). Enhancement of adenovirus growth in African green monkey kidney cell cultures by 8. V. 40. Proc. Sot. Exptl. Biol. Med. 116, 187-190. VALENTINE, R. C., ENGELHARDT, D. L., and ZINDER, N. D. (1964). Host-dependent mutants of bacteriophage f2. II. Rescue and complementation of mutants. Virology 23, 159-163. WECKER, E., and LEDERHILGER, G. (1964). Curtailment of the latent period by double-infection with polioviruses. Proc. Natl. Acad. Sci. U.S. 52, 246-251. WILSON, J. N., and COOPER, P. D. (1963). Aspects of the growth of poliovirus as revealed by the photodynamic effects of neutral red and acridine orange. Virology 21, 135-145.
VIROLOGY
Rescue
of One
Phenotype Mutants
in Mixed
Infections
of Type
1 Poliovirus
PETER Department
of Microbiology,
John Curtin
with
Heat-Defective
D. COOPER
School of Medical Research, Canberra, Australia
Australian
National
University,
Accepted November 20, 1964 When cells are mixedly infected with two heat-defective (hd) poliovirus mutants at temperatures above those that are optimal for growth of these mutants, the virus yield is 4-14 times the sum of the yields of each mutant when grown separately under identical conditions (“leak” yields). The mixed infection yields are only one thousandth of the yield of wild type (ho!+) under these conditions. The leak and mixed infection yields are 90-997, mutant rather than hdf, but only one of the mutants was detected in the mixed infection yields. Any rescue of the hd mutants by hd+ was too small to be detected. INTRODUCTION
Conditional lethal mutations of bacteriophage T4, which prevent its growth either in certain hosts or at higher temperatures, have been used with great effect (Epstein et al., 1963) to map the T4 genome and to indicate many of its gene functions. The discovery that 5-fluorouracil produces many heat defective (hd) mutants in poliovirus (Cooper, 1964) suggests the possibility of a similar approach to the genetic analysis of a small animal ribovirus. The hd mut,ants that have been isolated are now being examined in several ways. One way is to classify them by means of a complementation test, with the object of defining those groups of mutants which have defects in the same gene. This test showed that mixed infection at high temperature with many but not all pairs of hd mutants gave an enhanced yield. The enhancement was always very small, however. This paper describes the complementation test and reports the results of some experiments in which the interaction between two of the mutants is examined in some detail with the object of defining optimal conditions. It is shown that, for this particular pair at least, the enhancement was
not reciprocal within test.
the sensitivity
of the
METHODS
Temperature control. Well-circulated water baths controlled by Braun (Melsungen, Germany) Thermomix II mercury contact units maintained temperature to fO.05’; the most convenient monitors were clinical thermometers graduated in O.l”C. Tubes were stoppered and were immersed to a depth of 3 cm in open racks; maximum temperature was reached within 3 minutes. Plaque assays of virus at high temperatures were made with a bicarbonate medium (Cooper, 1961) contained in screw-capped bottles immersed inverted in water baths in open baskets, in such a way as not to impede water circulation. In some experiments (indicated in the text) petri plates were enclosed in a sealed box that was immersed in a water bath at a supraoptimal temperature, but this method was discontinued because poor initial temperature control produced a spuriously high plaque count with the hd mutants. Infectivity assays. The agar cell-suspension plaque method (Cooper, 1961) was used, with the human amnion strain U cells
431
432
COOPEIt.
