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Complementation between temperature-sensitive mutants of Sindbis virus
VIROLOGY
30, 214-223 (1966)
Complementation
Between
Temperature-sensitive
Mutants
of
Sindbis
Virus’ BOYCE W. BURGE” Department
of Bacteriology
and
Immunology,
E. R. PFEFFERKORN
AND
Harvard
Medical
School,
Boston,
Massachusetts 0.2116
June 1, 1966
Accepted
Eleven temperature-sensitive mutants of Sindbis virus were classified according to their ability to complement other mutants in pairwise mixed infections at a nonpermissive temperature. Five nonoverlapping complementation groups were thus defined. In two groups the temperature-sensitive function is required for viral RNA synthesis, while mutants of the other three groups are unable to carry out some essential step occurring after viral RNA synthesis. The progeny produced by complementation were found, in general, to include both the genomes used to initiate the mixed infection; these progeny particles were phenotypically mixed in a manner that suggests exclusion of mutationally altered protein from rescued particles.
RNA synthesis (RNA+). We have examined six RNA+ mutants and five randomly chosen RNA- mutants for their ability to complement in pairwise mixed infections. A nonoverlapping pattern of complementation is observed, and five complementation groups have been defined.
INTRODUCTION
When temperature-sensitive conditionallethal mutants of Sindbis virus (Burge and Pfefferkorn, 1966) are propagated at a nonpermissive temperature, certain pairs produce a much higher yield of virus from mixed infection than either mutant produces alone. This ability of each mutant to provide the function defective in the other allows classification
of mutants
into
discrete
functional
MATERIALS
or
complementation groups (Fincham, 1966). Thus, for a given population of mutants, a complementation group is defined as a set of mutants that do not complement each other but that do complement all other mutants in the population. In the preceding article of this issue (Burge and Pfefferkorn 1966) we described two classes of mutants: those blocked in viral RNA synthesis at nonpermissive temperatures (RNA-) and those blocked in some maturational event occurring after viral 1 This investigation was supported in whole by United States Public Health Service research grants AI-04531-04 and -05 from the Institute of Allergy and Infectious Diseases. 2 Supported by a predoctoral fellowship from the U. S. Public Health Service, number l-FlGM-31, 037-01.
AND
METHODS
Tissue culture and virological techniques are described in the preceding paper (Burge and Pfefferkorn, 1966). Temperature-sensitive conditional lethal mutants of Sindbis virus are designated ts-1, ts-2, etc. The heatstable strain from which all these mutants were derived is designated HR (Burge and Pfefferkorn, 1966). Multiplicities of infection were determined from the number of PFU adsorbed and the number of cells per monolayer. Replicate monolayers were dispersed with trypsin and the cells were counted in a hemocytometer. The temperature and time used for the adsorption step of routine titrations (Pfefferkorn and Hunter, 1963) were different from those used in complementation experiments. Therefore, the number of PFU adsorbed per monolayer was calculated from the number of plaques produced by suitable dilutions of
214
COMPLEMENTATION
WITH
the viral stocks after adsorption under the conditions used in studying complementation. RESULTS
Procedures for Measuring Complementation Mixed infections with pairs of conditionallethal mutants were begun by allowing approximately equal multiplicities of each parent (about 10 PFU/cell) to adsorb to precooled monolayers. After 45 minutes at 4’, the monolayers were rinsed with warm medium and were incubated at a nonpermissive temperature, usually 39”, for 4 hours. The monolayers were then drained and rinsed with prewarmed medium to remove uneclipsed parental virus. The monolayers were incubated for 2 more hours at the nonpermissive temperature, and progeny produced during this period was measured by plaque titration at 27”. We should like to emphasize here the necessity for washing the infected monolayers after several hours’ incubation at the nonpermissive temperature. Uneclipsed parental particles that cannot be removed by washing immediately after adsorption are eluted during the first few hours of infection. Similar behavior of Western equine encephalitis virus has been reported by Dulbecco and Vogt (1954). The presence of these eluted virus particles which do not represent viral growth can partially or completely mask the occurrence of complementation. Failure to recognize this factor accounts for the very low levels of complementation reported earlier (Burge and Pfeff erkorn, 1965). The yield produced in double infections between 4 and 6 hours was divided by the sum of the yields produced by the 2 mutants grown separately, under identical conditions at the same time. The resulting figure, called here the “complementation level,” is a numerical expression of the extent of complementation. Values of 2 or greater are considered positive results, whereas values of 1 or less are interpreted as absence of complementation. With complementing pairs of mutants the “complementation level” assumes a value between 2- and 300-fold. This value depends less on the yield from the double infection,
SINDBIS
MUTANTS
215
which is similar for all complementing pairs, than on the “background” produced by the two parents grown separately. This “background” is determined by mutant specific properties, such as reversion frequency and leakiness (Burge and Pfefferkorn, 1966)) and in consequence the complementation level for a given pair of mutants is also determined by mutant-specific properties. Noncomplementing pairs of mutants produce complementation levels of 1 or less. Values less than 1 may indicate interference by one mutant with growth of the other at nonpermissive temperatures. Manipulatable parameters such as multiplicity of infection and temperature of incubation during the mixed infection also determine the level of complementation. These latter parameters were examined with a pair of RNA+ mutants (ts-5 and ts-lo), chosen because of their ability to produce a high complementation level. Effect of Temperature on Complementation When compared with their HR ancestor, conditional-lethal mutants are markedly defective in growth at all temperatures between 35” and 41’ (Burge and Pfefferkorn, 1966). Thus, complementation might be expected to occur at all temperatures in this range, since the growth of each mutant is limited by its unique temperature-sensitive defect. Four identical sets of monolayers were infected with mutants ts-5, ts-10, or both ts-5 and ts-10, as described above, and each set of infected monolayers was incubated at a different temperature in the 35’-41” range. The yield of virus produced between hours 4 and 6 of infection is plotted in Fig. 1. Although the yields in both single and double infections are reduced by higher incubation temperatures, the complementation level remains high at all but the highest temperature examined, 41”. Experiments with other mutants have given similar results. It appears that complementation can be detected over the considerable temperature range in which both mutants are markedly defective in growth and the parental virus is able to grow well. Temperatures which limit the growth of the parental HR virus also limit the production of diffusible gene products by
BURGE
AND
PFEFFERKORN TABLE EFFECT
OF MULTIPLICITY
1 ON COMPLEMENTATION~:
RNA+ X Rh'A+ Multiplicity of infection (PFU/cell)
ts-5 12
3 0.75 0.16
Incubation
Temperature (“Cl
FIG. 1. Complement&ion yield as a function of temperature. The dashed line represents the sum of the two single infections. Above the solid line which plots the complementation yield [(>, (ts-5 Xts-lo)] is listed the complementation level at each temperature.
