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Isolation of an influenza virus temperature-sensitive mutant of a new recombination-complementation group
VIROLOGY
78,
365-374
(1977)
Isolation of an Influenza Virus Temperature-Sensitive Mutant of a New Recombination-Complementation Group K. NAKAJIMA Department
of Microbiology,
The Znstitute
AND
of Public Tokyo 108,
Accepted
January
A. SUGIURA Health, Japan
6-1, Shirokanedai
I-chome,
Minutoku,
3,1977
Wild-type WSN of influenza A virus which had been inactivated by ultraviolet (uv) irradiation was capable, to various extents, of rescuing temperature-sensitive (ts) mutants of the same virus, resulting in the production of wild-type recombinants at the nonpermissive temperature. The capability of wild-type virus to rescue ts mutants decreased with increasing uv dose following single-hit kinetics. Inactivation of rescue capability varied when mutants of different recombination-complementation (recombination) groups were compared, but was nearly identical to different members of one recombination group, except for Group IV mutants. The inactivation rate was significantly greater when it was determined with two mutants (ts-11 and ~9-60) than when determined with the rest of the Group IV mutants. Because the inactivation rate was expected to be greater for a double mutant than for virus strains having either one of two mutational lesions alone, it was suspected that ts-11 and ts-60 had an additional mutational lesion in a still undeiined gene. Segregation of a new mutant from ts-60 was, therefore, attempted by backcrossing it with wild-type WSN and screening progeny clones. A clone which had lost Group IV mutation during the process but still retained temperature sensitivity had been obtained. Genetic crossing of this segregant, ts-GOS, with mutants of all seven other recombination groups gave rise to recombinants of wildtype character, indicating that ts-60s was a mutant of unknown recombination group (Group VIII). Both ts-11 and ts-60 were confirmed to be double mutants (Groups IV + VIII). During the process of segregation, a number of progeny clones with a novel genotype were obtained which had not been manifest in the parent viruses, i.e., the mutation in either the group I or Group III gene, in addition ta the one in the Group VIII gene. It was found that these paradoxical phenomena were caused by mixed aggregates present in the parent viruses, b-11 and ts-60. Mixed aggregates contained, at least, triple mutants (either Groups I + IV + VIII or Groups III + IV + VIII) and the double mutant (Groups IV + VIII). The preparation of ts-11 predominantly showed aggregates which sedimented faster than wild-type virus particles and which could be dispersed by the treatment with neuraminidase. Aggregates were formed most likely as a result of incomplete desialylation due to low neuraminidase activity of Group IV mutants. INTRODUCTION
Our collection of temperature-sensitive (ts) mutants derived from the WSN strain of influenza A virus had been classified into seven recombination-complementation (recombination) groups (Sugiura et al., 1975). Since influenza A genome RNA was resolved into eight RNA species by electrophoresis (Pans, 1976; Bean and Simpson, 1976; Content, 1976; Palese and Schulman, 1976; McGeoch et al., 1976; Scholtissek et al., 19761, the eighth mutant Copyright All rights
0 197'7 by Academic Press, Inc. of reproduction in any form reserved.