(Pohjanpelto, 1961) grown in medium CSV.6 (Cooper et al., 1959). Virus strains. All virus strains were poliovirus type 1. Strains hd-5 and hd-19 are two of 26 heat-defective and doubly plaquepurified clones isolated from a stock of hd+ that was grown in the presence of 1 mM 5-fluorouracil (Cooper, 1964); hd+ is a clone grown at 40” from a large plaque picked from a 40” assay of strain VS.41, which itself is the Sabin vaccine strain LSc adapted by Dr. A. Lwoff to grow at 41”. Both the leak and reversion rates are low but not zero in both hd-5 and hd-19. Virus stocks. The mutant and hdf stocks used in these experiments were grown in monolayers infected with a multiplicity of 2-3 PFU/cell. After infection the cells were washed, overlaid with Eagle’s medium containing 10 % calf serum and 4 pg neutral red per milliliter, and incubated at 36” for 16 hours in darkness. All manipulations up to irradiation (see below) were done under a red safe light (Wilson and Cooper, 1963). Standard complementation test. Barely confluent monolayers of 3 X lo5 U cells were grown on the base of stoppered flatbottomed tubes (20 mm diameter) by overnight incubation in 1 ml medium CSV.6; the tubes were always used standing upright. These cultures provided reproducible replicates. After immersion in ice water and removal of the medium, each tube received 3 drops of virus dilutions from Pasteur pipettes calibrated to deliver drops of 0.02 ml. The tubes were shaken and stood for 90 minutes at 0”; the inocula were then removed, and the tubes were washed once with 2 ml of PBS. One milliliter of cold Eagle’s medium containing 10 % calf serum and 0.03% bicarbonate was added per tube, stoppers were replaced, and the tubes were plunged into a water bath at the appropriate temperature. It is estimated that the cells reached water bath temperature within 1 minute. After 2-3 hours the cultures were irradiated with white light without removing them from the water bath, a treatment which reduced noneclipsed infectivity to < 102 PFU per culture so as not to obscure the low yields obtained. After incubation for a further 4-5 hours the tubes were frozen for
assay of infectivity. Tubes were usually in duplicate, and each experiment included controls of cultures infected with each mutant alone plus medium, to give the same final inoculum dilution as in t’he mixed infections. Measurement of thymidine uptake by infected cells. Replicate tubes containing 3 X lo5 cells were completely infected with each virus stock in duplicate at 0”. The stocks used were clones isolated from a complementation experiment, and stocks of hd-5, hd-19, and hd+; some tubes were left uninfected to serve as controls. The cells were washed, overlaid with 1 ml of medium and duplicate tubes were incubated at 37.0” and at 39.5”. After 3.75 hours’ incubation, 2 equal drops of a solution of thymidine-2-C’* (New England Nuclear Corporation, 0.1 mc/0.96 mg) were added per tube to give about lo4 cpm per milliliter of culture; incubation was continued for a further 1.75 hours, when the cultures were abruptly chilled, the medium was drained off, and the cells were washed thoroughly 3 times with 2 ml PBS. Four ml of cold 10 70 trichloroacetic acid were then added to each tube, and after standing overnight at 4” the tubes were washed again with trichloroacetic acid. The tubes were then heated (20 minutes at 100’) and dried; the contents, dissolved at, 100” in 0.2 ml 0.2 N NaOH, were transferred to planchettes and counted in an end-window counter. Duplicate cultures all agreed within a few counts per minute. Terminology used. The leak refers to the ability of a mutant stock to produce some mutant progeny under the restrictive growth conditions, as distinct from reversion, which refers to the ability of purified mutant stocks to produce progeny that are wild type in respect of the character examined. RESULTS
The Thermal Growth Requirements of hd+, hd-5 and hd-19 Figure 1 compares the effect of temperature on the abilities of hd+, hd-5, and hd-19 to form plaques, and also shows the effect of temperature on the 6-hour yield of hdf in liquid medium. It is seen that plaque production by hd+ decreases above 39,0”,
COMPLEMENTATION
y
, 36’
,
370 380 TEMPERATU FE OF
BETWEEN
\,-., 39O 4o” INCUBATION
FIG. 1. Thermal growth requirements of the poliovirus strains described in this paper. Three curves (0, a, A) show the effect of temperature on plaque formation, plaque assay bottles of hd+, k-5 and h&19, respectively, being immersed for 3 days in water baths at the indicated t.emperatures. The fourth curve (0) shows the &hour yield of hd+ when grown in tube cultures at the indicated temperatures.