the mutants, and thus reduce the efficiency of complementation. E$ect of Multiplicity
o-n Complementation
One cannot confidently predict the effect of multiplicity of infection on the efficiency of complementation between virus mutants in animal cells. It is known that recombination between certain animal virus mutants can be easily detected only when clumps of virus are used to initiate mixed infection, thus assuring the proximity of the two mutants during the early stages of infection (Abel, 1962; Kirvaitis and Simon, 1965). Since chick fibroblasts have a volume lo3 times that of Escherichiu coli cells, in which most observations on viral complementation have been made, it might be supposed that a high multiplicity of infection would be required to produce intracellular proximity of the conditional-lethal mutants during growth and thus ensure successful complementation. The experiment recorded in Table 1 shows
k-10 2.8 0.7 0.18
0.045
Level Percentage of cells mixedly infected”
94 48 8.5 0.85
Yield due to complementation”
3.2 1.8
4.0 1.0
X x x x
10’ 106 105 106
,&plementation 80 55 87 38
0 Complementation was performed as described in the text, and multiplicities were determined by the procedure outlined in Materials and Methods. b The percentage of mixedly infected cells is (1 - t?) (1 - e-u) 100, where 2 is the multiplicity of one mutant (PFU/cell), and y is the multiplicity of the other. c The yield due to complementation is the yield obtained in the double infection minus the sum of the yields of the parents grown separately at the same time.
that this is not so. Complementation between mutants ts-5 and ts-10 was performed with sets of identical monolayers, using a different multiplicity of each virus for each set. From the multiplicity of infection the fraction of mixedly infected cells was calculated by the Poisson distribution. Table 1 shows that the viral yield due to complementation is an approximate linear function of the fraction of mixedly infected cells, even when this fraction is very small. Thus a given cell produces about the same complementation yield whether infected with only one PFU of each mutant or with 10 PFU of each mutant. E$ect of Multiplicity on Complementaticm When One Parent is RNAKSince both ts-5 and ts-10 are RNA+, their genomes continue to replicate at nonpermissive temperatures and they presumably produce essentially normal quantities of gene products. Diffusion of these gene products through the cytoplasm of infected cells allows complementation. A different result is possible when one of the parents in a complementation experiment is RNA-, and thus
COMPLEMENTATION TABLE EFFECT
OF MULTIPLICITY
RNA~t$i$~,’
ts-17
Percentage of cells rnixedly infected
19 3.2 0.53 0.09
100 89 14 0.56
(PFU/cell) ts- 10 15.8 2.6 0.44 0.07 a All
details
as in Table
WITH
TABLE
x RNA+
x X x 3 x
EFFECT
RNA-
ts-10 X ts-17 Level of yield due to complecowlemenmentation tation 1.8 2.5 1.3
217
MUTANTS
2 ON COMPLEMENTATION’:
10’ lo6 105 103
270 108 11.7 5
1 footnotes.
possibly incapable of extensive production of gene products. A similar experiment was therefore performed in which an RNA+ mutant (ts-10) was crossed with an RNA- mutant (ts-17). In this case, the yield of virus produced by complementation was not a linear function of the number of mixedly infected cells (Table 2). Increased multiplicities of infection beyond those required to infect nearly all the cells resulted in much greater complementation yields. The critical factor in this experiment was suspected to be the multiplicity of the RNA- mutants. This suggestion was confirmed by an experiment in which the multiplicity of the RNA+ mutant was held constant at 4 while the multiplicity of the RNA- mutant was varied. Again the yield of virus from complementation was markedly dependent on multiplicity (Table 3). Complementation
SINDBIS
Groups
Of the 23 conditional-lethal mutants isolated (Burge and Pfefferkorn, 1966), 11 were chosen for studies of complementation. These included 5 randomly chosen RNAmutants and 6 of the 7 RNA+ mutants. The seventh RNA+ mutant, ts-9, failed to complement any other mutant. Since it had no detectable reversion frequency (Burge and Pfefferkorn, 1966) and was thus suspected of being a double mutant or deletion, it was omitted from classification. By the complementation criteria outlined above, these 11 mutants were resolved into 5 nonoverlapping complementation groups (Table 4). As these groupings imply, RNA-
3
OF MULTIPLICITY ON COMPLEMENTATION”: X FIXED MULTIPLICITY OF RNA+
Multiplicity of infection (PFU/cell)
Percentage of cells mixedly infected
ts-10 X ts-17 yield due to comdementation
ts-10
ts-17
4 4 4 4
6.5 1.1 .18 .03
98 66 16 3
a All
details
as in Table
3.5 8.8 6.0 3.5
TABLE
RNA phenotype&
A B C D E
RNARNARNA+ RNAf RNA+
0 RNA+ mutants at the nonpermissive mutants cannot.
complementation
106 106 104 103
61.5 47 6.5 1.5
4 GROUPS Mutants
in group
ts-4, ts-17, ts-19, ts-11 ts-2, ts-5, ts-13 ts-10, ts-23 ts-20 are
of
1 footnotes.
COMPLEMENTATION Group
x X X x
Level
ts-21
able to make viral temperature while
RNA
RNA-
mutants always complement RNA+ mutants. Because we could not do all pairwise crosses required for the definition of these 5 groups in a single experiment, the above results were accumulated from several experiments, two of which are described below and recorded in Tables 5 and 6. In the experiment of Table 5 each complementation group is represented by at least one mutant. The yield produced in each double infection is listed together with the complementation level (in parentheses) for that cross. Complementation yields in this experiment were l-2 % of the HR yield under identical conditions of growth. Table 6 records the results of double infections with all RNA- mutants, including the 4 mutants of group A and the single mutant of group B, ts-11. It should be noted that no complementation occurs in crosses between mutants of group A (the definition of a complementation group), but that all group A mutants complement ts-11 (an ex-
218
BURGE
ANI)
PFEFFERKORN
TABLE
5
COMPLEMENTATION" Com-
plementation group
Yield from M utant single infection (PFU/ml) A ts-4
A
ts-4
5.5 x 104
A
ts-17
2.2 x 105
B
ts-11
1.1 X 104
C
ts-5
1.5 x 105
D
ts-10
1.0 x 106
E
ts-20 HR
4.3 x 105 6.0 x 108
-
Yield (PFU/ml) in mixed infection with A ts-17
B ts-11
c ts-5
1.0 x 106 (0.4) -
4.5 x 105 (7) 7.7 x 106 (30) -
9.0 x 106 (56) 1.1 X 10’ (30) 8.0 x 106 (50)
D ts-10
E ts-20
4.0 x 106 3.0 x 106
(56)
(6.2)
3.0 x 106 (9.5) 6.0 X lo6 (55) 2.5 X 10’ (100) -
3.5 x 106 (5.4) 1.9 X 10’ (44) 3.4 X lo6 (5) 1.8 X 10’ (34) -
a Below the yield in mixed infections, in parentheses, is the complementation level which is defined as the yield from mixed infections divided by the sum of the yields of each parent grown separately. TABLE COMPLEMENTATION
Cofnplementatlon group
Mutant
6
BETWEEN
RNA-
MUTANTS"
Yield in mixed infection with
Yield from single (PFU/ml)
A ts.4
A ts-17
A ts-19
A ts-21
B ts-11
-
5.0 x 106 (0.4) -
3.3 x 105 (0.5) 5.6 X lo6 (0.4)
1.7 x 106 (1) 1.8 X 105 (0.08) 4.0 x 105
1.5 x 106 (7) 3.0 x 10’ (30) 5.7 x ia7 (1‘Jo) 5.2 x lo7 (35) -
infection
A
ts-4
2.0 x 106
A
ts-17
1.0 X 106
A
ts-19
4.5 x 106
A
ts-21
1.5 X 106
B
ts-11 HR
2.5 X lo4 1.0 X 109
(0.2) -
a Details as in Table 5 footnot,e.