group was still to be found. Some mutants, although considered to be single at present, might possibly be double mutants involving the putative Group VIII gene. We tried, therefore, to test the singularity of mutation utilizing cross-reactivation. Multiplicity reactivation (Barry, 1961) and cross reactivation (Simpson and Hirst, 1961; Tumova and Pereira, 1965; Sugiura and Kilbourne, 1966; McCahon and Schild, 1971; Sugiura and Ueda, 1971) are the particular forms of genetic recombination
365 ISSN
0042-6822
366
NAKAJIMA
which involve parent virus inactivated by damage to the viral genome. A ts mutant would also be rescued by ultraviolet (uv)irradiated wild-type virus, when a gene in the latter that has escaped a uv hit replaces the corresponding gene in the mutant and thereby restores its reproductive capacity at the nonpermissive temperature. The ability of wild-type virus to rescue a particular mutant would be abolished by uv irradiation at the rate reflecting the target size of the mutated gene. The target size of the mutated portion of the genome in a double mutant is expected to be greater than that of constituent single mutants. The experiments described here show the validity of the assumption and revealed that two of the Group IV mutants might have an additional mutation in a still unidentified gene. This hypothesis has been verified by segregation of a mutant belonging to a new recombination group. We also describe a genetically anomalous behavior of Group IV mutants which could be attributed to the low neuraminidase activity of the mutants. MATERIALS
AND
METHODS
Cells and virus. MDBK
cells were used. The media employed for propagation and maintenance of cells have been described previously (Sugiura et al., 19721, except that the growth medium contained heatinactivated newborn calf serum, kindly provided by the Mitsubishi - Kasei Institute of Life Sciences, instead of fetal calf serum. The wild-type virus (ts+) and ts mutants of the WSN strain of influenza A virus have also been described elsewhere (Sugiura et al., 1972, 1975). Ultraviolet-irradiated virus. The ts+ was diluted tenfold in phosphate-buffered saline (PBS) containing 0.2% bovine serum albumin. The virus was distributed in 5ml amounts in lo-cm petri dishes and was irradiated by a 15-W germicidal lamp at a distance of 20 cm for various time periods, during which the open dishes were constantly rocked. Reactivation of ts mutants diated ts+ (uv-ts+) . Virus
with
uv-irra-
titration was performed by counting plaque-forming infective centers. Virus mixture containing
AND
SUGIURA
2.5 x lo4 PFU (calculated from preirradiation titer) of uv-ts+ and 5 x lo4 PF’U of a ts mutant in 0.2 ml was prepared. A suspension of 5 x lo5 freshly trypsinized cells in 0.2 ml was combined with the above mixture. Input multiplicity was 0.05 PFU/cell for uv-ts+ and 0.1 PFU/cell for a ts mutant. Virus adsorption to cells was allowed to proceed for 30 min at 37” with occasional shaking. Cells were washed and resuspended in fresh maintenance medium. Serial dilutions were prepared in fivefold steps and 0.1 ml of the cell suspension was deposited on drained monolayer cultures in 6-cm petri dishes. Inoculated cells were spread over the entire surface by shaking the dish. After incubation at 37” for 30 min for attachment of cells to the monolayer, agar overlay medium was gently added and the cultures were incubated at a nonpermissive temperature (39.5”). Plaques were counted after 4 days of incubation. Recombination test. The presence or absence of genetic recombination was determined by standard assay. Briefly, cells were infected with two mutants, both at a multiplicity of l-5 PFU/cell, and were incubated at 34”. At 3 hr, the cultures were treated with rabbit anti-WSN antiserum diluted to l:l,OOO for 10 min, washed three times with PBS containing 0.2% bovine serum albumin, and fed with fresh maintenance medium. Incubation was continued at 34”. The culture fluid was harvested at 10 hr after infection. Appropriate controls, namely, cultures singly infected with each mutant, were included. The progeny virus produced during one-step growth was titrated by plaque assay at two temperatures. Recombination frequency was calculated by the following formula (Sugiura et al., 1972):
where [AB3JS4 and [AB34]39.5are the titers of a mixed yield grown at 34” and assayed at 34” and 39.5”, respectively, and [A34139.5 and [B34139.5 are the titers of single yields grown at 34” and assayed at 39.5”. Mixed infection for simplified complementation-recombination.
cells were infected with mutants
test of
MDBK individ-
A NEW
INFLUENZA