and is only 25 % of the maximum value at 40.0”. The 6-hour yield of hd+ drops more rapidly and is only 4 % of the maximum value at 40.0”. The mutations that gave rise to hd-5 and to hd-19 have had a very large effect on growth at high temperatures: at 38.9”, the plaque formation by both mutant strains is 2 to 3 X 10e3 of the maximum value, while at 39.2” that of hd-5 is 10-3, and that of hd-19 is 2 to 3 X 1OV. The opposite differences in sensitivity of hd-5 and hd-19 above and below 38.9” are reproducible. The plaques produced by both mutants above 39” are small and indistinct; they are clearly distinguishable from those of hd+, which are large and sharp. Mixed Infection with hd-5 and hd-19 at High Temperatures-Effect of Multiplicity of Infection Table 1 shows the marked enhancement in yield that occurs when cells are mixedly infected with hd-5 and hd-19 and are then
POLIOVIRUS
MUTANTS
433
incubated at a temperature above the optimum for growth of these mutants. The replicate assays at 39.2” (in parentheses) show that the enhanced yields are predominantly mutant, not wild type. The yields from each mutant alone are also mainly mutant, and hence represent leak rather than content of wild-type revertant. As is to be expected, enhancement of yield is small where not all cells are infected. However, a phenomenon found in most experiments with many hd mutants is shown by the undiluted hd-19 stock; the yield is often less enhanced at higher multiplicities. The highest mixed infection yield was about 10-S of wild-type yields, and 4-6 times the sum of the leak yields. Table 1 also shows another consistent finding: the increase in multiplicity of infection of the mutant controls above that needed to infect all cells does not increase the leak yield. For this reason, and also because only 1 mutant may predominate in the mixed infection yield (see below), the true control value for comparing with mixed infection yields is probably less than the sum of the individual leak yields. However, since this “true value” cannot be arrived at, the sum will be used. The Proportion of Each Mutant Infection Yield
in the Mixed
In order to see whether the apparent complementation was fully reciprocal, i.e., whether both mutants appeared equally in the yield, a standard complementation test (duplicate tubes, 6 hours at 38.8”) was performed with approximately equal multiplicities of hd-5 and hd-19. The average mixed infection yield was 5.3 X lo* PFU per 3 x lo5 cells, while the single infection yields were 1.1 X lo* (hd-19) and 5 X lo3 (hd-5). Four-inch petri dishes were employed for the assays, and about 50 plaques per dish were present in the assay of the mixed infection yields; after spraying with stain (Cooper, 1964) plaques were picked at random and clones were grown from them. Wild-type poliovirus inhibits the uptake of thymidine by cells. Strain hd-5 is defective in this inhibition at 39.5” but not at 37.0”, while h&19 is not defective in this function
434
COOPER TABLE EFFECT
OF MULTIPLICITY
OF INFECTION hd-5 .\ND
1
ON MIXED GROWTH hd-19 AT 39.2””
OF Po~,~ovraas
STRAINS
hd-5 inoculum (PFU/cell) lzd-19 inoculum (PFU/cell)
20
24 (1)
54 (2)b 136 (5) 89 (7)
20 6 2 0.6 0
2
6
85 72 31 5
68 (2) 7 (3)
25 36 24 2 1
(3) (3) (0) (1)
0.6
(1) (0) (1) (3) (0)
0
18 (0)
10 (1)
21 (0)
12 (0)
13 (1) 0 (0) 0 (0)
5 (2) 1 (1) -
144 (15O)c
hd+ (20 PFU/cell)
a The conditions were those of the standard complementation test (see Methods), in tube cultures at 39.2”. Tubes were irradiated with white light at 2.5 hours, and frozen for infectivity assay at 6.5 hours. b The figures in the table are the plaque counts on plates containing 0.1 ml of 10-l dilution of the 6.5 hour yields and incubated at 37”; the figures in parentheses are plaque counts on replicate petri plates incubated in boxes immersed in a water bath at 39.2’. c Plaques from 0.1 ml of 10-d dilutions. TABLE EFFECT
OF INFECTION
WITH
CLONES
CELLULAR
Controls
Uninfected Uninfected hd-5 hd-5 hd-19 hd-19 hd+ hd+
Thymidine” uptake at 37.0”
39.5”
48 47 11 13 11 8 15 10
57 59 3 -2 29 23 26 24
2
ISOLATED UPTAKE
Clone no.
