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Physiological characterization of heat-defective (temperature-sensitive) poliovirus mutants: Preliminary classification
VIROLOGY
30, 638-649
Physiological
(1966)
Characterization sitive)
PETER Department
Poliovirus
D. COOPER, of Microbiology,
of Mutants:
RICHARD John
Heat-Defective
Curtin
Preliminary
T. JOHNSON; School of Medical Canberra, Australia
Accepted
July
(Temperature-SenClassification
AND
Research,
DAVID Australian
J. GARWES2 National
University,
18, 1966
Cells infected with ts mutants of poliovirus were incubated at temperatures (39.4”-39.6”) too high to support full production of infective virus, and were then examined for certain changes that are normally characteristic of virus growth. The changes reported here, which can be used as genetic characters of the virus, are denoted:-a+: the production of poliovirus antigen; pli f: the prevention of cellular incorporation of thymidine; ppilf, pp&+, pp&+: the prevention of a3P incorporation into, respectively, 28 S, 16 S or 4-10 S cellular RNA. The wild-type strain (is+) grew well at 39.6”; it also displayed all five characters at 39.6”, but most of the 29 mutants tested did not. Tests for the characters a+, pti+, and pp&+ revealed large temperature defects in many mutants, and were used to define four groups of mutants with the following combinations of characters: group A: a, ppip, pti; group B: a, ppia; group C: a; group D: all characters wild type. Other combinations were not found, and so these characters covary in an asymmetrical or polar fashion. Temperature shift experiments suggested that certain gene products are made and used throughout the growth cycle, although most appear to be associated with ‘late’ junctions. INTRODUCTION
Mutant strains of virus that cannot multiply under certain defined conditions (conditional lethal mutants) may be used to determine something of the number and function of the defective viral genes (Epstein et al., 1963). The temperature-sensitive (termed heat-defective, or hd) strains of poliovirus that were isolated for this purpose (Cooper, 1964) were induced by 5fluorouracil, and a complementation test was developed by which the mutants could be examined (Cooper, 1965); this examina1 Recipient of a Special Fellowship (BT 1009) from the National Institute of Neurological Disease and Blindness, U. S. Public Health Service. Present address: Division of Neurology, Cleveland Metropolitan General Hospital and Western Reserve University School of Medicine, Cleveland, Ohio. 2 Present address: Microbiological Research Establishment, Porton, Wills., England.
tion is still in progress. The present paper reports the results of a preliminary analysis of the temperature sensitive (ts) mutants by another method, namely in terms of the physiological defects that appear under the restrictive conditions. This paper embodies a change in terminology. The term “temperature-sensitive” (ts) as originally applied to poliovirus was used for strains whose virions were thermolabile (Dulbecco, 1960; Papaevangelou and Youngner, 1961; McBride, 1962). To avoid confusion, a different term (heat defective, or hd) was therefore coined for poliovirus strains whose growth was defective at higher temperatures (Cooper, 1964, 1965, 1966; Pohjanpelto and Cooper, 1965). However, the subsequent wide usage of “ts” for such strains of other viruses obliges us to discard "ha? and to use ‘Ys” instead. It must accordingly be noted that the ts poliovirus mutants referred to below and subsequently are formally analogous to the ts bacteriophage
CLASSIFICATION
OF
mutants of Epstein et al. (1963) and different from the ts poliovirus mutants of Dulbecco Papaevangelou and Youngner (196’3, (1961), and McBride (1962).
POLIOVIRUS
ts MUTANTS
639
mutants. Accordingly, some relatively leaky mutants (with plating efficiencies between 0.1 and 0.01) were included in case a stringent selection for nonleaky st,rains produced only double mutants. MATERIALS AND METHODS Infection of ring cultures and staining with Cell strain. Human amnion cells of strain Jluorescent antibody. Semiconfluent monoU (Pohjanpelto, 1961) were used throughout layers (2 X lo5 cells) were formed overnight and were routinely subcultured in medium in ring cultures (Cairns, 1960) placed on the CSV.6 (Cooper et al., 1959). ends of narrow microscope slides. The culVirus assay. Infectivity was assayed by tures were chilled at 4”, the medium was rethe agar cell-suspension plaque method moved, and 3 standard 0.02 ml drops of a (Cooper, 1961) using U cells. Assays at 39” cold undiluted virus stock (containing lo6 to or above were incubated in water baths 10’ PFU) or of normal medium were added (Cooper, 1965). to each of two cultures. After 90 minutes at Isolation of temperature-sensitive mutants. 4”, the inocula and the rings were removed Mutant strains ts-2 to -23 were among the and incubation was begun abruptly by 26 mutants isolated at a rate of 10% after deeply immersing the cells in 15 ml of growth of the wild-type poliovirus strain Eagle’s medium (pH 7.3) containing 1 mg hd+ (now termed ts+) in presence of 1 mM bovine plasma albumin per millilitre, but no 5-fluorouracil (Cooper, 1964). Mutants bicarbonate. This medium was held in ts-28 to -155 were isolated similarly except capped wide-necked tubes in water baths that a lower dose of mutagen (0.25 mM, maintained either at 37.0” or at 39.4”, both giving an isolation rate of 2-3 %) was used &0.05”. One of each pair of duplicate culto minimize the occurrence of double tures was incubated at each temperature. mutants. The two sets of mutants had a After 5.5 hours, the slides were washed twice similar distribution of physiological defects with PBS at 4” and dried under a fan in a and of efficiencies of plating. The mutagen room at 36”. They were then washed twice was employed to ensure t’hat most isolates with reagent grade acetone, dried, and were independently derived. The assay treated for 20 minutes with monkey antiplates of the stock subcultured from that serum, obtained from animals that had been grown in 5-Auorouracil were incubated for throat-painted twice at an interval of 10 2 days at 36”, then transferred to an incudays with type 1 Sabin vaccine virus. The bator at 40” for 2 days; small plaques were indirect method of staining, involving picked, grown up in tube cultures, and spotfluorescein-labelled goat anti-monkey ytested. Temperature-sensitive strains were globulin (Microbiological Associates, Beassayed at 37” and 39.2”; strains with an thesda, Maryland), was then used as deefficiency of plating at 39.2” of 0.1 or less scribed by Johnson (1964). were recloned twice and given two or three Effect of virus on cellular incorporation of one-step subcultures t’o prepare stocks of thymidineJ4C. Monolayers of 2 X lo5 U cells 30-60 ml which were stored in 3-ml aliquots formed on the base of flat-bottomed tubes at -70”. Stocks whose efficiency of plating were chilled to 2” and given 3 standard 0.02 at 39.2” had increased during this procedure ml drops, either of virus stocks diluted to 1 were rejected. The remainder ($$o of the to 2 X lo6 PFU per 3 drops, or of normal original isolates), plus 10 mutants recloned medium (Cooper, 1965). Four or six replicate and grown up by this procedure from the tubes were infected with each virus stock, first 26 isolates, comprised 29 mutants. Their and the replicates were divided into two efficiencies of plating at 39.2”, together with equal groups held in separate metal racks. some other properties, are given in Table 1 The cells were washed twice with PBS, and (see below). The mode of the efficiencies of 0.5 ml of Eagle’s medium (containing 1 mg plating at 39.2” (the maximum temperature of bovine plasma albumin and 0.28 mg of at which ts+ was unaffected) was about 0.1 NaHC03 per millilitre) was added to each for the original isolates of both sets of tube. The tubes were left unstoppered, and
640
COOPER,
JOHNSON,
AND
TABLE
1
SUMMARY OF RESULTS OF PHYSIOLOGICAL
TESTS
Character Group
Wild A
ts
type
+ 5 20 23 81 99 18 22 28 44 46 104 123 19 37 89 2 3 8
B
C
D
Unclassified
Proportion defectives
9 38 50 63 65 94 96 149 150 151 155 of
a+ 39.4"
P&f 39.6"
i+
+ + +
+ + + + + +
-I+ + + + + + + + + + + +
+
f
+j+
OF POLIOVIRUS
ppi1+ 39.6"
1 + + +
+ + +
ND + -I+ f + f : + + -I+ + i-
ND + + + + + + + + + + -I+ + -I+
Late Late ND ND ND Late ND Late Late ND Late -
0.001 0.001 0.05 0.1 v.d. 0.1
ND -
I?& ND ND ND ND ND ND
-I+ ND -IND ND ND ND ND ND ND
24/27
13/19
5/28
4/20
o/20
-
e.o.p.b 39.2" 1 0.001 0.001 d. 0.001
+ f + -If f f + + ND +
f -IND
Critical period of cycle None Late Late Early Late ND ND Late Late ND Late Late ND Late Late Late ND ND ND
ND ND ND ND ND ND ND ND ND ND
ND
ts MUTANTS”
and test temperature PPiP’ 39.6"
$6"
GARWES
a The significance of the characters and the details of the tests are given in the text. a+ = of antigen; pti+, = prevention of incorporation of thymidine; pp&+, ppis+, pp&+ = prevention phorus incorporation into RNA fractions 1, 2, and 3, respectively. In the body of the table, type response; - = markedly defective; i = slightly but significantly defective; ND = mined. b Equals the efficiency of plating at 39.2”, expressed as a ratio of plaques formed at 39.2” formed in concurrent assays at 37”; d., v.d. = delayed, very delayed. c ‘(wt” = not usually distinguishable from wild-type; s = smaller than wild-type; L = wild-type.