ample of nonoverlapping complementation) . The yields in this experiment are slightly higher than those in Table 5, because a slightly lower incubation temperature was used (38”). Phenotypic Properties of Rescued Particles We have shown that efficient phenotypic mixing occurs during mixed infection with several related group A arboviruses (Burge and Pfeff erkorn, unpublished results). That is, the coat of a single progeny particle may contain structural units determined by each infecting virus. Complementation between 2
RNA+ mutants, with temperature-sensitive defects in different structural proteins, might be supposed to occur by a process akin to phenotypic mixing, in which normal proteins would appear in rescued particles while proteins of abnormal conformation would be excluded by the principles of self-assembly (Caspar, 1963). We have shown that RNA+ mutants generally are much more heat labile than the HR virus from which they were derived and have suggested that this property is largely a consequence of a conditionallethal mutation in a coat protein (Burge and Pfefferkorn, 1966). If this is true, the parti-
COMPLEMENTATION
WITH
SINDBIS
219
MUTANTS TABLE
IDENTIFICATION Complementation
b-5
(RNA+) 17 (RNA-) ts-10 (RNA’)
pair
x
7 OF GENOTYPES”
Complementation levelb
Ratio
of genotypes in progeny
ts-
31
X ts-
17
7 ts-10:2
100
10 ts-5:2
17 (RNA--) b-5 (RNA+) x ts-
13 ts-5:7
ts-17 ts-17 ts-10
10 (RNA+)
2.5 Minutes FIG. 2. Heat ts-10, HR virus, complementation.
5.0 at
SO0
inactivation curves for ts-5, and the progeny of ts-5 X ts-10
cles produced by complementation should be relatively heat stable. This prediction was tested by determining the heat stability of the progeny produced by complementation between mutants ts-5 (group C) and ts-10 (group D). Figure 2 shows that the progeny of the mixed infection were markedly more resistant to heating than either parent, and only slightly less heat resistant than the ancestral HR virus. Since 99.9 % of the complementation progeny were genotypically mutant (i.e., could not produce plaques at 39”) and were therefore not recombinants, the heat resistance gained during mixed infection appeared to have been only a phenotypic property. This conclusion was confirmed by determining the heat stability genotype of the viruses produced by complementation. Ten plaques produced by the survivors of 5 minutes’ heating provided the inocula for separate clones grown at 27”. These were all found to be very heat labile, proving that the heat stability was only a transient phenotypic property. The phenotypic properties of progeny from an RNA- X RNA+ complementation (ts-17 X ts-10) were also examined. In this
o Progeny of complementation were cloned and identified either by ability to stimulate uridine1% incorporation into actinomycin-treated monolayers at 39” (RNA+ X RNA-), or by their ability to complement known stocks of one or the other parent (RNA+ X RNA+). b Complementation level is the yield from mixed infections divided by the sum of t,he yields of each parent grown separately.
cross, the RNA- parent (ts-17) is heat stable, but the RNA+ parent (ts-10) is heat labile and is the majority genotype rescued by complementation. (See the nest section for a description of the identification of rescued genotype.) An artificial mixture of the 2 parental strains (ts-17 and ts-10) present in the proportion suggested by the ratio of rescued genotypes in complementation yields (Table 7) would produce a two-component inactivation curve on 60’ heating, as described by the dashed line in Fig. 3. The actual complementation yield showed only a single component, which was heat resistant. This result demonstrates that phenotypic heat resistance of “genotypically” heatlabile mutants is characteristic of RNA- X RNA+ complementation, as well as the RNA+ X RNA+ complementation described above. Genotypes of Rescued Particles Since recombination cannot be detected under our experimental conditions, each virion produced during complementation should be of one of the parental genotypes. We therefore determined in which proportions the two mutant genotypes were represented. With complementation between RNA+ and RNA- mutants this was done by
BURGE
220
AND
PFEFFE tants
KKORN is not
restricted
to the
RT\rA
strand
from which it is transcribed, but may also less frequently replicate RNA- genomes dur0
ing mixed
Absence of Detectable Recombination Conditional-Lethal Mutants
HR
& i-l: c $ 0 ?! ki 0 f?
IO
ts-17 ts-17
x ts-IO
2.0
3.0
-I
4.0
50 25 Minutes
5.0 at
infection.
60’
FIG. 3. Heat inactivation curves for ts-10, ts-17, HR virus, and the progeny of b-10 X ts-17 complementation. The dashed line represents the two-component inactivation curve expected for an artificial mixture of ts-10 and ts-17 in the proportions suggested by Table 7.
cloning the progeny and determing their ability to stimulate uptake of uridine-2J4C into actinomycin D-treated monolayers at, a nonpermissive temperature (Burge and Pfefferkorn, 1966). In a complementation between two RNA+ mutants, of course, all progeny will be able to stimulate uridine uptake. In this case, clones can be identified only by their ability to complement with known stocks of one or the other mutant. The results of genotype identification for 3 complementing pairs are listed in Table 7. In all cases both genomes appear. The RNA+ genome predominates in RNA+ X RNAinfections. Since the RNA+ genome is capable of extensive multiplication at the nonpermissive temperature, significant representation of the RNAgenome in the complementation progeny would not, be expected unless it, too, were replicated. Thus these results suggest that the functional RNA polymerase produced by RNA+ mu-
between
Since we were able to demonstrate that progeny produced by complementation retained the conditional-lethal character (i.e., inability to produce plaques at 39”), it was apparent that increased yields from double infections could not be explained in terms of the formation and outgrowth of recombinant genomes. However, since recombination has been reported with RNA animal virus mutants (reviewed by Fenner and Sambrook, 1964), experiments were carried out, to determine whether any recombination was occurring in mixed infections with Sindbis virus mutants. Various pairs of conditional-lethal mutants with low reversion frequencies were used to produce mixed infections at permissive temperatures (27”33”). The yields from such double infections were examined for an increase in the frequency of particles able to form plaques at 39” (i.e., particles that behave like recombinants). Although the progeny from similar double infections exhibited phenotypic mixing, proving that mixed inTABLE ABSENCE Mutant(s) used in infection
b-5 alone ts-10 alone ts-16 alone ts-5 x ts-10 ts-5 X ts-16 ts-10 X ts-16
8
OF RECOMBINATION~ Reversion
39” Titer (PFU/ml) 1.0 1.0 3.0 1.0 7.0 2.5
x x x x x x
108 103 103 103 102 lo3
27” Titer (PFU/ml) 9.0 8.0
x x
10’
1.2 1.2 1.0 1.0
x x x x
10s 108 10s 10s
107
frequency (39” titer/ 27” titer) 1.1 1.5 2.5 8.5 7.0 2.5
x 10-s x 10-6 X W6
x lo-6 x 10-e x lO+
a Monolayers were infected with about 10 PFU/cell of each mutant and incubated at 32”. After 2 hours monolyares were washed, and the progeny produced between 2 and 8 hours after infection were scored for ability to produce plaques at 27” and 39”. ts-5 and te-10 are RNA+ mutants, and thus defective in a cistron different from that of ts-16, an RNA- mutant.