1 2 3 4 5 6 7 8
FRO&~ A COWLEMENTATION
TEST
ON
OF THYMIDINE~
Thymidineh uptake at 37.0”
39.5”
17 15 13 9 13 17 8 20
21 17 21 20 15 14 20 17
Clone no.
9 10 11 12 13 14 15 16
Thymidineb uptake at 37.0”
39.5”
8 16 9 10 18 12 8 3
18 24 13 20 31 19 15 20
a Procedure described in Methods. b Thymidine Cl4 cpm/3 X lo5 cells taken up in 1.75 hours; uninfected values represent observed uptakes, other values represent the differences between observed uptakes and the mean uninfected values at the respective temperature.
at either temperature (fuller details of these findings will be presented elsewhere). A test for the ability to inhibit thymidine uptake at 39.5” should therefore distinguish hd-5 and hd-19 genotypes in the mixed infection yields. Accordingly, 16 of the clones described above were tested for their effect upon thymidine uptake at 37.0” and 39.5”, as described in the Methods. The results are shown in Table 2, which gives the thymidine uptake by uninfected controls and expresses the inhibition of up-
take caused by hd+, hd-5, hd-19, and the clones as the difference between the uptakes by the infected cultures and the average uptake by uninfected cultures. It can be seen that the ratio of the inhibition of uptake at 39.5” to the inhibition of uptake at 37” is similar for M-19 (2.7), hd+ (2.0), and the clones (mean ratio = 1.88). Strain hd-5 gives a normal inhibition at 37” but is virtually completely defective in thymidine uptake inhibition at 39.5”; none of the clones is at all similar to hd-5. Independent assays
COMPLEMENTATION
BETWEEN
of the efficiencies of plating at 39.3” of these clones gave values of 1 to 6 X lop5 (with two exceptions of 10h4, which contained some wild-type plaques). Thus although the clones were wild type in thymidine uptake inhibition, they were nevertheless highly heat defective, having the plating efficiency of h&19 (2 X 10h5) rather than that of hd-5 (10e3). Furthermore, Ad-5 forms small plaques at 37.0” whereas hd-19 plaques are wild type, and few of the plaques in the mixed infection yield could be regarded as typical of hd-5. It is therefore concluded that the apparent complementation observed was not as effective for one mutant as for the other and that hd-19 predominated in the yield. The number of clones tested was insufficient to indicate that hd-5 was entirely excluded from the yield. More extensive testing for this point is likely to be inconclusive because of the leakage of hd-5.
2
3
4
POLIOVIRUS
MUTANTS
Effect of Time of Incubation hancement
435 on Yield En-
Figure 2 compares the one-step growth at 39.2” of hd-5 and hd-19, alone and in mixed infection, with that of hd+. The conditions were those of the standard complementation test described in the Methods, except that tubes were removed and frozen at intervals for assay. The maximum enhancement in yield in mixed infections of hd-5 and hd-19 occurred between 6 and 7 hours after infection, being 3 to 4 times the sum of the individual mutant yields and 0.5 X 1O-3 of hdf yields. The growth of the mixed yield followed roughly the same time course as the leak of both mutants (e.g., the 50% maximum yields all occurred at the same time). However, growth of all three mutants was delayed by almost one hour when compared with that of hd+ under the same conditions. All plaques in the 39.2”
5
6
HOURS
AT
7
8
9
39.2’
FIG. 2. One-step growth at 39.2” of poliovirus strains hd+ (A), k-5 alone (o), h&19 alone (A), and hd-5 plus ho!-19 in mixed infection (0) : the sum of the mutant yields when grown on their own is given by 0. The ha!+ curve is related to the left-hand ordinate, the others to the right. These five curves were obtained by assay at 37”. The sixth curve (0) shows the assay at 39.2” of the hat-5 plus k-19 mixed infection cultures (for this assay, petri plates in sealed boxes were immersed in a water bath at 39.2” for 3 days). The growth conditions were those of the complementation test (see Methods); replicate tubes were removed at intervals and frozen for assay.