the pH remained at about 7.3. At zero time the tubes were immersed in a bath, one rack at 37.0” f 0.05” and the duplicate rack at 39.6” f 0.05. After 3.75 hours’ incubation 2 standard drops containing 2500 cpm of
PlaqueC size 37" wt S wt wt wt wt wt wt wt
L L wt S
wt L wt S S
wt L L wt wt S
wt wt wt L L wt -
production of phos+ = wildnot deterto plaques larger
than
thymidineJ4C were rapidly added to each tube without removing it from the bath. After a further 1.75 hours’ incubation, the cells were chilled and washed at O”, twice with 2 ml PBS, twice with 2 ml 10 % tri-
CLASSIFICATION
OF
POLIOVIRUS
chloroacetic acid, and once with 1% acetic acid. The residues were dried, dissolved in 0.2 ml of 0.2 N NaOH, transferred to planchettes, and counted in an end-window counter with an efficiency of about 7 %. E$ect of virus mu incorporation of 32P into cellular RNA. Freshly harvested U cells were incubated at 36” overnight at 5 X lo5 cells/ml in a spinner culture containing Eagle’s medium plus 10 % calf serum and 0.7 mg NaHCO&l, then chilled and resuspended to lo7 cells/ml in normal growth medium, or in volumes of ts+ or mutant stocks equivalent to 5-10 PFU/cell. After 90 minutes’ rocking at 2”, the cells were washed twice in 0.14 M NaCl and abruptly added (giving 2.5 X lo6 cells/ml) to 50-ml Ehrlenmeyer flasks containing 10 ml of protein-free Eagle’s medium in which the phosphate had been replaced by 1 mM sodium citrate. The flasks, already immersed in a bath at 39.6 f 0.05”, were rotated to keep the cells in suspension. After 1.5 hours, orthophosphate-32P was added to each flask to 25 PC/ml, and after a further 2.5 hours the cells were chilled and washed twice. The RNA was then extracted and fractionated by sucrose density gradient centrifugation as described by Fenwick (1963). The efficiency of counting was 60 %. The RNA cont)ent is derived from the optical density, assuming that 1 pg/ml gives an optical density at 260 mp of 0.025. Isotopes. Thymidine-2-14C (25.3 mC/ mmole) was obtained from New England Nuclear Corporation, Boston, Massachusetts. Sodium phosphate (““P) injection B.P. was from the Radiochemical Centre, Amersham; its specific activity at the time of use wa’s 0.25-l mC/mg. RESULTS
ifhmmay of Properties Mutants
of Poliovirus
ts
A summary of all the present experimental results is given in Table 1, which will be referred to throughout this paper. Production of Viral Antigen Fluorescent Antibody
Reacting
with
Late in the normal growth cycle of poliovirus, material appears in the cytoplasm that
ts
641
MUTANTS
binds fluorescent poliovirus antibody (Buekley, 1956; Levy, 1961; Mayor, 1961). Its quantity and its site and time of synthesis suggest that this material is viral capsid protein. Mutants were examined for their production of such antigen by the procedure described in Materials and Methods, in which ring cultures of U cells were used. About 10 mutants were tested at one time, and each experiment included two uninfected cultures and two infected with ts+. Infected cells before incubation, and all uninfected cells, appeared normal and showed minimal background fluorescence. Cells infected with ts+ showed characteristic virus effects: after 3-4 hours at both 37” and 39.4” almost all cells showed a marked cytoplasmic fluorescence, which was most intense 5-7 hours after infection. The results of the tests are summarized in Table 1: they fell into two classes. The majority of the mutants (designated a) showed no fluorescence at 39.4”, infected and uninfected cells looking alike in this property. Three mutants (designated a+) produced amounts of fluorescence at 39.4” that were indistinguishable from that of wild type. All mutants produced a response at 37” that was the same as that of wild type. The three a+ mutants (ts-2, -3, and -8) were reexamined using inocula that were more dilute. The most dilute inocula infected only 10 % of the cells, but, as before, cells were either fully fluorescent or completely nonreactive, and the numbers of cells wit,h wild-type response were approximately equal at 37.0” and 39.4”. Thus the wild-type response was not due to a trace of wild-type revertant virus, nor to leak of the mutant in a small proportion of the virus-cell encounters. Prevention of Thymicline
Incorporation
Poliovirus prevents the net increase of cellular DNA and also the incorporation of labelled precursors into the DNA without initially affecting the integrity of the existing material (Goldfine et al., 1958; Salzman et al., 1959; Levy, 1961; Holland, 1962; Holland and Peterson, 1964). The onset of the prevention of DNA synthesis is detectable 3-4 hours after infection.
642
COOPER,
JOHNSON,
AND
GARWES
37.2”
2
4
6
0
2
4
6
HOURS
FIG. 1. Time course of incorporation of thymidine-14C by uninfected U cells (0) and by cells infected with poliovirus strains k-19 (0) and k-20 (A). The procedure is described in Materials and Methods; replicate tube cultures of U cells were infected at 4” and incubated in baths at 37.2” or 39.6” with medium containina thvmidine-14C. At the indicated times, cultures were chilled, treated with trichloroacetic acid and-assayed for IQ.
The ability of ts mutants to prevent cellular DNA synthesis was examined by means
at 39.6”, whereas the remainder (designated ptif) were not significantly different from wild type. The possibility that the pti character may have resulted from someheat defect in the initiation of infection is made unlikely by the wild-type response of these strains in five other tests (tests for pp&+, and pp&+, seebelow, and three tests not described in this paper). The similar responseof pti and pti+ strains at 37” shows that the pti strains are not normally defective in penetration.
Table 2 shows a typical experiment, which illustrates the way in which the data have been considered. The values for prevention of thymidine incorporation at 39.6” by pti mutants were each less than the mean of all values at 39.6” minus one standard deviation, and lessthan the mean of pti+ values at 39.6” minus three times their standard deviation. Analysis by means of the standard error of the difference, using either the origi-
CLASSIFICATIOX
OF
POLIOVIRUS
nal results in terms of counts per minute (cpm) or the pti values themselves, indicated t,hat the difference between the means of the ptif and the @i strains at 39.6” was very highly significant (P < 10-l’)). The same type of analysis also showed that the difference between the mean of all values at 37.2” and the mean of pti values at 37.2”, although not large, was nevertheless highly significant (P < 10p5). Thus the pti strains were also slightly defective at 37.2”. Prevention of Cellular RNA
643
ts MUTANTS
Figure 2 depicts two typical experiments. The optical densit.y readings show that the RNA has been separated into the three fractions that are presumed to correspond to 28 S ribosomal RNA (fraction I), to 16 S ribosomal RNA (fraction 2), and to the rapidly labelled RNA of 4-10 S (fraction 3) (Darnell et al., 1963). As expected, tsf grown at 39.6” almost eliminated net RNA synthesis in fractions 1 and 2. Synthesis of fraction 3 was reduced by 50 % ; this may reflect prevention of mRNA synt,hesis, but
Synthesis
Previous reports do not agree on the very early effects of poliovirus on cellular RNA synthesis. Ackermann and co-workers (Maassab et al., 1957; Ackermann et al., 1959) found a lasting stimulation of RNA synthesis, but quite marked although more transient stimulations (disappearing by 2-3 hours after infection) have been reported by Hydbn (1947), Miroff et al. (1957), Contreras et al. (1959), Levy (1961) and Toha et al. (1961). On the other hand, cellular RNA synthesis is often depressed (Goldfine et al., 1958; Salzman et al., 1959; Fenwick, 1963; Zimmerman et al., 1963; Holland, 1963; Holland and Peterson, 1964; Bablanian et al., 1965), in some cases quite markedly so by 1.5-3 hours after infection. Finally, in one experiment of Salzman et al. (1959), and in another of Zimmerman et al. (1963), no change in cellular RNA synthesis was detectable within the first 2-3 hours. Presumably the effect of poliovirus on cellular RNA synthesis is complex and depends upon the physiological state of the cells; there are other suggestions that this is the case (Munyon, 1964; Tershak, 1964; Cooper, 1966). However, most reports agree that poliovirus eventually decreases the net rate of cellular RNA synthesis. Preliminary tests, similar to those of Table 2 but with uridine 14C showed this effect, and revealed several muiants (namely ts-5, -23, -99, -123) with rather small defects. Since poliovirus affects at least three species of RNA (see references given above) the ts mutants were reexamined with a system in which t’he RNA species could be separated. This system involved U cells from a spinner culture, and is described in Materials and Met’hods.