ually or in combination. The multiplicity was 0.2 PFU/cell for each mutant. After virus adsorption for 30 min at 37” cultures received maintenance medium and were immersed in the water bath maintained at 39.5” (*O.lO). At 22-24 hr, culture fluids were harvested and titrated for hemagglutination (HA) activity (Sugiura et al., 1975). The criterion of positive complementation-recombination was an HA titer at least eightfold higher in the mixedly infected culture than the sum of the HA titers in singly infected cultures. Activity of HA and neuraminidase was measured as previously described (Sugiura et al., 1972). Velocity sedimentation of virus. MDBK cells in 6-cm petri dishes were infected with either ts+ or ts-11 at a multiplicity of 1 PFU/cell at 34”. At 3 hr, 10 $i of L3Hluridine (42.5 Ci/mmole, New England Nuclear) was added per dish, and incubation was continued at 34” overnight. After the culture fluid was clarified at 6000 rpm for 20 min at 4”, the supernatant was treated with neuraminidase. The supernatant was layered onto the lo-40% (w/v) linear sucrose gradient in 0.1 M NaCl, 0.001 it4 EDTA, 0.01 M Tris-HCI (pH 7.21, and was centrifuged at 24,000 rpm for 50 min at 4” in a Spinco SW 27 rotor. Fractions of 1.4 ml each were collected from the bottom. One-milliliter aliquots were used for determination of acid-insoluble radioactivity in an Aloka Model LSC-601 scintillation spectrometer. The remaining portion was tested for infectivity by plaque assay at the permissive temperature. Neuraminidase treatment of ts-11. One milliliter of virus preparation was incubated with 50 units of V. cholerae neuraminidase (Behringwerke, Marburg, Germany) for 30 min at 37”. RESULTS
Rescue of ts Mutants and uv-Irradiated
When
with ts +
Unirradiated
cells infected with unirradiated for infective centers, the number of plaques formed was 57% of input PFU (5 x lo5 cells infected with ts+ at a multiplicity of 0.05 PFU/cell should have given 25,000 infective centers; Table 1). ts+ were plated
367
ts MUTANT
Since cultures for infective center assay were incubated at the nonpermissive temperature, at which temperature the plaquing efficiency of ts+ was between 25 and 40% of that at the permissive temperature (Sugiura et al., 19721, adsorption of input virus to cells in suspension was nearly complete. Coinfection with ts-3 resulted in a 3.4-fold increase in the number of infective centers, although the single infection with this mutant at the same multiplicity yielded only a small number of infective centers at the nonpermissive temperature. Most mutants caused a variable degree of increment, up to fivefold. Additional infective centers must .have been initiated by ts+-type recombinants resulting from interaction between non-plaque-forming, but genetically competent virus particles contained by ts+ and those contained by a mutant virus preparation (Hirst, 1973). Throughout this study, cells were infected with ts mutants at the multiplicity of 0.1 PFU/cell. With many mutants, a higher multiplicity led to the reduction in infective centers among mixedly infected cells. A few mutants (ts-52 and ts-57) depressed the number of infective centers by 20 to 40% even at a multiplicity of 0.1 PFU/cell. These results indicated that the multiplicity employed was a saturating dose of nonTABLE NUMBER OR MIXEDLY
Time bed
OF INFECTIVE
CENTERS
INFECTED CELLS W-IRRADIATION
Number
FORMED
BY SINGLY
AT VARIOUS OF t.S+ a
of infective centers lo5 cells ts+ b
None 10 20 30 60
1
14,300 (100) 11 (0.77) -
DOSES
OF
per 5 x
ts+ b + ts-3’ 49,400 24,000 8,892 4,400 450
(1001 (48.6) (18.0) (8.9) (0.9)
(1 Singly or mixedly infected cells were plated on MDBK monolayers as described in Materials and Methods. Plaque-forming infective centers were counted after I-day incubation at the nonpermissive temperature (39.5”). b Input multiplicity:0.05 PFU/cell (calculated from preirradiation titer). c Input multiplicity:O. 1 PFU/cell. Single infection with ts-3 at this multiplicity yielded 50 plaqueforming infective centers per 5 x lo5 cells plated.
368
NAKAJIMA
AND
plaque-forming but genetically competent virus particles, but the exact mechanism for the reduction of infective centers was not known. Because some mutants exerted a strong interfering effect upon the growth of ts+ virus at the nonpermissive temperature (Sugiura et al., 1975), the interference might be partially responsible for the observed inhibition. When cells were infected with the mixture of ts-3 and ts+ irradiated for various periods, the number of infective centers decreased with increasing uv dose, but at a much slower rate than the loss of infectivity of ts+. Deviation of the former from the latter is accounted for by the cross-reactivation of uv-ts+ by the portion of ts-3 viral genome that was functional at the nonpermissive temperature, or stated otherwise, by the replacement of the mutated gene of ts-3 genome by the corresponding gene of ts+ that had escaped a uv hit. Thus the decrease in I
I
SUGIURA
infective centers among mixedly infected cells represented the inactivation of the apability of ts+ to rescue ts-3 at the nonpermissive temperature. When the survival rate is plotted on a logarithmic scale against uv dose, it can be seen that the inactivation of both infectivity and rescue capability proceeds with single-hit kinetics (Fig. 1). The capability of ts+ to rescue ts-4 (Group II) and ts-15 (Group III) also declined as a single-hit curve, but the slope of the inactivation curve was steeper than for ts-3 (Group IV). It was examined, therefore, whether the difference in the inactivation rate was not accidental, but was based upon the genotypic difference among three recombination groups. Two to five mutant strains were chosen from each of seven recombination groups defined previously (Sugiura et al., 1975) and similar rescue experiments were repeated. The results presented in Fig. 2 show that the slopes of inactivation curves were reasonably close together for mutants of the same group, and that the inactivation rate was characteristic of each group of mutants. The only exception to the former conclusion was Group IV mutants. Comparison of Znactivation Kinetics for the Rescue of Single and Double Mutan ts