436
COOPER
assay of the mixed yield (lowest curve) were small and indistinct, characteristic of mutant rather than hd+; similar assays at 39.2” of the growth cycles of each mutant alone gave plaques of similar number and character. Hence again (a) these growth curves of h,d-5 and M-19 by themselves represent leak at 39.2” and not growth of contaminant wild type, and (b) less than 1OP of the enhanced yield is wild type.
easier to detect at the higher temperatures. Compared with wild-type yields, however, the enhanced yields (calculated as excess over combined leaks) decreased from 2 X 1OV to 3 X 10e4 of hdf yield with increase in temperature from 38.4” to 39.6”. At temperatures above 39” it is evident that the drop with increased temperature in leak, and presumably also in mixed infection yields, reflects some defect in the parental hd+ or in the host cell as much as the defect peculiar to the mutant.
E$ect of Temperature on Yield Enhancement Several standard 6-hour complementation tests were performed concurrently in water baths at several different temperatures. Figure 3 shows that the excess of the mixed infection yields over the combined leak increased with the temperature over the range studied. At 38.4’ the mixed infection yield was less than twice the sum of the individual mutant yields, whereas at 39.6” the mixed infection was 14 times the combined leaks. Hence enhancement of yield is
ii s IO2
I
38O TEMPERATURE
E$ect of pH on Yield Enhancement Four concurrent complementation tests done at pH values of 6.9, 7.1, 7.3, and 7.5, respectively, showed that the degree of enhancement was no greater at other pH values that covered the range optimal for growth of poliovirus. The mixed infection yields were 3.9, 4.3, 4.2, and 3.4 times the sum of individual mutant yields, and about
,
A
39O OF INCUBATION
I 4o”
FIG. 3. The effect of temperature on mixed infection of poliovirus strains hd-5 and hd-19. The procedure was that of the standard complementation test (see Methods); sets of tubes were incubated for 6 hours at the indicated temperatures. The points give the average yield of 4 replicate cultures of hd-5 alone (0) and h&19 alone (A), and 8 replicate cultures of hd-5 plus h&19 in mixed infection (a); 0 equals the sum of the mutant yields on their own, while k equals hd+ yield (right-hand ordinate).