TABLE EFFECT
OF TE~UPERATURE
CELLULAR THYMIDINE BY POLIOVIRUS
Strain
ts
PREVENTION
Prevention of thymidine incorporation (%)b 39.6”
None 5 20 23 81 99
0 56 65 50 52 51
ts+
75
2
78 56 78 70 80 54 81 63 75 65.6 11.3
0 13.8 3.9 0 18.4 20.4 79 71 53 77 G9 65 61 75 62 62 48.8 27.6
22 38 44 89 104 123 150 deviations
OF
INCORPORATIOS ts MUTANTS~
37.2”
8
Means Standard
2 ON THE
Character
pti pti pti pti
pti pti+ pti+ pti+ pti+ Qlif
pti+ pti+ pti+ pti+ pti+ -
a Four tube cultures of U cells were infected with each of the virus strains indicated and incubated in water baths at the given temperatures, as described in Materials and Methods. After 3.75 hours of incubation the cells were given a 1.75 hour pulse of thymidineJ4C then were chilled and washed with trichloroacetic acid and the isotope content was assayed. Uninfected culturer incorporated 396 and 391 cpm at 37.2”, and 427 and 455 cpm at 39.6”. b Equals the difference between the mean counts per minute (cpm) for each mutant and the mean uninfected cpm, expressed as a percentage of the mean uninfected cpm.
644
COOPER,
JOHNSON,
AND
GARWES
a
TUBE
NUMBER
FIG. 2. The effect of poliovirus infection on orthophosphate-s*P incorporation into the RNA of U cells. The procedure is described in Materials and Methods. Cells from a spinner culture were infected at 2” with ts+ or the mutants indicated, or were treated with normal medium (‘(uninfected”), then were abruptly raised to 39.6” and incubated in suspension. Radioactive phosphate was added after 1.5 hours’ incubation, and the RNA was extracted after a further 2.5 hours and fractionated by sucrose gradient centrifugation. Two similar experiments (a and b) are shown. In experiment a, actinomycin D was added at commencement of incubation to 2 pg/ml to one uninfected culture. Tube number one corresponds with the bottom of the sucrose gradient.
direct evidence for this is not available. Actinomycin D had an effect similar to that of ts+. Strains ts-2 and h-9 had a wild-type effect on all three fractions, whereas ts-18, -20, -22, -28, and -99 were markedly defective in their effect on fraction 3. All these mutants had a wild-type effect on fractions
1 and 2, except k-22, which was markedly defective in its effect on fraction 2. Table 3 shows the way in which the da&a have been considered. The mutants were compared in terms both of the total cpm per fraction, and of the specific activity of that fraction, but both methods gave the same
CLASSIFICATION
OF
TABLE EFFECT
RKA
fraction property
1. cpm m RNA cpm/e 2. cpm /.e RNA wm/!4 3. cpm I*g RNA cpm/e
OF POLIOVIRUS
and
ts
2,410 45.2 53.3 2,640 31.0 85.2 7,030 19.4 363
645
MUTANTS
3
MUTARTS ON 32P INCORPORATIOS FRACTIONS AT 39,60a Mutant
Uninfected
ts
POLIOVIRUS
strain
ts+
k-9
1,160 + 38.1 30.6+ 1,230 + 30.3 40.6+ 3,990 + 18.5 216 +
566 + 45.8 12.4+ 851 + 31.2 27.3+ 3,750 + 18.4 204 +
INTO
and character ts-20 682 f 42.5 16.0+ 1,080 + 30.2 35.7+ 5,730 14.2 404 -
CELLULAR
RNA
designationb ts-22 1,150 44.0 ZS.l+ 2,770 35.0 79.15,270 20.3 260
h-99 +
-
f
772 + 39.7 19.4+ 1,270 + 31.6 40.1+ 6,780 18.4 369 -
Q The procedure is described in Materials and Methods. U cells from a spinner culture were either infected at 2” with the virus strains or treated in parallel with normal medium, and were then incubated at 39.6” in suspension; orthophosphate-32P was added after 1.5 hours of incubation, and the RNA was extracted after a further 2.5 hours for fractionation by sucrose gradient centrifugation. This experiment is the same as that shown in Fig. 2b. 6 + = wild type, - = defective, and ZIZ = slightly defective.
HOURS FIG. 3. Temperature-shift -28. Replicate tube cultures time zero. At the indicated hours for assay of infectivity at intervals for determination
(“step-up”) experiments with poliovirus mutants k-5, -19, -22, and of U cells were identically infected at 2”, and immersed in a 37.0” bath at times, some tubes were transferred to a bath at 39.6”, and were frozen at 8 (“step-up” experiments, 0). The remainder of the 37” tubes were frozen of one-step growth at 37” (0).
646
COOPER,
JOHNSON,
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GARWES
results. In Table 3, all mutants have been scored as wild type in effect on fraction 1 and all except ts-22 as wild type in effect on fraction 2, but only ts-9 as wild type in effect on fraction 3. The results of all the tests are summarized in Table 1. A majority of the mutants (13/19, designated ppi3) behaved like ts-18, -20, -22, -28, and -99, the permitted incorporation of 32P into fraction 3 being nearer uninfected than t&infected values. The values for the remainder, (ts-2, -3, -8, -19, -37, and -89, designated pp&+) were close to those of wild type. Although k-9 was pp&+ in the experiment of Fig. 2, its response in the repeat experiment was less certainly wild t,ype and so it is designated as “not determined” for the time being. The wild-type effects on RNA of fractions 1 and 2 again provided controls to show that the defective actions on fraction 3 were unlikely to result from some deficiency in the initiation of infection. The wild-type effects on thymidine incorporation also provided similar controls in the majority of cases. Most mutants, designated ppiz+, gave a wild-type response for fraction 2, but four (designated ppi&) were somewhat defective. However, in the cases of ts-44, -46, and -63, this apparent defect was small and probably reflected contamination with fraction 3. The action of ts-22 on fraction 2 was markedly defective in two experiments (one of which is shown), but was not appreciably defective in a third experiment, and so its significance must await further work. All 20 mutants tested (designated ppG+) had a wild-type effect on RNA of fraction 1.
37” at intervals. Some of these tubes were left at 37” (“step-down” experiment) and some were replaced at 39.5” after 5 minutes at 37” (“pulse-of-release” experiment), but in both cases the tubes were frozen after 8 hours as before. Figures 3 and 4 show five typical experiments; the results of all experiments are summarized in Table 1. For 16 out of 17 mutants, growth was blocked in the step-up experiment if the temperature was increased at any time before maturation began (Fig.