1 \ ,t
10
/
20
I
30
UV IRRADIATION
40 TIME.
50
60
SECONDS
FIG. 1. Kinetics of uv inactivation of the capability of wild-type WSN (ts+) to rescue ts mutants. The UV-ts+ was mixed with each ts mutant as described in the legend to Table 1. The number of plaqueforming infective centers on plating cells mixedly infected with unirradiated ts+ and a ts mutant was taken as 100% (see Table 11. Values for mixtures consisting of uv-ts+ and mutants were plotted on a logarithmic scale on the ordinate against uv dose on the abscissa. The dotted line marked as ts+ shows the inactivation of ts+ infectivity. Throughout all figures in this paper G is the abbreviation for Group. G-II (ts-4), x; G-III (ts-15), 0; G-IV (ts-3), 0.
The finding that the capability of ts+ to rescue all mutants belonging to a particular recombination group was lost at the same rate suggested that the inactivation rate reflected the target size of the mutated gene to UV irradiation. If this is the case, the inactivation curve should be significantly steeper when a double mutant is rescued than when mutants containing either one of the mutated genes alone are tested. The ts-2 which possessed mutation in both Group II and Group III genes and single mutants, ts-4 (Group II) and ts-15 (Group III), were rescued with uv-ts+. The results presented in Fig. 3 show that the capability of ts+ to rescue the double mutant was more vulnerable to uv irradiation than that for either of the constituent single mutants, as expected. Group IV mutants were delineated into two classes showing different slopes. One class com-
A NEW 100
INFLUENZA
369
ts MUTANT
a G-I
c G-III
d G-N
f G-VI
g G-W
a,
/
-i?&-&
i0 UV IRRADIATION
20 30 10 TIME, SECONDS
20
30
FIG. 2. Kinetics of uv inactivation of the capability of wild-type WSN to rescue ts mutants of seven recombination groups. See the legend to Fig. 1. (a) G-I: ts-6, 0; ts-8, 0; h-54, x. (b) G-II: ts-4, 0; ts-7, 0; ts52, X; ts-57, A; ts-59, 0. (c) G-III: ts-5, 0; ts-15, 0; ts-101, x. (d) G-IV: ts-3, 0; ts-11, 0; ts-60, x; ts-64, A. (e) G-V: ts-12, 0; ts-56, 0. (f) G-VI: ts-GlS, 0; ts-104, 0. (g) G-VII: ts-51, 0; ts-62, 0.
prising h-3 and ts-64 was characterized by a significantly slower inactivation rate than the other consisting of La-11 and ts-60. From the relationship between single mutants and a double mutant (Fig. 31, it was suggested that ts-11 and ts-60 contained at least a second mutational lesion, in addition to the one in the Group IV gene, and that, since both ts-11 and ts-60 crossed readily with any mutants of the other six recombination groups, the second mutation resided in a still undefined gene (Group VIII). Segregation of a Single Mutant of a New Recombination-Complementation (Recombination) Group from Double Mutants We attempted, therefore, to segregate a single mutant of putative Group VIII from these double mutants, essentially follow-
ing the method employed by Ueda and Kilbourne (1976). However, a clone was considered to be a Group VIII single mutant if recombination and complementation occurred in its cross with Group IV single mutants (ts-3 and ts-64) and were absent when it was crossed with the parent double mutants (ts-11 and ts-60). MDBK cells were infected with the mixture of ts+ (1-3 PFU/cell) and either ts-11 or ts-60 (3 PFU/cell). The culture fluid harvested after 20 hr of incubation at the permissive temperature was plated on monolayer cultures of MDBK cells. Well-isolated plaques formed at the permissive temperature were picked. Eighty-one clones were collected from a cross between ts-11 and ts+, and 77 clones from two separate crosses between ts-60 and ts+. These clones were inoculated into MDBK cells in duplicate, one set of cultures being incu-
370
NAKAJIMA
AND
bated at the permissive temperature (34”) and the other at the nonpermissive temperature (39.5”) for 22 hr. Culture fluids were tested for HA activity. A clone was regarded as possessing ts character when 34” fluid titered 1:128 or higher, while 39.5 fluid titered 1:8 or less. Twenty out of 158 clones turned out to be ts. The 20 ts clones were then screened against the mutation in the Group IV gene in a simplified test of
UV IRRADIATION
TIME,
SECONDS
FIG. 3. Comparison of inactivation kinetics of ts+ rescue capability for single and double mutants. k-4 (G-II), x; ts-15 (G-III), 0; h-2 (G-II + III), 0.