COMPLEMENTATION
BETWEEN
10d3 of that expected for hd+. The leak yields were also not much affected by pH. Attempted Rescue of hd-5 and hd-19 by hd+ Previous sections of this paper indicate that the hd-5 and hd-19 stocks used contained few wild-type revertants. Accordingly, if wild type enhances mixed infection yields with as low efficiency as the mutants, then the enhancement described above between hd-5 and hd-19 is most unlikely to result from some interaction between the mutants and traces of wild-type virus contained in the mutant st,ocks. The efficiency of enhancement with wild type was tested for by mixedly infecting cells with several high multiplicities of infection (4 to 20 PFU/cell) of either hd-5 or hd-19, together with hd+ in multiplicities from 10 PFU/cell to 1 PFU per lo4 cells. At the lowest multiplicities the yield of hd+ when grown on its own was about the same as the mutant leak rates. The experimental procedure was that of the standard complementation t,est performed at 39.0”, and the 6.25 hour yields were assayed at 37” and 39.3”. The mutant hd-19 interfered markedly with hd+; investigations of this interference will be described elsewhere (Pohjanpelto and Cooper, 1965). However, in all cases any excess of the titre obtained by the 37” assay over that of the 39.3” assay (an excess presumably equal to the content of’ mutant virus) was no greater than the leak of mutant alone (2 X lo3 PFU per 2 X lo5 cells). It is therefore concluded that a.ny rescue of mutant by wildtype virus is too small to be detected and hence cannot play a significant part in the reaction between hd-5 and hd-19 described above. DISCUSSION
These data show that hd-5 and hd-19 interact during growth at high temperature so as partially t’o overcome the heat defect of at least one of them. In evaluating this complementation of hd-19 by hd-5, two main aspects must be considered. The first aspect is that complementation in other systems is usually reciprocal; i.e., both mutants appear in the enhanced yield. Only hd-19 was detected in the interaction
POLIOVIRUS
MUTANTS
437
between hd-5 and hd-19 under the conditions studied. The reasons for this are being investigated; one very likely reason is the st,rong interference manifest by hd-19 at high temperatures, for which a number of models can be constructed (Pohjanpelto and Cooper, 1965). However, in other systems the yields of complementing genotypes may be grossly unequal (Pittenger et al., 1955; Valentine et al., 1964), and in our case there may have been a small undetected enhancement of hd-5. Whether or not hd-5 and h&19 interact reciprocally, some kind of cooperative gene function is likely to be the cause of the enhanced yields. Such effects are found frequently in many kinds of living things. Among animal viruses, guanidine-sensitive and guanidine-dependent polioviruses can cooperate in growth (Cords and Holland, 1964; Wecker and Lederhilger, 1964; Ago1 and Shirman, 1965) ; other reports of rescue or yield enhancement of animal viruses exist (Kumagai et al., 1961; Hermodsson, 1963; Hanafusa et al., 1963; Rabson et al., 1964). Among plant viruses, a defective virus with a very small genome is found with, and depends upon the growth of, the unreIated tobacco necrosis virus (Kassanis, 1962). The second aspect is that presented by the very low efficiency of the rescue process. Several factors might account for this. First, the mutants may contain overlapping deletions or alterations which reduce or eliminate the possibility of complementation. However, the reversion to wild type of both hd-5 and hd-19 is small but detectable, and this together with the type of mutagen used to induce them (5-fluorouracil) and the type of defect (in which competent gene products are made at 37”) indicate that they contain point mutations rather than extensive changes. Second, hd-5 and hd-19 may contain defects in the same gene, and interallelic complementation is usually of low efficiency. However, these mutants are defective in different physiological functions (for example, the inhibition of thymidine uptake described above) ; hence at least one of the mutants would have to contain two mutations, each leading to a gross defect at
438
COOPER
39.2”, and one defect would have to be common to both mutants. Since crosses between hd-5 and hd-19 at 37” reproducibly yield enhanced yields of infective units that’ are negligibly defective at 39.2” (Cooper, unpublished), this possibility is also unlikely. Stronger evidence against interallelic complementation as the cause of the inefficient interaction between the hd mutants is that in no case was the enhancement of yield more efficient among over 100 “complementing” (i.e., yield-enhancing) pairs of hd mutants now studied (Cooper, unpublished) ; indeed the pair h,d-5 and hd-19 was selected for further work for its relatively good performance. This study involved 31 different mutants that were isolated independently at a mutation rate of 10 % (Cooper, 1964) or 2-3 % and in which different physiological defects are often demonstrable (Cooper, Garwes, and Johnson, unpublished). Third, the low efficiency, like the inequality in the yields, may reflect the extensive interference which exists between hd+ and many of the hd mutants at high temperatures (Cooper, unpublished). Strain hd-19 happens to be a strong interfering agent, and the interference by hd-19 results from a hindrance to some event occurring after uncoating of the challenge virus and before its RNA synthesis (Pohjanpelto and Cooper, 1965). Fourth, possible effects of sequential gene function, coupled with sequestration of gene products or their dispersion through a large cell, and/or possible needs for some synchrony of gene function, might well reduce complementation efficiencies. The inefficient rescue of hd-5 by hd+ (a cis control of a kind) would imply some such case [hd-5 happens not to interfere with hd+ at high temperatures (Cooper, unpublished)]. REFERENCES AGOL, V. I., and SHIRMAN, G. A. (1964). Interaction of guanidine-sensitive and guanidine dependent variants of poliovirus in mixedly infected cells. Biochem. Biophys. Res. Commun. 17, 28-33. COOPER, P. D. (1961). An improved agar cellsuspension plaque assay for poliovirus: some factors affecting efficiency of plating. Virology 13, 153-157.