Temperature
FIG. 4. Temperature shift (“step-up” and “pulse-of-release”) experiments with poliovirus mutant k-23. Replicate tube cultures of U cells were identically infected at 2” and immersed at time zero either in a bath at 37.0” or in another at 39.6”. Some tubes were transferred from the 37” to the 39.6” bath at the indicated times, and were frozen at 8 hours for assay of infectivity (step-up experiment, 0). The remainder of the 37” tubes were frozen at intervals for determination of onestep growth at 37” (0). The tubes initially incubated at 39.6” were transferred to the 37” bath at the indicated times, and after 5 minutes at 37” were replaced at 39.6” and frozen at 8 hours (pulseof-release experiment, A)
Xhift Experiments
Experiments were done to find the temperature-sensitive period of the growth cycle of some ts mutants. Replicate tube cultures were infected at 4” with 1 PFU per lo-100 cells, and were washed and immersed in a 37” bath at time zero. At intervals some tubes were frozen for assay of one-step growth, and others were transferred to a bath at 39.5” and frozen after a total of 8 hours’ incubation (“step-up” experiment). In some experiments replicate infected cultures incubated at 39.5” were transferred t.o
dL----22 0
12
3
4
5
6
7
CLASSIFICATION
OF
POLIOVIRUS
3). These mutants appear to have a critical period late in the cycle. The critical period for ts-23 seemed to be earlier (Fig. 4). The results of the step-up and step-down tests for any given mutant were identical, i.e., late or early rises in the step-up curves were mirrored by late or early falls in the stepdown curves, respectively. The step-down curves are not shown. The pulse-of-release experiments were done with strains k-9, -19, -23, -28, and -46; the one shown in Fig. 4 is typical of all. In contrast to the step-up and step-down experiments, the pulse-of-release experiments failed to show a critical period of the cycle for any mutant; this apparent paradox is considered in the Discussion. These results seem to contradict some of Lwoff (1962)) but the Lwoffs’ strain was not related to the present ones; it may have contained more than one heat defect, or its defective gene may differ from those of our mutant’s, and so no direct comparisons can be drawn. DISCUSSION
Those mutants defined in terms of the characters pti+, p&+, and a+ have been classified into the four groups shown in Table 1. Each mutant has unique properties and hence was independently derived. Group A mutants produced no detectable antigen and were defective in preventing synthesis both of DNA and of fraction 3 RNA. Group B mutants did not produce antigen and were defective in preventing fraction 3 RNA synthesis. Group C mutants did not produce antigen. Group D mutants gave wild-type responses in all three tests; they were t,he only mutants to produce detectable antigen under the restrictive conditions, and probably have defects in late stages of growth, such as maturation or assembly (D. McCahon 1966, unpublished data). The way in which the defective characters were distributed has some intrinsic interest. The characters pti+ and ppi3+ were not covariant and therefore reflect always distinct functions, but they were both defective in the five mutants of group A; it is most unlikely that all five were double mutants. Mutants of the class pti, pp&+ have not been
ts MUTANTS
647
found. Thus, in the limited number of mutants tested, a defect in pti+ was always associated with a defect, in ppi3+, but the converse was not true, so that the covariation between pti+ and ppi3+ was asymmetrical. An asymmetrical covariation also occurred between a+ and pp$+, and between a+ and pti+, so that these three characters and the four groups defined by them can be arranged in the matrix shown in the upper left-hand quadrant of Table 1. More mutants are needed to confirm the significance of this finding, but it seems likely that the temperature-sensitive mutations can have a pleiotropism that is polar in nature. Such polarity could explain the inefficient and asymmetrical nature of the complementation that occurs between k-5 and ts-19 (Cooper, 1965). Tessman (1965) describes a conditional lethal mutant of phage S13 that did not seem to be a double mutant but that was defective in two distinct gene functions. Asymmetrical covariation in the poliovirus system may result from a variety of causes, either genetic, or physiological, or both. Whatever the cause, however, one implication is that the mutants in each group of Table 1 are not necessarily allelic. Further work is therefore needed to determine the primary defect of these mutants. The temperature shift experiments were not useful for classifying the mutants, because similar results were obtained for all strains but one. Tessman (1965) found much the same for step-up experiments with phage S13. However, it should be said that temperature shift experiments on their own cannot show whether given functions are in fact “early” or “late.” This is because heatdefective gene products may (Horowitz and Fling, 1953) or may not be thermolabile, and such uncertainty strongly affects the interpretations of temperature shift experiments. Thus the mutated gene products of all the mutants (except b-23) may or may not be thermostable and therefore may or may not be made or function early in the cycle; one can only say that their function still helps to make new progeny at a time when maturation has already begun. On the other hand, the defective gene product of k-23 may be
648
COOPER,
JOHNSON,
relatively thermostable once it is made, and appears to be made throughout the cycle; it may or may not still be needed at a late time. In the pulse-of-release experiments, a brief release at any time increased the final yield somewhat, but no period was especially important to any mutant. Strain k-23 behaved exactly like the others in this respect. The simplest interpretation of the temperature shift data is probably that the defective gene products are all made throughout the cycle, that their continued functioning is needed for further replication even after some maturation has begun, but that some products are stable and others unstable at 39.6”. The prevention of cellular DNA synthesis and the prevention of (presumably) mRNA synthesis are shown to result from at least two distinct gene functions of poliovirus. Mutants of group C indicate at least one more function, whose defect is undefined. In addition, tests for prevention of synthesis of the two ribosomal RNA’s and for several characters not reported here such as prevention of cellular protein synthesis, have so far failed to reveal significantly defective mutants. These characters are therefore controlled by a gene or genes distinct from those controlling pti+ and pp&+, and from the gene(s) defective in group C mutants, bringing the minimum total of gene functions to four. Summers et al. (1965) have distinguished some four structural and ten nonstructural poliovirus proteins in the infected cell, and the present work provides physiologica evidence for the presence of at least four. Since several characters have not yet been tested, and since the presence of asymmetrical covariation may conceal several defects within one apparently homogeneous group of mutants, it is likely that evidence for more will be found. ACKNOWLEDGMENTS We are grateful to Dr. C. Mims for preparing the monkey antiserum, and to Mrs. M. Boyden for her reliable help. REFERENCES ACKERMANN, W. W., LOH, (1959). Studies of the
P. C., and PAYNE, F. E. biosynthesis of protein
AND
GARWES
and ribonucleic acid in HeLa cells infected wit1 poliovirus. Viiirology 7, 170-183. B~BLANIBN, R., EGGERS, H. J., and TAMM, I (1965). Studies on the mechanism of poliovirus. induced cell damage. I. The relation betweec poliovirus-induced metabolic and morphological alterations in cultured cells. Virology 26, lOO113. BUCKLEY, S. M. (1956). Visualization of polio myelitis virus by fluorescent antibody. Arch Ges. Vimsforsch. 6, 388-400. CAIRIXS, J. (1960). The initiation of vaccinia infec. tion. Virology 11, 603-623. COXTRERAS, G., ToH~, J., and OHLBALJM A. (1959). Preliminary study on the kinetics ol P3% incorporation into proteins and RNA oj HeLa cells infected with poliovirus. Biochim Biophys. Acta 35, 268-269. COOPER, P. D. (1961). An improved agar cell. suspension plaque assay for poliovirus: some factors affecting efficiency of plating. Virology 13, 153-157. COOPER, P. D. (1964). The mutation of poliovirus by 5-fluorouracil. Virology 22, 186-192. COOPER, P. D. (1965). Rescue of one phenotype in mixed infections with heat-defective mutants of type 1 poliovirus. Virology 25, 431-438. COOPER, P. D. (1966). The inhibition of poliovirus growth by actinomycin D and the prevention of the inhibition by pretreatment of the cells with serum or insulin. Virology 28, 663-678. COOPER, P. D., WILSON, J. N., and BURT, A. M. (1959). The bulk growth of animal cells in continuous suspension culture. J. Gen. Microbial. 21, 702-720. DARNELL, J. E., PENMAN, S., SCHERRER, K., and BECKER, Y. (1963). A description of various classes of RNA from HeLa cells. Cold Spring Harbor Symp. Quant. Biol. 28, 211-214. DULBECCO, R. (1960). Discussion in Second Inlern. Conf. Live Poliovirus Vaccines, W.H.O., Washington, pp. 4749. EPSTEIN, R. H., BOLLE, A., STEINBERG, C. M.: KELLENBERGER, E., BOY DE LA TOUR, E., CHEVALLEY, R., EDGAR, R. S., SUSMAN, M.; DENHARDT, G. H., and LIELAUSIS, A. (1963). Physiological studies of conditional lethal mutads of bacteriophage T4D. CoEd Spring Harbor Sywap. Qua&. Biol. 28, 375-394. FENWICK, M. L. (1963). The influence of poliovirus infection on RNA synthesis in mammalian cells. Virology 19, 241-249. GOLDFINE, H., KOPPELMAN, R., and EVANS, E. A. (1958). Nucleoside incorporation into HeLa cells infected with poliomyelitis virus. J. Biol. Chem. 232, 577-588. HOLLAND, J. J. (1962). Altered base ratios in RNA synthesized during enterovirus infection of
CLASSIFICATION
OF
POLIOVIRUS
human cells. Proc. Natl. Acad. Xci. U. S. 48, 2044-2051. HOLLAND, J. J. (1963). Depression of host-controlled RNA synthesis in human cells during poliovirus infection. Proc. Natl. Acad. Sci. U. X. 49, 23-28. HOLLAND, J. J., and PETERSON, J. A. (1964). Nucleic acid and protein synthesis during poliovirus infection of human cells. J. Mol. Biol. 8, 556-573. HOROWITZ, N. H., and FLING, M. (1953). Genetic determination of tyrosinase thermostability in Neurospora. Genetics 38, 360-374. HYD~N, H. (1947). The nucleoproteins in virus reproduction. Cold Spring Harbor Xymp. Quant. Biol. 12, 104-114. JOHNSON, R. T. (1964). The pathogenesis of herpesvirus encephalitis. I. Virus pathways to the nervous system of suckling mice demonstrated by fluorescent antibody staining. J. Exptl. Med. 119, 343-356. LEVY, H. B. (1961). Intracellular sites of poliovirus reproduction. Virology 15, 173-184. LWOFF, A. (1962). The thermosensitive critical event of the viral cycle. Cold Spring Harbor Symp. Quant. Biol. 27, 159-172. Maass~~, H. F., LOH, P. C., and ACKERMANN, W. W. (1957). Growth characteristics of poliovirus in HeLa cells: nucleic acid metabolism. J. Exptl. Med. 106, 641-648. MAYOR, H. D. (1961). Cytochemical and fluorescent antibody studies on the growth of poliovirus in tissue culture. Texas Rept. Biol. Med. 19, 106-122. MCBRIDE, W. D. (1962). Biological significance of poliovirus mutants of altered cystine requirement. Virology 18, 118-130. MIROFF, G., CORNATZER, W.E., and FISCHER, R.