COMPLEMENTATION
WITH
fections had been estabished (Burge and Pfeff erkorn, unpublished data), no increase in the frequency of revertant genotypes was observed. Since mutants with reversion frequencies of about 10e5 were used a recombination frequency of lo-4 would easily have been detected. Details of typical experiments are given in Table 8. While these experiments by no means rule out recombination in arboviruses, they establish that recombination cannot explan the observed complementation. DISCUSSION
One of the principal motives for investigating conditional lethal mutants is the hope that each complementation group will correspond to a specific biochemical defect in the sequence of infection, the identification of which will lead to a more precise understanding of discrete events in viral multiplication. At present we can discriminate mutants biochemically only by their ability, or inability, to produce viral RNA at nonpermissive temperatures. None of the RNA+ mutants produce noninfectious hemagglutinating particles at 39”. We plan to examine thin sections of mutant-infected cells with the expectation of finding aberrations in the normal morphological sequence of arbovirus infection described by Morgan et al. (1961). In the absence of biochemical or morphological defects which correspond to individual complementation groups, it is important to examine the evidence arising from complementation per se that our complementation groups correspond to viral cistrons. If each mutant in the population can be unambiguously assigned to one and only one such group, then the complementation pattern is said to be nonoverlapping. A nonoverlapping pattern observed with a sufficiently large number of mutants provides prima facie evidence that each complementation group corresponds to one cistron, all members of the group representing mutations of that cistron. When the protein product of a cistron is made up of two or more identical subunits another possibility exists, and mutants within that cistron may complement each other (Fincham and Pateman, 1957). Such
SINDBIS
MUTANTS
221
intracistronic complementation gives rise to an overlapping complementation pattern and, as a consequence, resolution of cistrons is made more difficult. We have found that 11 independent mutants (each characterized by independent origin and a unique combination of heat lability, reversion frequency, and leakiness of growth) fall into 5 complementation groups that do not overlap. Were intracistronic complementation occurring among these mutants, some overlapping might be expected. The complementation reported here represents only a few per cent of the yield of the ancestral HR virus at 39”. With temperature-sensitive mutants of bacteriophage T4, intercistronic complementation is very efficient (50% of the wild-type burst) whereas intracistronic complementation is much less efficient (usually < 10% of the wild-type burst) (Edgar et al., 1964). However, since both types of complementation in the T4-E. coli system are more efficient than the complementation we observe, few conclusions can be drawn. More can be gained, perhaps, by an examination of the complementation results for conditional-lethal mutants of RNA viruses. Cooper (1965) studied complementation between temperature-sensitive mutants of poliovirus in human amnion cells. Complementation in this system is quite inefficient. Although 31 independently isolated mutants were studied, the highest complementation levels recorded were less than 10, a value corresponding to 0.1% of the wild-type yield under identical conditions. Since a biochemical investigation revealed that these mutants differed in their temperature-sensitive defects, it appears that low efficiency is characteristic of intercistronic complementation with poliovirus. ValenGne et al. (1964) have studied complementation between conditional-lethal mutants of the RNA bacteriophage fz. These authors found that when two amber mutants of different cistrons mixedly infected a nonpermissive host, the burst was only l-3% as large as that of wild-type fz. This efficiency is similar to that observed for conditional-lethal mutants of Sindbis virus complementing in a cell 103 the size of E. coli;
222
BURGE
AND
thus low efficiency may simply be a characteristic of complementation between RNA virus mutant’s Additional evidence that we are studying intercistronic complementation comes from comparison of complementation yields produced by different combinations of the two RNA phenotypes. Complementation between RNA+ and RNA- mutants, which is certainly complementation between cistrons, produces yields no greater than the yields observed in RNA+ X RNA+ crosses, or RNA- X RNA- crosses. If the usual relation between kid of complementation and efficiciency holds here (that is, a much lower efficiency for intracistronic than for intercistronic complementation) then this complementation would appear to be intercistronic. It is also possible that the complementation observed occurs with a high efficiency, but in only a small proportion of mixedly infected cells. Examination of the virus yield of single cells will be used to test this suggestion. The complementation between RNA- mutants (Table 5) is especially interesting because it implies that two cistrons are required for the synthesis of viral RNA. Since there is no agreement at present as to the number of enzymes required for viral RNA synthesis (Spiegelman et al., 1965; Lodish and Zinder, 1966), and since it is not certain that bacterial and animal RNA viruses will employ identical modes of RNA replication, our observation is open to several interpretations. Two enzymes may be essential, one to form a double-stranded “replicative form” from the input strand, and one to produce new progeny strands. Alternatively, one enzyme may suffice for replication, but be composed of two identical polypeptides as are many bacterial enzymes (Yanofsky and Crawford, 1959). Finally, one cistron may be required to neutralize a cellular activity prejudicial to viral RNA synthesis (e.g., RNase) while the second cistron provides polymerase. At least the first of these interpretations is open to experimental examination. It predicts that the RNA of mutants in only one RNAcomplementation group should be converted to a double-strand form during infection at the nonpermissive temperature.
PFEFFERKORN ACKNOWLEDGMENT We wish to thank Mrs. Helen skillful technical assistance.
M. Coady
for
REFERENCES P. (1962). Topography in vaccinia genetics. Virology 16, 347-348. BURGE, B. W., and PFEFFERKORN, E. R. (1965). Conditional-lethal mutants of an animal virus: Identification of two cistrons. Science 148, 959-
ABEL,
960. BURGE,
B. W., and PFEFFERKORN, E. R. (1966). Isolation and characterization of conditionallet,hal mutants of Sindbis virus. Virology 30, 204-213. CASPAR, D. L. D. (1963). Assembly and stability of the tobacco mosaic virus particle. Advan. Protein Chem. COOPER, P.
18,37-123.
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J. R. S. (1966). “Genetic ComplementaBenjamin, New York. FINCHAM, J. R. S., and PATEMAN, J. A. (1957). Formation of an enzyme through complementary action of mutant “alleles” in separate nuclei in a heterocaryon. Nature 179, 741. KIRVAITIS, J., and SIMON, E. H. (1965). A radiobiological study of the development of Newcastle disease virus. Virology 26, 545-553. LODISH, H. F., and ZINDER, N. D. (1966). Replication of the RNA of bacteriophage fz. Science tion.”