COOPER, P. I>. (1964). The mutation of poliovirus by 5.fluorouracil. Virology 22, 186-192. COOPER, P. D., WILSON, J. N., and BURT, A. M. (1959). The bulk growth of animal cells in continuous suspension culture. J. Gen. ~~~crobiol. 21, 702-720. CORDS, C. E., and HOLLAND, J. J. (1964). Replication of poliovirus RNA induced by heterologous virus. Proc. Natl. Acad. Sci. U. S. 51, 1080-1082. EPSTEIN, R. H., BOLLE, A., STEINBERG, C. M., KELLENBERGER, E., BOY DE LA TOCR, E., CHEVALLY, R., EDGAR, R. S., SUSMAN, M., DENHARDT, G. H., and LIELAUSIS, A. (1963). Physiological studies of conditional lethal mutants of bacteriophage T4D. Cold Spring Harbor Symp. Quant. Biol. 28, 375-394. HANAFUSA, H., HANAFUSA, I., and RUBIN, H. (1963). The defectiveness of Rous sarcoma virus. Proc. Natl. Acad. Sci. U.S. 49, 572-580. HERMODSSON, S. (1963). Inhibition of interferon by an infection with parainfluenza virus type 3 (PIV-3). Virology 20, 333-343. KASSANIS, B. (1962). Properties and behaviour of a virus depending for its multiplication on another. J. Gen. Microbial. 27, 477-488. KUMAGAI, T., SHIMIZU, T., IKEDA, S., and MATUMOTO, M. (1961). A new in vitro method (END) for detection and measurement of hog cholera virus and its antibody by means of effect of HC virus on Newcastle disease virus in swine tissue culture. J. Immunol. 87, 245-256. PITTENGER, T. H., KIMBALL, A. W., and ATTWOOD, K. C. (1955). Control of nuclear ratios in Neurospora heterokaryons. Am. J. Botany 42, 954958. POHJANPELTO, P. (1961). Response of enteroviruses to cystine. Vz%oZogy 15, 225-230. POHJANPELTO, P., and COOPER, P. D. (1965). Interference between polioviruses induced by strains that cannot multiply. Virology, 25, 350357, this issue. RABSON, A. S., O’CONNOR, G. T., BEREZESKY, I. K., and PAUL, F. J. (1964). Enhancement of adenovirus growth in African green monkey kidney cell cultures by 8. V. 40. Proc. Sot. Exptl. Biol. Med. 116, 187-190. VALENTINE, R. C., ENGELHARDT, D. L., and ZINDER, N. D. (1964). Host-dependent mutants of bacteriophage f2. II. Rescue and complementation of mutants. Virology 23, 159-163. WECKER, E., and LEDERHILGER, G. (1964). Curtailment of the latent period by double-infection with polioviruses. Proc. Natl. Acad. Sci. U.S. 52, 246-251. WILSON, J. N., and COOPER, P. D. (1963). Aspects of the growth of poliovirus as revealed by the photodynamic effects of neutral red and acridine orange. Virology 21, 135-145.