ts >IUTANTS
649
G. (1957). Effect of poliomyelitis virus type 1 (Mahoney strain) on the phosphorus metabolism of t,he HeLa cell. J. Biol. Chem. 228, 22.5 262. MUNYON, W. (1964). Inhibition of poliovirus by Z$diaminopurine. Virology 22, 15-22. PAPAEVANGELOU, G. J., and YOUNGNER, J. S. (1961). Correlation between heat-resistance of polioviruses and other genetic markers. Proc. Sot. Exptl. Biol. Med. 108, 5055507. POHJANPELTO, P. (1961). Response of enteroviruses to cystine. Virology 15, 225-230. POHJANPELTO, P. and COOPER, P. D. (1965). Interference between polioviruses induced by strains that cannot multiply. Virology 25, 350-357. SALZMAN, N. P., LOCKART, R. Z., JR., and SEBRING, E. D. (1959). Alterations in the HeLa cell metabolism resulting from poliovirus infection. Virology 9, 244-259. SUMMERS, D.F., MAIZEL, J.V., JR., and D,4RNELL, J. E., JR. (1965). Evidence for virus-specific noncapsid proteins in poliovirus-infected HeLa cells. Proc. Natl. Acad. Sci. V. S. 54, 505-513. TERSHAH, D. R. (1964). Effect of 5-fluorouracil on poliovirus growth. Virology 24, 26%269. TESSIMAN, E. S. (1965). Complementation groups in phage S13. Virology 25, 303-321. ToH~, J., CONTRERBS, G., and OHLBAUM, A. (1961). Study of some biochemical changes related to poliovirus multiplication in HeLa cells. I. Kinetics of P32 incorporation into phosphoproteins and RNA and the inhibitory action of chloramphenicol. Biochim. Biophys. Acta 47, 158-163. ZIMMERMAN, E. F., HEETER, M., and DARNELL, J. E. (1963). RNA synthesis in poliovirusinfected cells. Virology 19, 400-408.
30, 638-649
Physiological
(1966)
Characterization sitive)
PETER Department
Poliovirus
D. COOPER, of Microbiology,
of Mutants:
RICHARD John
Heat-Defective
Curtin
Preliminary
T. JOHNSON; School of Medical Canberra, Australia
Accepted
July
(Temperature-SenClassification
AND
Research,
DAVID Australian
J. GARWES2 National
University,
18, 1966
Cells infected with ts mutants of poliovirus were incubated at temperatures (39.4”-39.6”) too high to support full production of infective virus, and were then examined for certain changes that are normally characteristic of virus growth. The changes reported here, which can be used as genetic characters of the virus, are denoted:-a+: the production of poliovirus antigen; pli f: the prevention of cellular incorporation of thymidine; ppilf, pp&+, pp&+: the prevention of a3P incorporation into, respectively, 28 S, 16 S or 4-10 S cellular RNA. The wild-type strain (is+) grew well at 39.6”; it also displayed all five characters at 39.6”, but most of the 29 mutants tested did not. Tests for the characters a+, pti+, and pp&+ revealed large temperature defects in many mutants, and were used to define four groups of mutants with the following combinations of characters: group A: a, ppip, pti; group B: a, ppia; group C: a; group D: all characters wild type. Other combinations were not found, and so these characters covary in an asymmetrical or polar fashion. Temperature shift experiments suggested that certain gene products are made and used throughout the growth cycle, although most appear to be associated with ‘late’ junctions. INTRODUCTION
Mutant strains of virus that cannot multiply under certain defined conditions (conditional lethal mutants) may be used to determine something of the number and function of the defective viral genes (Epstein et al., 1963). The temperature-sensitive (termed heat-defective, or hd) strains of poliovirus that were isolated for this purpose (Cooper, 1964) were induced by 5fluorouracil, and a complementation test was developed by which the mutants could be examined (Cooper, 1965); this examina1 Recipient of a Special Fellowship (BT 1009) from the National Institute of Neurological Disease and Blindness, U. S. Public Health Service. Present address: Division of Neurology, Cleveland Metropolitan General Hospital and Western Reserve University School of Medicine, Cleveland, Ohio. 2 Present address: Microbiological Research Establishment, Porton, Wills., England.
tion is still in progress. The present paper reports the results of a preliminary analysis of the temperature sensitive (ts) mutants by another method, namely in terms of the physiological defects that appear under the restrictive conditions. This paper embodies a change in terminology. The term “temperature-sensitive” (ts) as originally applied to poliovirus was used for strains whose virions were thermolabile (Dulbecco, 1960; Papaevangelou and Youngner, 1961; McBride, 1962). To avoid confusion, a different term (heat defective, or hd) was therefore coined for poliovirus strains whose growth was defective at higher temperatures (Cooper, 1964, 1965, 1966; Pohjanpelto and Cooper, 1965). However, the subsequent wide usage of “ts” for such strains of other viruses obliges us to discard "ha? and to use ‘Ys” instead. It must accordingly be noted that the ts poliovirus mutants referred to below and subsequently are formally analogous to the ts bacteriophage
CLASSIFICATION
OF
mutants of Epstein et al. (1963) and different from the ts poliovirus mutants of Dulbecco Papaevangelou and Youngner (196’3, (1961), and McBride (1962).
POLIOVIRUS
ts MUTANTS
639
mutants. Accordingly, some relatively leaky mutants (with plating efficiencies between 0.1 and 0.01) were included in case a stringent selection for nonleaky st,rains produced only double mutants. MATERIALS AND METHODS Infection of ring cultures and staining with Cell strain. Human amnion cells of strain Jluorescent antibody. Semiconfluent monoU (Pohjanpelto, 1961) were used throughout layers (2 X lo5 cells) were formed overnight and were routinely subcultured in medium in ring cultures (Cairns, 1960) placed on the CSV.6 (Cooper et al., 1959). ends of narrow microscope slides. The culVirus assay. Infectivity was assayed by tures were chilled at 4”, the medium was rethe agar cell-suspension plaque method moved, and 3 standard 0.02 ml drops of a (Cooper, 1961) using U cells. Assays at 39” cold undiluted virus stock (containing lo6 to or above were incubated in water baths 10’ PFU) or of normal medium were added (Cooper, 1965). to each of two cultures. After 90 minutes at Isolation of temperature-sensitive mutants. 4”, the inocula and the rings were removed Mutant strains ts-2 to -23 were among the and incubation was begun abruptly by 26 mutants isolated at a rate of 10% after deeply immersing the cells in 15 ml of growth of the wild-type poliovirus strain Eagle’s medium (pH 7.3) containing 1 mg hd+ (now termed ts+) in presence of 1 mM bovine plasma albumin per millilitre, but no 5-fluorouracil (Cooper, 1964). Mutants bicarbonate. This medium was held in ts-28 to -155 were isolated similarly except capped wide-necked tubes in water baths that a lower dose of mutagen (0.25 mM, maintained either at 37.0” or at 39.4”, both giving an isolation rate of 2-3 %) was used &0.05”. One of each pair of duplicate culto minimize the occurrence of double tures was incubated at each temperature. mutants. The two sets of mutants had a After 5.5 hours, the slides were washed twice similar distribution of physiological defects with PBS at 4” and dried under a fan in a and of efficiencies of plating. The mutagen room at 36”. They were then washed twice was employed to ensure t’hat most isolates with reagent grade acetone, dried, and were independently derived. The assay treated for 20 minutes with monkey antiplates of the stock subcultured from that serum, obtained from animals that had been grown in 5-Auorouracil were incubated for throat-painted twice at an interval of 10 2 days at 36”, then transferred to an incudays with type 1 Sabin vaccine virus. The bator at 40” for 2 days; small plaques were indirect method of staining, involving picked, grown up in tube cultures, and spotfluorescein-labelled goat anti-monkey ytested. Temperature-sensitive strains were globulin (Microbiological Associates, Beassayed at 37” and 39.2”; strains with an thesda, Maryland), was then used as deefficiency of plating at 39.2” of 0.1 or less scribed by Johnson (1964). were recloned twice and given two or three Effect of virus on cellular incorporation of one-step subcultures t’o prepare stocks of thymidineJ4C. Monolayers of 2 X lo5 U cells 30-60 ml which were stored in 3-ml aliquots formed on the base of flat-bottomed tubes at -70”. Stocks whose efficiency of plating were chilled to 2” and given 3 standard 0.02 at 39.2” had increased during this procedure ml drops, either of virus stocks diluted to 1 were rejected. The remainder ($$o of the to 2 X lo6 PFU per 3 drops, or of normal original isolates), plus 10 mutants recloned medium (Cooper, 1965). Four or six replicate and grown up by this procedure from the tubes were infected with each virus stock, first 26 isolates, comprised 29 mutants. Their and the replicates were divided into two efficiencies of plating at 39.2”, together with equal groups held in separate metal racks. some other properties, are given in Table 1 The cells were washed twice with PBS, and (see below). The mode of the efficiencies of 0.5 ml of Eagle’s medium (containing 1 mg plating at 39.2” (the maximum temperature of bovine plasma albumin and 0.28 mg of at which ts+ was unaffected) was about 0.1 NaHC03 per millilitre) was added to each for the original isolates of both sets of tube. The tubes were left unstoppered, and
640
COOPER,
JOHNSON,
AND
TABLE
1
SUMMARY OF RESULTS OF PHYSIOLOGICAL
TESTS
Character Group
Wild A
ts
type
+ 5 20 23 81 99 18 22 28 44 46 104 123 19 37 89 2 3 8
B
C
D
Unclassified
Proportion defectives
9 38 50 63 65 94 96 149 150 151 155 of
a+ 39.4"
P&f 39.6"
i+
+ + +
+ + + + + +
-I+ + + + + + + + + + + +
+
f
+j+
OF POLIOVIRUS
ppi1+ 39.6"
1 + + +
+ + +
ND + -I+ f + f : + + -I+ + i-
ND + + + + + + + + + + -I+ + -I+
Late Late ND ND ND Late ND Late Late ND Late -
0.001 0.001 0.05 0.1 v.d. 0.1
ND -
I?& ND ND ND ND ND ND
-I+ ND -IND ND ND ND ND ND ND
24/27
13/19
5/28
4/20
o/20
-
e.o.p.b 39.2" 1 0.001 0.001 d. 0.001
+ f + -If f f + + ND +
f -IND
Critical period of cycle None Late Late Early Late ND ND Late Late ND Late Late ND Late Late Late ND ND ND
ND ND ND ND ND ND ND ND ND ND
ND
ts MUTANTS”
and test temperature PPiP’ 39.6"
$6"
GARWES
a The significance of the characters and the details of the tests are given in the text. a+ = of antigen; pti+, = prevention of incorporation of thymidine; pp&+, ppis+, pp&+ = prevention phorus incorporation into RNA fractions 1, 2, and 3, respectively. In the body of the table, type response; - = markedly defective; i = slightly but significantly defective; ND = mined. b Equals the efficiency of plating at 39.2”, expressed as a ratio of plaques formed at 39.2” formed in concurrent assays at 37”; d., v.d. = delayed, very delayed. c ‘(wt” = not usually distinguishable from wild-type; s = smaller than wild-type; L = wild-type.