GENETIC
CHARACTERIZATION
OF
CLONES
SUGIURA
complementation-recombination with ts64 (Group IV). Seven clones (A to G) complemented ts-64 and were, therefore, free from Group IV mutation. They were further plaque-purified and their genotypes were determined by pairwise crosses with mutants of known recombination groups. Surprisingly, all clones were found to have a mutation that had never been detected in their parents (Table 2). Four clones, B, C, D, and G, were double mutants of Group III and Group VIII. Clone E was a single mutant of Group III, and clone F was a double mutant of Group I and Group VIII. The reason for the unexpected appearance of Group I or Group III mutation was investigated in the experiment described in the following section. By whatever mechanism these additional mutations had arisen, they had to be eliminated by further segregation in order to obtain a Group VIII single mutant. Backcrossing of clone C (Group III + VIII) with ts+ and screening of progeny clones were conducted essentially in the same way as had been described. Nineteen clones were of ts character among 49 clones examined. They were screened by a simplified complementation-recombination test with each of ts-15 (Group III), ts-64 (Group IV), and ts-60 (Group IV + VIII). Three clones complemented ts-15 and ts-64, but not ts60. They belonged, therefore, to neither Group III nor Group IV, but still retained
TABLE 2 BY RECOMBINATION
Clone@
WITH PROTOTYPE
STRAINS”
Groups
A B C D E F G
I h-6
II h-4
III ts-15
IV k-64
V ts-12
fC + + + + -
+ + + + + + +
-d -
+ + + + + + +
+ + + + + + +
+
+ -
a The presence or absence of genetic recgmbination Materials and Methods. b Clones A and B were derived from ts-11; the other c Recombination frequency of 1% or higher. d Recombination frequency of less than 0.1%. e Not tested.
was clones
tested from
VI ts-61s
VII h-51
+ + + + + + + by ts-60.
standard
+ + + + + + + assay
IV + VIII b-60 n.d.’ + as described
in
A NEW
INFLUENZA
Group VIII mutation and were candidates for Group VIII single mutants. One of these clones with the least leakiness was plaque-purified, grown up into virus stocks, and designated as ts-GOS, because it had been derived from ts-60 by two cycles of segregation. Properties
of ts-60s
Plaquing efficiency of ts-60s at 39.5” was less than 5 x 10e6 of that at 34”. The genetic character was examined either by recombination test with mutants of other recombination groups or by simplified complementation-recombination test with other mutants of Group IV. Recombination occurred between ts-6OS and mutants of all other groups except ts-60 (Table 3); ts-6OS complemented ts-3 and ts-64 (Group IV) but not ts-11 and ts-60 (data not shown). From these results, we concluded that ts60s did not belong to any of preexisting seven recombination groups, but shared a mutational lesion only with ts-11 and ts60. The presence of a new recombination group, Group VIII, has now been confirmed. Both ts-11 and ts-60 were double mutants of Group IV and Group VIII and ts-60s was a segregant from ts-60 which had inherited only the latter mutation. The ultraviolet (uv) irradiation study has also confirmed the above conclusion. Target size of Group IV, VIII, and Group IV + VIII genes was compared by cross reactivaor uv-irradition with ts+, unirradiated ated. A more gradual decline of the capability of ts+ to rescue ts-60s (Group VIII) than ts-60 (Group IV + VIII) indicated the decreased target size of the mutated gene in the former (data not shown). The ts-60s also differed from its parent ts-60 in physiological characteristics. Unlike ts-60, TABLE RECOMBINATION
3
FREQUENCY AND
OTHER
(%)
BETWEEN
ts-60s
MUTANTS’
Groups I
II ts-4
III ts-15
IV ts-64
ts-6
,s:2
1.8
3.0
6.0
2.5
8.0
tsk
,::i
‘;I; ts-60
n Recombination scribed in Materials
10.0
16.0
frequency was calculated and Methods.