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COMPLEMENTATION
SPIEGELMAN,HOLLAND, S., HARUNA, I., I., BEAUDREAU, A., and MILLS, D. (1965). Synthesis of a self-propagating and infectious nucleic acid with a purified enzyme. Proc. Natl. Acad. Sci. u. s. 54.919927. VALENTINE, R. C., ENGLEHARDT,D. L., and ZINDER,N. D. (1964). Host-dependent mutants
WITH
SINDBIS
223
MUTANTS
of the bacteriophage fz. II. Rescue and complementation of mutants. Virology 23,159-163. YANOFSKY,C., and CRAWFORD, I. P. (1959). The effects of deletions, point mutations, reversions and suppressor mutations on the two components of the tryptophan synthetase of Escherichia
coli.
Proc.
Natl.
Acad.
Sci.
U. S. 45,1016.
30, 214-223 (1966)
Complementation
Between
Temperature-sensitive
Mutants
of
Sindbis
Virus’ BOYCE W. BURGE” Department
of Bacteriology
and
Immunology,
E. R. PFEFFERKORN
AND
Harvard
Medical
School,
Boston,
Massachusetts 0.2116
June 1, 1966
Accepted
Eleven temperature-sensitive mutants of Sindbis virus were classified according to their ability to complement other mutants in pairwise mixed infections at a nonpermissive temperature. Five nonoverlapping complementation groups were thus defined. In two groups the temperature-sensitive function is required for viral RNA synthesis, while mutants of the other three groups are unable to carry out some essential step occurring after viral RNA synthesis. The progeny produced by complementation were found, in general, to include both the genomes used to initiate the mixed infection; these progeny particles were phenotypically mixed in a manner that suggests exclusion of mutationally altered protein from rescued particles.
RNA synthesis (RNA+). We have examined six RNA+ mutants and five randomly chosen RNA- mutants for their ability to complement in pairwise mixed infections. A nonoverlapping pattern of complementation is observed, and five complementation groups have been defined.
INTRODUCTION
When temperature-sensitive conditionallethal mutants of Sindbis virus (Burge and Pfefferkorn, 1966) are propagated at a nonpermissive temperature, certain pairs produce a much higher yield of virus from mixed infection than either mutant produces alone. This ability of each mutant to provide the function defective in the other allows classification
of mutants
into
discrete
functional
MATERIALS
or
complementation groups (Fincham, 1966). Thus, for a given population of mutants, a complementation group is defined as a set of mutants that do not complement each other but that do complement all other mutants in the population. In the preceding article of this issue (Burge and Pfefferkorn 1966) we described two classes of mutants: those blocked in viral RNA synthesis at nonpermissive temperatures (RNA-) and those blocked in some maturational event occurring after viral 1 This investigation was supported in whole by United States Public Health Service research grants AI-04531-04 and -05 from the Institute of Allergy and Infectious Diseases. 2 Supported by a predoctoral fellowship from the U. S. Public Health Service, number l-FlGM-31, 037-01.
AND
METHODS
Tissue culture and virological techniques are described in the preceding paper (Burge and Pfefferkorn, 1966). Temperature-sensitive conditional lethal mutants of Sindbis virus are designated ts-1, ts-2, etc. The heatstable strain from which all these mutants were derived is designated HR (Burge and Pfefferkorn, 1966). Multiplicities of infection were determined from the number of PFU adsorbed and the number of cells per monolayer. Replicate monolayers were dispersed with trypsin and the cells were counted in a hemocytometer. The temperature and time used for the adsorption step of routine titrations (Pfefferkorn and Hunter, 1963) were different from those used in complementation experiments. Therefore, the number of PFU adsorbed per monolayer was calculated from the number of plaques produced by suitable dilutions of
214
COMPLEMENTATION
WITH
the viral stocks after adsorption under the conditions used in studying complementation. RESULTS
Procedures for Measuring Complementation Mixed infections with pairs of conditionallethal mutants were begun by allowing approximately equal multiplicities of each parent (about 10 PFU/cell) to adsorb to precooled monolayers. After 45 minutes at 4’, the monolayers were rinsed with warm medium and were incubated at a nonpermissive temperature, usually 39”, for 4 hours. The monolayers were then drained and rinsed with prewarmed medium to remove uneclipsed parental virus. The monolayers were incubated for 2 more hours at the nonpermissive temperature, and progeny produced during this period was measured by plaque titration at 27”. We should like to emphasize here the necessity for washing the infected monolayers after several hours’ incubation at the nonpermissive temperature. Uneclipsed parental particles that cannot be removed by washing immediately after adsorption are eluted during the first few hours of infection. Similar behavior of Western equine encephalitis virus has been reported by Dulbecco and Vogt (1954). The presence of these eluted virus particles which do not represent viral growth can partially or completely mask the occurrence of complementation. Failure to recognize this factor accounts for the very low levels of complementation reported earlier (Burge and Pfeff erkorn, 1965). The yield produced in double infections between 4 and 6 hours was divided by the sum of the yields produced by the 2 mutants grown separately, under identical conditions at the same time. The resulting figure, called here the “complementation level,” is a numerical expression of the extent of complementation. Values of 2 or greater are considered positive results, whereas values of 1 or less are interpreted as absence of complementation. With complementing pairs of mutants the “complementation level” assumes a value between 2- and 300-fold. This value depends less on the yield from the double infection,
SINDBIS
MUTANTS
215
which is similar for all complementing pairs, than on the “background” produced by the two parents grown separately. This “background” is determined by mutant specific properties, such as reversion frequency and leakiness (Burge and Pfefferkorn, 1966)) and in consequence the complementation level for a given pair of mutants is also determined by mutant-specific properties. Noncomplementing pairs of mutants produce complementation levels of 1 or less. Values less than 1 may indicate interference by one mutant with growth of the other at nonpermissive temperatures. Manipulatable parameters such as multiplicity of infection and temperature of incubation during the mixed infection also determine the level of complementation. These latter parameters were examined with a pair of RNA+ mutants (ts-5 and ts-lo), chosen because of their ability to produce a high complementation level. Effect of Temperature on Complementation When compared with their HR ancestor, conditional-lethal mutants are markedly defective in growth at all temperatures between 35” and 41’ (Burge and Pfefferkorn, 1966). Thus, complementation might be expected to occur at all temperatures in this range, since the growth of each mutant is limited by its unique temperature-sensitive defect. Four identical sets of monolayers were infected with mutants ts-5, ts-10, or both ts-5 and ts-10, as described above, and each set of infected monolayers was incubated at a different temperature in the 35’-41” range. The yield of virus produced between hours 4 and 6 of infection is plotted in Fig. 1. Although the yields in both single and double infections are reduced by higher incubation temperatures, the complementation level remains high at all but the highest temperature examined, 41”. Experiments with other mutants have given similar results. It appears that complementation can be detected over the considerable temperature range in which both mutants are markedly defective in growth and the parental virus is able to grow well. Temperatures which limit the growth of the parental HR virus also limit the production of diffusible gene products by
BURGE
AND
PFEFFERKORN TABLE EFFECT
OF MULTIPLICITY
1 ON COMPLEMENTATION~:
RNA+ X Rh'A+ Multiplicity of infection (PFU/cell)
ts-5 12
3 0.75 0.16
Incubation
Temperature (“Cl
FIG. 1. Complement&ion yield as a function of temperature. The dashed line represents the sum of the two single infections. Above the solid line which plots the complementation yield [(>, (ts-5 Xts-lo)] is listed the complementation level at each temperature.