the pH remained at about 7.3. At zero time the tubes were immersed in a bath, one rack at 37.0” f 0.05” and the duplicate rack at 39.6” f 0.05. After 3.75 hours’ incubation 2 standard drops containing 2500 cpm of
PlaqueC size 37" wt S wt wt wt wt wt wt wt
L L wt S
wt L wt S S
wt L L wt wt S
wt wt wt L L wt -
production of phos+ = wildnot deterto plaques larger
than
thymidineJ4C were rapidly added to each tube without removing it from the bath. After a further 1.75 hours’ incubation, the cells were chilled and washed at O”, twice with 2 ml PBS, twice with 2 ml 10 % tri-
CLASSIFICATION
OF
POLIOVIRUS
chloroacetic acid, and once with 1% acetic acid. The residues were dried, dissolved in 0.2 ml of 0.2 N NaOH, transferred to planchettes, and counted in an end-window counter with an efficiency of about 7 %. E$ect of virus mu incorporation of 32P into cellular RNA. Freshly harvested U cells were incubated at 36” overnight at 5 X lo5 cells/ml in a spinner culture containing Eagle’s medium plus 10 % calf serum and 0.7 mg NaHCO&l, then chilled and resuspended to lo7 cells/ml in normal growth medium, or in volumes of ts+ or mutant stocks equivalent to 5-10 PFU/cell. After 90 minutes’ rocking at 2”, the cells were washed twice in 0.14 M NaCl and abruptly added (giving 2.5 X lo6 cells/ml) to 50-ml Ehrlenmeyer flasks containing 10 ml of protein-free Eagle’s medium in which the phosphate had been replaced by 1 mM sodium citrate. The flasks, already immersed in a bath at 39.6 f 0.05”, were rotated to keep the cells in suspension. After 1.5 hours, orthophosphate-32P was added to each flask to 25 PC/ml, and after a further 2.5 hours the cells were chilled and washed twice. The RNA was then extracted and fractionated by sucrose density gradient centrifugation as described by Fenwick (1963). The efficiency of counting was 60 %. The RNA cont)ent is derived from the optical density, assuming that 1 pg/ml gives an optical density at 260 mp of 0.025. Isotopes. Thymidine-2-14C (25.3 mC/ mmole) was obtained from New England Nuclear Corporation, Boston, Massachusetts. Sodium phosphate (““P) injection B.P. was from the Radiochemical Centre, Amersham; its specific activity at the time of use wa’s 0.25-l mC/mg. RESULTS
ifhmmay of Properties Mutants
of Poliovirus
ts
A summary of all the present experimental results is given in Table 1, which will be referred to throughout this paper. Production of Viral Antigen Fluorescent Antibody
Reacting
with
Late in the normal growth cycle of poliovirus, material appears in the cytoplasm that
ts
641
MUTANTS
binds fluorescent poliovirus antibody (Buekley, 1956; Levy, 1961; Mayor, 1961). Its quantity and its site and time of synthesis suggest that this material is viral capsid protein. Mutants were examined for their production of such antigen by the procedure described in Materials and Methods, in which ring cultures of U cells were used. About 10 mutants were tested at one time, and each experiment included two uninfected cultures and two infected with ts+. Infected cells before incubation, and all uninfected cells, appeared normal and showed minimal background fluorescence. Cells infected with ts+ showed characteristic virus effects: after 3-4 hours at both 37” and 39.4” almost all cells showed a marked cytoplasmic fluorescence, which was most intense 5-7 hours after infection. The results of the tests are summarized in Table 1: they fell into two classes. The majority of the mutants (designated a) showed no fluorescence at 39.4”, infected and uninfected cells looking alike in this property. Three mutants (designated a+) produced amounts of fluorescence at 39.4” that were indistinguishable from that of wild type. All mutants produced a response at 37” that was the same as that of wild type. The three a+ mutants (ts-2, -3, and -8) were reexamined using inocula that were more dilute. The most dilute inocula infected only 10 % of the cells, but, as before, cells were either fully fluorescent or completely nonreactive, and the numbers of cells wit,h wild-type response were approximately equal at 37.0” and 39.4”. Thus the wild-type response was not due to a trace of wild-type revertant virus, nor to leak of the mutant in a small proportion of the virus-cell encounters. Prevention of Thymicline
Incorporation
Poliovirus prevents the net increase of cellular DNA and also the incorporation of labelled precursors into the DNA without initially affecting the integrity of the existing material (Goldfine et al., 1958; Salzman et al., 1959; Levy, 1961; Holland, 1962; Holland and Peterson, 1964). The onset of the prevention of DNA synthesis is detectable 3-4 hours after infection.
642
COOPER,
JOHNSON,
AND
GARWES
37.2”
2
4
6
0
2
4
6
HOURS
FIG. 1. Time course of incorporation of thymidine-14C by uninfected U cells (0) and by cells infected with poliovirus strains k-19 (0) and k-20 (A). The procedure is described in Materials and Methods; replicate tube cultures of U cells were infected at 4” and incubated in baths at 37.2” or 39.6” with medium containina thvmidine-14C. At the indicated times, cultures were chilled, treated with trichloroacetic acid and-assayed for IQ.
The ability of ts mutants to prevent cellular DNA synthesis was examined by means
at 39.6”, whereas the remainder (designated ptif) were not significantly different from wild type. The possibility that the pti character may have resulted from someheat defect in the initiation of infection is made unlikely by the wild-type response of these strains in five other tests (tests for pp&+, and pp&+, seebelow, and three tests not described in this paper). The similar responseof pti and pti+ strains at 37” shows that the pti strains are not normally defective in penetration.
Table 2 shows a typical experiment, which illustrates the way in which the data have been considered. The values for prevention of thymidine incorporation at 39.6” by pti mutants were each less than the mean of all values at 39.6” minus one standard deviation, and lessthan the mean of pti+ values at 39.6” minus three times their standard deviation. Analysis by means of the standard error of the difference, using either the origi-
CLASSIFICATIOX
OF
POLIOVIRUS
nal results in terms of counts per minute (cpm) or the pti values themselves, indicated t,hat the difference between the means of the ptif and the @i strains at 39.6” was very highly significant (P < 10-l’)). The same type of analysis also showed that the difference between the mean of all values at 37.2” and the mean of pti values at 37.2”, although not large, was nevertheless highly significant (P < 10p5). Thus the pti strains were also slightly defective at 37.2”. Prevention of Cellular RNA
643
ts MUTANTS
Figure 2 depicts two typical experiments. The optical densit.y readings show that the RNA has been separated into the three fractions that are presumed to correspond to 28 S ribosomal RNA (fraction I), to 16 S ribosomal RNA (fraction 2), and to the rapidly labelled RNA of 4-10 S (fraction 3) (Darnell et al., 1963). As expected, tsf grown at 39.6” almost eliminated net RNA synthesis in fractions 1 and 2. Synthesis of fraction 3 was reduced by 50 % ; this may reflect prevention of mRNA synt,hesis, but
Synthesis
Previous reports do not agree on the very early effects of poliovirus on cellular RNA synthesis. Ackermann and co-workers (Maassab et al., 1957; Ackermann et al., 1959) found a lasting stimulation of RNA synthesis, but quite marked although more transient stimulations (disappearing by 2-3 hours after infection) have been reported by Hydbn (1947), Miroff et al. (1957), Contreras et al. (1959), Levy (1961) and Toha et al. (1961). On the other hand, cellular RNA synthesis is often depressed (Goldfine et al., 1958; Salzman et al., 1959; Fenwick, 1963; Zimmerman et al., 1963; Holland, 1963; Holland and Peterson, 1964; Bablanian et al., 1965), in some cases quite markedly so by 1.5-3 hours after infection. Finally, in one experiment of Salzman et al. (1959), and in another of Zimmerman et al. (1963), no change in cellular RNA synthesis was detectable within the first 2-3 hours. Presumably the effect of poliovirus on cellular RNA synthesis is complex and depends upon the physiological state of the cells; there are other suggestions that this is the case (Munyon, 1964; Tershak, 1964; Cooper, 1966). However, most reports agree that poliovirus eventually decreases the net rate of cellular RNA synthesis. Preliminary tests, similar to those of Table 2 but with uridine 14C showed this effect, and revealed several muiants (namely ts-5, -23, -99, -123) with rather small defects. Since poliovirus affects at least three species of RNA (see references given above) the ts mutants were reexamined with a system in which t’he RNA species could be separated. This system involved U cells from a spinner culture, and is described in Materials and Met’hods.
TABLE EFFECT
OF TE~UPERATURE
CELLULAR THYMIDINE BY POLIOVIRUS
Strain
ts
PREVENTION
Prevention of thymidine incorporation (%)b 39.6”
None 5 20 23 81 99
0 56 65 50 52 51
ts+
75
2
78 56 78 70 80 54 81 63 75 65.6 11.3
0 13.8 3.9 0 18.4 20.4 79 71 53 77 G9 65 61 75 62 62 48.8 27.6
22 38 44 89 104 123 150 deviations
OF
INCORPORATIOS ts MUTANTS~
37.2”
8
Means Standard
2 ON THE
Character
pti pti pti pti
pti pti+ pti+ pti+ pti+ Qlif
pti+ pti+ pti+ pti+ pti+ -
a Four tube cultures of U cells were infected with each of the virus strains indicated and incubated in water baths at the given temperatures, as described in Materials and Methods. After 3.75 hours of incubation the cells were given a 1.75 hour pulse of thymidineJ4C then were chilled and washed with trichloroacetic acid and the isotope content was assayed. Uninfected culturer incorporated 396 and 391 cpm at 37.2”, and 427 and 455 cpm at 39.6”. b Equals the difference between the mean counts per minute (cpm) for each mutant and the mean uninfected cpm, expressed as a percentage of the mean uninfected cpm.
644
COOPER,
JOHNSON,
AND
GARWES
a
TUBE
NUMBER
FIG. 2. The effect of poliovirus infection on orthophosphate-s*P incorporation into the RNA of U cells. The procedure is described in Materials and Methods. Cells from a spinner culture were infected at 2” with ts+ or the mutants indicated, or were treated with normal medium (‘(uninfected”), then were abruptly raised to 39.6” and incubated in suspension. Radioactive phosphate was added after 1.5 hours’ incubation, and the RNA was extracted after a further 2.5 hours and fractionated by sucrose gradient centrifugation. Two similar experiments (a and b) are shown. In experiment a, actinomycin D was added at commencement of incubation to 2 pg/ml to one uninfected culture. Tube number one corresponds with the bottom of the sucrose gradient.
direct evidence for this is not available. Actinomycin D had an effect similar to that of ts+. Strains ts-2 and h-9 had a wild-type effect on all three fractions, whereas ts-18, -20, -22, -28, and -99 were markedly defective in their effect on fraction 3. All these mutants had a wild-type effect on fractions
1 and 2, except k-22, which was markedly defective in its effect on fraction 2. Table 3 shows the way in which the da&a have been considered. The mutants were compared in terms both of the total cpm per fraction, and of the specific activity of that fraction, but both methods gave the same
CLASSIFICATION
OF
TABLE EFFECT
RKA
fraction property
1. cpm m RNA cpm/e 2. cpm /.e RNA wm/!4 3. cpm I*g RNA cpm/e
OF POLIOVIRUS
and
ts
2,410 45.2 53.3 2,640 31.0 85.2 7,030 19.4 363
645
MUTANTS
3
MUTARTS ON 32P INCORPORATIOS FRACTIONS AT 39,60a Mutant
Uninfected
ts
POLIOVIRUS
strain
ts+
k-9
1,160 + 38.1 30.6+ 1,230 + 30.3 40.6+ 3,990 + 18.5 216 +
566 + 45.8 12.4+ 851 + 31.2 27.3+ 3,750 + 18.4 204 +
INTO
and character ts-20 682 f 42.5 16.0+ 1,080 + 30.2 35.7+ 5,730 14.2 404 -
CELLULAR
RNA
designationb ts-22 1,150 44.0 ZS.l+ 2,770 35.0 79.15,270 20.3 260
h-99 +
-
f
772 + 39.7 19.4+ 1,270 + 31.6 40.1+ 6,780 18.4 369 -
Q The procedure is described in Materials and Methods. U cells from a spinner culture were either infected at 2” with the virus strains or treated in parallel with normal medium, and were then incubated at 39.6” in suspension; orthophosphate-32P was added after 1.5 hours of incubation, and the RNA was extracted after a further 2.5 hours for fractionation by sucrose gradient centrifugation. This experiment is the same as that shown in Fig. 2b. 6 + = wild type, - = defective, and ZIZ = slightly defective.
HOURS FIG. 3. Temperature-shift -28. Replicate tube cultures time zero. At the indicated hours for assay of infectivity at intervals for determination
(“step-up”) experiments with poliovirus mutants k-5, -19, -22, and of U cells were identically infected at 2”, and immersed in a 37.0” bath at times, some tubes were transferred to a bath at 39.6”, and were frozen at 8 (“step-up” experiments, 0). The remainder of the 37” tubes were frozen of one-step growth at 37” (0).
646
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results. In Table 3, all mutants have been scored as wild type in effect on fraction 1 and all except ts-22 as wild type in effect on fraction 2, but only ts-9 as wild type in effect on fraction 3. The results of all the tests are summarized in Table 1. A majority of the mutants (13/19, designated ppi3) behaved like ts-18, -20, -22, -28, and -99, the permitted incorporation of 32P into fraction 3 being nearer uninfected than t&infected values. The values for the remainder, (ts-2, -3, -8, -19, -37, and -89, designated pp&+) were close to those of wild type. Although k-9 was pp&+ in the experiment of Fig. 2, its response in the repeat experiment was less certainly wild t,ype and so it is designated as “not determined” for the time being. The wild-type effects on RNA of fractions 1 and 2 again provided controls to show that the defective actions on fraction 3 were unlikely to result from some deficiency in the initiation of infection. The wild-type effects on thymidine incorporation also provided similar controls in the majority of cases. Most mutants, designated ppiz+, gave a wild-type response for fraction 2, but four (designated ppi&) were somewhat defective. However, in the cases of ts-44, -46, and -63, this apparent defect was small and probably reflected contamination with fraction 3. The action of ts-22 on fraction 2 was markedly defective in two experiments (one of which is shown), but was not appreciably defective in a third experiment, and so its significance must await further work. All 20 mutants tested (designated ppG+) had a wild-type effect on RNA of fraction 1.
37” at intervals. Some of these tubes were left at 37” (“step-down” experiment) and some were replaced at 39.5” after 5 minutes at 37” (“pulse-of-release” experiment), but in both cases the tubes were frozen after 8 hours as before. Figures 3 and 4 show five typical experiments; the results of all experiments are summarized in Table 1. For 16 out of 17 mutants, growth was blocked in the step-up experiment if the temperature was increased at any time before maturation began (Fig.