the mutants, and thus reduce the efficiency of complementation. E$ect of Multiplicity
o-n Complementation
One cannot confidently predict the effect of multiplicity of infection on the efficiency of complementation between virus mutants in animal cells. It is known that recombination between certain animal virus mutants can be easily detected only when clumps of virus are used to initiate mixed infection, thus assuring the proximity of the two mutants during the early stages of infection (Abel, 1962; Kirvaitis and Simon, 1965). Since chick fibroblasts have a volume lo3 times that of Escherichiu coli cells, in which most observations on viral complementation have been made, it might be supposed that a high multiplicity of infection would be required to produce intracellular proximity of the conditional-lethal mutants during growth and thus ensure successful complementation. The experiment recorded in Table 1 shows
k-10 2.8 0.7 0.18
0.045
Level Percentage of cells mixedly infected”
94 48 8.5 0.85
Yield due to complementation”
3.2 1.8
4.0 1.0
X x x x
10’ 106 105 106
,&plementation 80 55 87 38
0 Complementation was performed as described in the text, and multiplicities were determined by the procedure outlined in Materials and Methods. b The percentage of mixedly infected cells is (1 - t?) (1 - e-u) 100, where 2 is the multiplicity of one mutant (PFU/cell), and y is the multiplicity of the other. c The yield due to complementation is the yield obtained in the double infection minus the sum of the yields of the parents grown separately at the same time.
that this is not so. Complementation between mutants ts-5 and ts-10 was performed with sets of identical monolayers, using a different multiplicity of each virus for each set. From the multiplicity of infection the fraction of mixedly infected cells was calculated by the Poisson distribution. Table 1 shows that the viral yield due to complementation is an approximate linear function of the fraction of mixedly infected cells, even when this fraction is very small. Thus a given cell produces about the same complementation yield whether infected with only one PFU of each mutant or with 10 PFU of each mutant. E$ect of Multiplicity on Complementaticm When One Parent is RNAKSince both ts-5 and ts-10 are RNA+, their genomes continue to replicate at nonpermissive temperatures and they presumably produce essentially normal quantities of gene products. Diffusion of these gene products through the cytoplasm of infected cells allows complementation. A different result is possible when one of the parents in a complementation experiment is RNA-, and thus
COMPLEMENTATION TABLE EFFECT
OF MULTIPLICITY
RNA~t$i$~,’
ts-17
Percentage of cells rnixedly infected
19 3.2 0.53 0.09
100 89 14 0.56
(PFU/cell) ts- 10 15.8 2.6 0.44 0.07 a All
details
as in Table
WITH
TABLE
x RNA+
x X x 3 x
EFFECT
RNA-
ts-10 X ts-17 Level of yield due to complecowlemenmentation tation 1.8 2.5 1.3
217
MUTANTS
2 ON COMPLEMENTATION’:
10’ lo6 105 103
270 108 11.7 5
1 footnotes.
possibly incapable of extensive production of gene products. A similar experiment was therefore performed in which an RNA+ mutant (ts-10) was crossed with an RNA- mutant (ts-17). In this case, the yield of virus produced by complementation was not a linear function of the number of mixedly infected cells (Table 2). Increased multiplicities of infection beyond those required to infect nearly all the cells resulted in much greater complementation yields. The critical factor in this experiment was suspected to be the multiplicity of the RNA- mutants. This suggestion was confirmed by an experiment in which the multiplicity of the RNA+ mutant was held constant at 4 while the multiplicity of the RNA- mutant was varied. Again the yield of virus from complementation was markedly dependent on multiplicity (Table 3). Complementation
SINDBIS
Groups
Of the 23 conditional-lethal mutants isolated (Burge and Pfefferkorn, 1966), 11 were chosen for studies of complementation. These included 5 randomly chosen RNAmutants and 6 of the 7 RNA+ mutants. The seventh RNA+ mutant, ts-9, failed to complement any other mutant. Since it had no detectable reversion frequency (Burge and Pfefferkorn, 1966) and was thus suspected of being a double mutant or deletion, it was omitted from classification. By the complementation criteria outlined above, these 11 mutants were resolved into 5 nonoverlapping complementation groups (Table 4). As these groupings imply, RNA-
3
OF MULTIPLICITY ON COMPLEMENTATION”: X FIXED MULTIPLICITY OF RNA+
Multiplicity of infection (PFU/cell)
Percentage of cells mixedly infected
ts-10 X ts-17 yield due to comdementation
ts-10
ts-17
4 4 4 4
6.5 1.1 .18 .03
98 66 16 3
a All
details
as in Table
3.5 8.8 6.0 3.5
TABLE
RNA phenotype&
A B C D E
RNARNARNA+ RNAf RNA+
0 RNA+ mutants at the nonpermissive mutants cannot.
complementation
106 106 104 103
61.5 47 6.5 1.5
4 GROUPS Mutants
in group
ts-4, ts-17, ts-19, ts-11 ts-2, ts-5, ts-13 ts-10, ts-23 ts-20 are
of
1 footnotes.
COMPLEMENTATION Group
x X X x
Level
ts-21
able to make viral temperature while
RNA
RNA-
mutants always complement RNA+ mutants. Because we could not do all pairwise crosses required for the definition of these 5 groups in a single experiment, the above results were accumulated from several experiments, two of which are described below and recorded in Tables 5 and 6. In the experiment of Table 5 each complementation group is represented by at least one mutant. The yield produced in each double infection is listed together with the complementation level (in parentheses) for that cross. Complementation yields in this experiment were l-2 % of the HR yield under identical conditions of growth. Table 6 records the results of double infections with all RNA- mutants, including the 4 mutants of group A and the single mutant of group B, ts-11. It should be noted that no complementation occurs in crosses between mutants of group A (the definition of a complementation group), but that all group A mutants complement ts-11 (an ex-
218
BURGE
ANI)
PFEFFERKORN
TABLE
5
COMPLEMENTATION" Com-
plementation group
Yield from M utant single infection (PFU/ml) A ts-4
A
ts-4
5.5 x 104
A
ts-17
2.2 x 105
B
ts-11
1.1 X 104
C
ts-5
1.5 x 105
D
ts-10
1.0 x 106
E
ts-20 HR
4.3 x 105 6.0 x 108
-
Yield (PFU/ml) in mixed infection with A ts-17
B ts-11
c ts-5
1.0 x 106 (0.4) -
4.5 x 105 (7) 7.7 x 106 (30) -
9.0 x 106 (56) 1.1 X 10’ (30) 8.0 x 106 (50)
D ts-10
E ts-20
4.0 x 106 3.0 x 106
(56)
(6.2)
3.0 x 106 (9.5) 6.0 X lo6 (55) 2.5 X 10’ (100) -
3.5 x 106 (5.4) 1.9 X 10’ (44) 3.4 X lo6 (5) 1.8 X 10’ (34) -
a Below the yield in mixed infections, in parentheses, is the complementation level which is defined as the yield from mixed infections divided by the sum of the yields of each parent grown separately. TABLE COMPLEMENTATION
Cofnplementatlon group
Mutant
6
BETWEEN
RNA-
MUTANTS"
Yield in mixed infection with
Yield from single (PFU/ml)
A ts.4
A ts-17
A ts-19
A ts-21
B ts-11
-
5.0 x 106 (0.4) -
3.3 x 105 (0.5) 5.6 X lo6 (0.4)
1.7 x 106 (1) 1.8 X 105 (0.08) 4.0 x 105
1.5 x 106 (7) 3.0 x 10’ (30) 5.7 x ia7 (1‘Jo) 5.2 x lo7 (35) -
infection
A
ts-4
2.0 x 106
A
ts-17
1.0 X 106
A
ts-19
4.5 x 106
A
ts-21
1.5 X 106
B
ts-11 HR
2.5 X lo4 1.0 X 109
(0.2) -
a Details as in Table 5 footnot,e.