Temperature
FIG. 4. Temperature shift (“step-up” and “pulse-of-release”) experiments with poliovirus mutant k-23. Replicate tube cultures of U cells were identically infected at 2” and immersed at time zero either in a bath at 37.0” or in another at 39.6”. Some tubes were transferred from the 37” to the 39.6” bath at the indicated times, and were frozen at 8 hours for assay of infectivity (step-up experiment, 0). The remainder of the 37” tubes were frozen at intervals for determination of onestep growth at 37” (0). The tubes initially incubated at 39.6” were transferred to the 37” bath at the indicated times, and after 5 minutes at 37” were replaced at 39.6” and frozen at 8 hours (pulseof-release experiment, A)
Xhift Experiments
Experiments were done to find the temperature-sensitive period of the growth cycle of some ts mutants. Replicate tube cultures were infected at 4” with 1 PFU per lo-100 cells, and were washed and immersed in a 37” bath at time zero. At intervals some tubes were frozen for assay of one-step growth, and others were transferred to a bath at 39.5” and frozen after a total of 8 hours’ incubation (“step-up” experiment). In some experiments replicate infected cultures incubated at 39.5” were transferred t.o
dL----22 0
12
3
4
5
6
7
CLASSIFICATION
OF
POLIOVIRUS
3). These mutants appear to have a critical period late in the cycle. The critical period for ts-23 seemed to be earlier (Fig. 4). The results of the step-up and step-down tests for any given mutant were identical, i.e., late or early rises in the step-up curves were mirrored by late or early falls in the stepdown curves, respectively. The step-down curves are not shown. The pulse-of-release experiments were done with strains k-9, -19, -23, -28, and -46; the one shown in Fig. 4 is typical of all. In contrast to the step-up and step-down experiments, the pulse-of-release experiments failed to show a critical period of the cycle for any mutant; this apparent paradox is considered in the Discussion. These results seem to contradict some of Lwoff (1962)) but the Lwoffs’ strain was not related to the present ones; it may have contained more than one heat defect, or its defective gene may differ from those of our mutant’s, and so no direct comparisons can be drawn. DISCUSSION
Those mutants defined in terms of the characters pti+, p&+, and a+ have been classified into the four groups shown in Table 1. Each mutant has unique properties and hence was independently derived. Group A mutants produced no detectable antigen and were defective in preventing synthesis both of DNA and of fraction 3 RNA. Group B mutants did not produce antigen and were defective in preventing fraction 3 RNA synthesis. Group C mutants did not produce antigen. Group D mutants gave wild-type responses in all three tests; they were t,he only mutants to produce detectable antigen under the restrictive conditions, and probably have defects in late stages of growth, such as maturation or assembly (D. McCahon 1966, unpublished data). The way in which the defective characters were distributed has some intrinsic interest. The characters pti+ and ppi3+ were not covariant and therefore reflect always distinct functions, but they were both defective in the five mutants of group A; it is most unlikely that all five were double mutants. Mutants of the class pti, pp&+ have not been
ts MUTANTS
647
found. Thus, in the limited number of mutants tested, a defect in pti+ was always associated with a defect, in ppi3+, but the converse was not true, so that the covariation between pti+ and ppi3+ was asymmetrical. An asymmetrical covariation also occurred between a+ and pp$+, and between a+ and pti+, so that these three characters and the four groups defined by them can be arranged in the matrix shown in the upper left-hand quadrant of Table 1. More mutants are needed to confirm the significance of this finding, but it seems likely that the temperature-sensitive mutations can have a pleiotropism that is polar in nature. Such polarity could explain the inefficient and asymmetrical nature of the complementation that occurs between k-5 and ts-19 (Cooper, 1965). Tessman (1965) describes a conditional lethal mutant of phage S13 that did not seem to be a double mutant but that was defective in two distinct gene functions. Asymmetrical covariation in the poliovirus system may result from a variety of causes, either genetic, or physiological, or both. Whatever the cause, however, one implication is that the mutants in each group of Table 1 are not necessarily allelic. Further work is therefore needed to determine the primary defect of these mutants. The temperature shift experiments were not useful for classifying the mutants, because similar results were obtained for all strains but one. Tessman (1965) found much the same for step-up experiments with phage S13. However, it should be said that temperature shift experiments on their own cannot show whether given functions are in fact “early” or “late.” This is because heatdefective gene products may (Horowitz and Fling, 1953) or may not be thermolabile, and such uncertainty strongly affects the interpretations of temperature shift experiments. Thus the mutated gene products of all the mutants (except b-23) may or may not be thermostable and therefore may or may not be made or function early in the cycle; one can only say that their function still helps to make new progeny at a time when maturation has already begun. On the other hand, the defective gene product of k-23 may be
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relatively thermostable once it is made, and appears to be made throughout the cycle; it may or may not still be needed at a late time. In the pulse-of-release experiments, a brief release at any time increased the final yield somewhat, but no period was especially important to any mutant. Strain k-23 behaved exactly like the others in this respect. The simplest interpretation of the temperature shift data is probably that the defective gene products are all made throughout the cycle, that their continued functioning is needed for further replication even after some maturation has begun, but that some products are stable and others unstable at 39.6”. The prevention of cellular DNA synthesis and the prevention of (presumably) mRNA synthesis are shown to result from at least two distinct gene functions of poliovirus. Mutants of group C indicate at least one more function, whose defect is undefined. In addition, tests for prevention of synthesis of the two ribosomal RNA’s and for several characters not reported here such as prevention of cellular protein synthesis, have so far failed to reveal significantly defective mutants. These characters are therefore controlled by a gene or genes distinct from those controlling pti+ and pp&+, and from the gene(s) defective in group C mutants, bringing the minimum total of gene functions to four. Summers et al. (1965) have distinguished some four structural and ten nonstructural poliovirus proteins in the infected cell, and the present work provides physiologica evidence for the presence of at least four. Since several characters have not yet been tested, and since the presence of asymmetrical covariation may conceal several defects within one apparently homogeneous group of mutants, it is likely that evidence for more will be found. ACKNOWLEDGMENTS We are grateful to Dr. C. Mims for preparing the monkey antiserum, and to Mrs. M. Boyden for her reliable help. REFERENCES ACKERMANN, W. W., LOH, (1959). Studies of the
P. C., and PAYNE, F. E. biosynthesis of protein
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human cells. Proc. Natl. Acad. Xci. U. S. 48, 2044-2051. HOLLAND, J. J. (1963). Depression of host-controlled RNA synthesis in human cells during poliovirus infection. Proc. Natl. Acad. Sci. U. X. 49, 23-28. HOLLAND, J. J., and PETERSON, J. A. (1964). Nucleic acid and protein synthesis during poliovirus infection of human cells. J. Mol. Biol. 8, 556-573. HOROWITZ, N. H., and FLING, M. (1953). Genetic determination of tyrosinase thermostability in Neurospora. Genetics 38, 360-374. HYD~N, H. (1947). The nucleoproteins in virus reproduction. Cold Spring Harbor Xymp. Quant. Biol. 12, 104-114. JOHNSON, R. T. (1964). The pathogenesis of herpesvirus encephalitis. I. Virus pathways to the nervous system of suckling mice demonstrated by fluorescent antibody staining. J. Exptl. Med. 119, 343-356. LEVY, H. B. (1961). Intracellular sites of poliovirus reproduction. Virology 15, 173-184. LWOFF, A. (1962). The thermosensitive critical event of the viral cycle. Cold Spring Harbor Symp. Quant. Biol. 27, 159-172. Maass~~, H. F., LOH, P. C., and ACKERMANN, W. W. (1957). Growth characteristics of poliovirus in HeLa cells: nucleic acid metabolism. J. Exptl. Med. 106, 641-648. MAYOR, H. D. (1961). Cytochemical and fluorescent antibody studies on the growth of poliovirus in tissue culture. Texas Rept. Biol. Med. 19, 106-122. MCBRIDE, W. D. (1962). Biological significance of poliovirus mutants of altered cystine requirement. Virology 18, 118-130. MIROFF, G., CORNATZER, W.E., and FISCHER, R.
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G. (1957). Effect of poliomyelitis virus type 1 (Mahoney strain) on the phosphorus metabolism of t,he HeLa cell. J. Biol. Chem. 228, 22.5 262. MUNYON, W. (1964). Inhibition of poliovirus by Z$diaminopurine. Virology 22, 15-22. PAPAEVANGELOU, G. J., and YOUNGNER, J. S. (1961). Correlation between heat-resistance of polioviruses and other genetic markers. Proc. Sot. Exptl. Biol. Med. 108, 5055507. POHJANPELTO, P. (1961). Response of enteroviruses to cystine. Virology 15, 225-230. POHJANPELTO, P. and COOPER, P. D. (1965). Interference between polioviruses induced by strains that cannot multiply. Virology 25, 350-357. SALZMAN, N. P., LOCKART, R. Z., JR., and SEBRING, E. D. (1959). Alterations in the HeLa cell metabolism resulting from poliovirus infection. Virology 9, 244-259. SUMMERS, D.F., MAIZEL, J.V., JR., and D,4RNELL, J. E., JR. (1965). Evidence for virus-specific noncapsid proteins in poliovirus-infected HeLa cells. Proc. Natl. Acad. Sci. V. S. 54, 505-513. TERSHAH, D. R. (1964). Effect of 5-fluorouracil on poliovirus growth. Virology 24, 26%269. TESSIMAN, E. S. (1965). Complementation groups in phage S13. Virology 25, 303-321. ToH~, J., CONTRERBS, G., and OHLBAUM, A. (1961). Study of some biochemical changes related to poliovirus multiplication in HeLa cells. I. Kinetics of P32 incorporation into phosphoproteins and RNA and the inhibitory action of chloramphenicol. Biochim. Biophys. Acta 47, 158-163. ZIMMERMAN, E. F., HEETER, M., and DARNELL, J. E. (1963). RNA synthesis in poliovirusinfected cells. Virology 19, 400-408.