ample of nonoverlapping complementation) . The yields in this experiment are slightly higher than those in Table 5, because a slightly lower incubation temperature was used (38”). Phenotypic Properties of Rescued Particles We have shown that efficient phenotypic mixing occurs during mixed infection with several related group A arboviruses (Burge and Pfeff erkorn, unpublished results). That is, the coat of a single progeny particle may contain structural units determined by each infecting virus. Complementation between 2
RNA+ mutants, with temperature-sensitive defects in different structural proteins, might be supposed to occur by a process akin to phenotypic mixing, in which normal proteins would appear in rescued particles while proteins of abnormal conformation would be excluded by the principles of self-assembly (Caspar, 1963). We have shown that RNA+ mutants generally are much more heat labile than the HR virus from which they were derived and have suggested that this property is largely a consequence of a conditionallethal mutation in a coat protein (Burge and Pfefferkorn, 1966). If this is true, the parti-
COMPLEMENTATION
WITH
SINDBIS
219
MUTANTS TABLE
IDENTIFICATION Complementation
b-5
(RNA+) 17 (RNA-) ts-10 (RNA’)
pair
x
7 OF GENOTYPES”
Complementation levelb
Ratio
of genotypes in progeny
ts-
31
X ts-
17
7 ts-10:2
100
10 ts-5:2
17 (RNA--) b-5 (RNA+) x ts-
13 ts-5:7
ts-17 ts-17 ts-10
10 (RNA+)
2.5 Minutes FIG. 2. Heat ts-10, HR virus, complementation.
5.0 at
SO0
inactivation curves for ts-5, and the progeny of ts-5 X ts-10
cles produced by complementation should be relatively heat stable. This prediction was tested by determining the heat stability of the progeny produced by complementation between mutants ts-5 (group C) and ts-10 (group D). Figure 2 shows that the progeny of the mixed infection were markedly more resistant to heating than either parent, and only slightly less heat resistant than the ancestral HR virus. Since 99.9 % of the complementation progeny were genotypically mutant (i.e., could not produce plaques at 39”) and were therefore not recombinants, the heat resistance gained during mixed infection appeared to have been only a phenotypic property. This conclusion was confirmed by determining the heat stability genotype of the viruses produced by complementation. Ten plaques produced by the survivors of 5 minutes’ heating provided the inocula for separate clones grown at 27”. These were all found to be very heat labile, proving that the heat stability was only a transient phenotypic property. The phenotypic properties of progeny from an RNA- X RNA+ complementation (ts-17 X ts-10) were also examined. In this
o Progeny of complementation were cloned and identified either by ability to stimulate uridine1% incorporation into actinomycin-treated monolayers at 39” (RNA+ X RNA-), or by their ability to complement known stocks of one or the other parent (RNA+ X RNA+). b Complementation level is the yield from mixed infections divided by the sum of t,he yields of each parent grown separately.
cross, the RNA- parent (ts-17) is heat stable, but the RNA+ parent (ts-10) is heat labile and is the majority genotype rescued by complementation. (See the nest section for a description of the identification of rescued genotype.) An artificial mixture of the 2 parental strains (ts-17 and ts-10) present in the proportion suggested by the ratio of rescued genotypes in complementation yields (Table 7) would produce a two-component inactivation curve on 60’ heating, as described by the dashed line in Fig. 3. The actual complementation yield showed only a single component, which was heat resistant. This result demonstrates that phenotypic heat resistance of “genotypically” heatlabile mutants is characteristic of RNA- X RNA+ complementation, as well as the RNA+ X RNA+ complementation described above. Genotypes of Rescued Particles Since recombination cannot be detected under our experimental conditions, each virion produced during complementation should be of one of the parental genotypes. We therefore determined in which proportions the two mutant genotypes were represented. With complementation between RNA+ and RNA- mutants this was done by
BURGE
220
AND
PFEFFE tants
KKORN is not
restricted
to the
RT\rA
strand
from which it is transcribed, but may also less frequently replicate RNA- genomes dur0
ing mixed
Absence of Detectable Recombination Conditional-Lethal Mutants
HR
& i-l: c $ 0 ?! ki 0 f?
IO
ts-17 ts-17
x ts-IO
2.0
3.0
-I
4.0
50 25 Minutes
5.0 at
infection.
60’
FIG. 3. Heat inactivation curves for ts-10, ts-17, HR virus, and the progeny of b-10 X ts-17 complementation. The dashed line represents the two-component inactivation curve expected for an artificial mixture of ts-10 and ts-17 in the proportions suggested by Table 7.
cloning the progeny and determing their ability to stimulate uptake of uridine-2J4C into actinomycin D-treated monolayers at, a nonpermissive temperature (Burge and Pfefferkorn, 1966). In a complementation between two RNA+ mutants, of course, all progeny will be able to stimulate uridine uptake. In this case, clones can be identified only by their ability to complement with known stocks of one or the other mutant. The results of genotype identification for 3 complementing pairs are listed in Table 7. In all cases both genomes appear. The RNA+ genome predominates in RNA+ X RNAinfections. Since the RNA+ genome is capable of extensive multiplication at the nonpermissive temperature, significant representation of the RNAgenome in the complementation progeny would not, be expected unless it, too, were replicated. Thus these results suggest that the functional RNA polymerase produced by RNA+ mu-
between
Since we were able to demonstrate that progeny produced by complementation retained the conditional-lethal character (i.e., inability to produce plaques at 39”), it was apparent that increased yields from double infections could not be explained in terms of the formation and outgrowth of recombinant genomes. However, since recombination has been reported with RNA animal virus mutants (reviewed by Fenner and Sambrook, 1964), experiments were carried out, to determine whether any recombination was occurring in mixed infections with Sindbis virus mutants. Various pairs of conditional-lethal mutants with low reversion frequencies were used to produce mixed infections at permissive temperatures (27”33”). The yields from such double infections were examined for an increase in the frequency of particles able to form plaques at 39” (i.e., particles that behave like recombinants). Although the progeny from similar double infections exhibited phenotypic mixing, proving that mixed inTABLE ABSENCE Mutant(s) used in infection
b-5 alone ts-10 alone ts-16 alone ts-5 x ts-10 ts-5 X ts-16 ts-10 X ts-16
8
OF RECOMBINATION~ Reversion
39” Titer (PFU/ml) 1.0 1.0 3.0 1.0 7.0 2.5
x x x x x x
108 103 103 103 102 lo3
27” Titer (PFU/ml) 9.0 8.0
x x
10’
1.2 1.2 1.0 1.0
x x x x
10s 108 10s 10s
107
frequency (39” titer/ 27” titer) 1.1 1.5 2.5 8.5 7.0 2.5
x 10-s x 10-6 X W6
x lo-6 x 10-e x lO+
a Monolayers were infected with about 10 PFU/cell of each mutant and incubated at 32”. After 2 hours monolyares were washed, and the progeny produced between 2 and 8 hours after infection were scored for ability to produce plaques at 27” and 39”. ts-5 and te-10 are RNA+ mutants, and thus defective in a cistron different from that of ts-16, an RNA- mutant.
COMPLEMENTATION
WITH
fections had been estabished (Burge and Pfeff erkorn, unpublished data), no increase in the frequency of revertant genotypes was observed. Since mutants with reversion frequencies of about 10e5 were used a recombination frequency of lo-4 would easily have been detected. Details of typical experiments are given in Table 8. While these experiments by no means rule out recombination in arboviruses, they establish that recombination cannot explan the observed complementation. DISCUSSION
One of the principal motives for investigating conditional lethal mutants is the hope that each complementation group will correspond to a specific biochemical defect in the sequence of infection, the identification of which will lead to a more precise understanding of discrete events in viral multiplication. At present we can discriminate mutants biochemically only by their ability, or inability, to produce viral RNA at nonpermissive temperatures. None of the RNA+ mutants produce noninfectious hemagglutinating particles at 39”. We plan to examine thin sections of mutant-infected cells with the expectation of finding aberrations in the normal morphological sequence of arbovirus infection described by Morgan et al. (1961). In the absence of biochemical or morphological defects which correspond to individual complementation groups, it is important to examine the evidence arising from complementation per se that our complementation groups correspond to viral cistrons. If each mutant in the population can be unambiguously assigned to one and only one such group, then the complementation pattern is said to be nonoverlapping. A nonoverlapping pattern observed with a sufficiently large number of mutants provides prima facie evidence that each complementation group corresponds to one cistron, all members of the group representing mutations of that cistron. When the protein product of a cistron is made up of two or more identical subunits another possibility exists, and mutants within that cistron may complement each other (Fincham and Pateman, 1957). Such
SINDBIS
MUTANTS
221
intracistronic complementation gives rise to an overlapping complementation pattern and, as a consequence, resolution of cistrons is made more difficult. We have found that 11 independent mutants (each characterized by independent origin and a unique combination of heat lability, reversion frequency, and leakiness of growth) fall into 5 complementation groups that do not overlap. Were intracistronic complementation occurring among these mutants, some overlapping might be expected. The complementation reported here represents only a few per cent of the yield of the ancestral HR virus at 39”. With temperature-sensitive mutants of bacteriophage T4, intercistronic complementation is very efficient (50% of the wild-type burst) whereas intracistronic complementation is much less efficient (usually < 10% of the wild-type burst) (Edgar et al., 1964). However, since both types of complementation in the T4-E. coli system are more efficient than the complementation we observe, few conclusions can be drawn. More can be gained, perhaps, by an examination of the complementation results for conditional-lethal mutants of RNA viruses. Cooper (1965) studied complementation between temperature-sensitive mutants of poliovirus in human amnion cells. Complementation in this system is quite inefficient. Although 31 independently isolated mutants were studied, the highest complementation levels recorded were less than 10, a value corresponding to 0.1% of the wild-type yield under identical conditions. Since a biochemical investigation revealed that these mutants differed in their temperature-sensitive defects, it appears that low efficiency is characteristic of intercistronic complementation with poliovirus. ValenGne et al. (1964) have studied complementation between conditional-lethal mutants of the RNA bacteriophage fz. These authors found that when two amber mutants of different cistrons mixedly infected a nonpermissive host, the burst was only l-3% as large as that of wild-type fz. This efficiency is similar to that observed for conditional-lethal mutants of Sindbis virus complementing in a cell 103 the size of E. coli;
222
BURGE
AND
thus low efficiency may simply be a characteristic of complementation between RNA virus mutant’s Additional evidence that we are studying intercistronic complementation comes from comparison of complementation yields produced by different combinations of the two RNA phenotypes. Complementation between RNA+ and RNA- mutants, which is certainly complementation between cistrons, produces yields no greater than the yields observed in RNA+ X RNA+ crosses, or RNA- X RNA- crosses. If the usual relation between kid of complementation and efficiciency holds here (that is, a much lower efficiency for intracistronic than for intercistronic complementation) then this complementation would appear to be intercistronic. It is also possible that the complementation observed occurs with a high efficiency, but in only a small proportion of mixedly infected cells. Examination of the virus yield of single cells will be used to test this suggestion. The complementation between RNA- mutants (Table 5) is especially interesting because it implies that two cistrons are required for the synthesis of viral RNA. Since there is no agreement at present as to the number of enzymes required for viral RNA synthesis (Spiegelman et al., 1965; Lodish and Zinder, 1966), and since it is not certain that bacterial and animal RNA viruses will employ identical modes of RNA replication, our observation is open to several interpretations. Two enzymes may be essential, one to form a double-stranded “replicative form” from the input strand, and one to produce new progeny strands. Alternatively, one enzyme may suffice for replication, but be composed of two identical polypeptides as are many bacterial enzymes (Yanofsky and Crawford, 1959). Finally, one cistron may be required to neutralize a cellular activity prejudicial to viral RNA synthesis (e.g., RNase) while the second cistron provides polymerase. At least the first of these interpretations is open to experimental examination. It predicts that the RNA of mutants in only one RNAcomplementation group should be converted to a double-strand form during infection at the nonpermissive temperature.
PFEFFERKORN ACKNOWLEDGMENT We wish to thank Mrs. Helen skillful technical assistance.
M. Coady
for
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COMPLEMENTATION
SPIEGELMAN,HOLLAND, S., HARUNA, I., I., BEAUDREAU, A., and MILLS, D. (1965). Synthesis of a self-propagating and infectious nucleic acid with a purified enzyme. Proc. Natl. Acad. Sci. u. s. 54.919927. VALENTINE, R. C., ENGLEHARDT,D. L., and ZINDER,N. D. (1964). Host-dependent mutants
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SINDBIS
223
MUTANTS
of the bacteriophage fz. II. Rescue and complementation of mutants. Virology 23,159-163. YANOFSKY,C., and CRAWFORD, I. P. (1959). The effects of deletions, point mutations, reversions and suppressor mutations on the two components of the tryptophan synthetase of Escherichia
coli.
Proc.
Natl.
Acad.
Sci.
U. S. 45,1016.