0.02
as de-
ts MUTANT
371
which failed to induce a detectable level of HA and neuraminidase activities at the nonpermissive temperature (Sugiura et al., 1975), infection of ts-60s resulted in the appearance of both activities at 39.5”, about tenfold lower than at 34”. Presence of Mixed Aggregates minidase-Defective Mutants
in Neura-
The backcross of either ts-11 or ts-60 with ts+ gave rise to progeny clones of novel genotype, Group I or Group III mutation, unrecognized in the parents (Table 2). In order to explain this puzzling finding, we made the following postulate. Group IV mutants were defective in neuraminidase, and virus particles budding from infected cells formed large aggregates at the nonpermissive temperature (Palese et al., 1974). Since these mutant vu-ions had a very low neuraminidase activity even when grown at the permissive temperature (Sugiura et al., 19721, it was conceivable that virus preparations consisted predominantly of aggregates of various sizes. If an additional mutation had been present or had occurred during subsequent passages in some virions, it would have passed unrecognized as long as it was complemented by virions of original genotype present in an aggregate. Although ts11 and ts-60 had both undergone several plaque-to-plaque passages, the predominance of aggregates would have virtually precluded effective cloning, thereby masking and perpetuating the presence of the additional mutation. The validity of this postulate was then tested, first by examining whether and to what extent the preparation of a neuraminidase-defective mutant contained aggregates by the method employed by Hirst (1973) and Hirst and Pons (1973). Mutant ts-11 was grown in the presence of [3Hluridine at the permissive temperature. Virus-containing culture fluid, either before or after the neuraminidase treatment, was immediately layered onto and centrifuged through a sucrose gradient. As shown in Fig. 4, both 3H label and the infectivity of ts-11 sedimented faster and in a broader peak than those of ts +, indicating the predominance of aggregates in the population. The treatment of
372
NAKAJIMA
b
AND
~-11
SUGIURA
(NAI
c
ts’
‘5-
-7
O-
-5
5-
-2
c 4 1 FRACTION
NO
IV + VIII) viruses labeled with FIG. 4. Velocity sedimentation patterns of ts+ and ts-11 (Group PHluridine. Both ts-11, either before (a) or after (b) the treatment with neuraminidase, and ts+ (c) viruses were centrifuged through sucrose gradients. Infectivity and radioactivity were determined for each fraction. Experimental details were described in Materials and Methods. TABLE ANALYSIS OF CLONES OR UNTREATED Neuraminidase treatment
+
4
DERIVED FROM b-11, WITH NEURAMINIDASE”
Number of clones isolated
24 23
TREATED
Number of clones not complementing ts-15” 0 3
a k-11 was treated with neuraminidase as described in Materials and Methods. b Examined by simplified complementation-recombination test as described in Materials and Methods.
ts-11 with bacterial neuraminidase restored the sedimentation pattern to the one similar to that of ts+, demonstrating the effectiveness of neuraminidase in dispersing aggregates. We examined, next, whether some virions in ts-I1 population contained a hidden mutational lesion which might be unmasked through dispersal with neuraminidase treatment. Stock virus of ts-11 was treated with either bacterial neuraminidase or PBS and plated for plaque isolation. Clones were grown once in the fluid medium and tested for the presence of
Group III mutation by a complementationrecombination test with ts-15 (Group III) (Table 4). All clones descended from untreated ts-11 complemented ts-15, while three out of 23 clones derived from neuraminidase-treated sample failed to complement ts-15. Such clones as these three must have been responsible for the results in Table 2 showing that the backcross of ts-11 with ts+ gave rise to clones containing Group III mutation. The above experiments have shown the presence of mixed aggregates composed at least of double (Group IV + VIII) and triple (Group III + IV + VIII) mutants in ts-11 population. DISCUSSION
Ultraviolet-irradiated ts+ was capable of replacing mutated genes of ts mutants and of giving rise to progeny virus of wild-type character. The inactivation of this ability by uv irradiation followed a single-hit kinetics, and was fairly constant when different virus strains of the same recombination group were rescued, whereas it varied when mutants of different groups were tested. Furthermore, the inactivation occurred more rapidly when a double mutant
A NEW
INFLUENZA
373
ts MUTANT
was rescued than when either of its constituent mutants was rescued. The inactivation rate is, thus, a measure of radiation sensitivity of a rescuing gene, and, as a consequence, indirectly reflects the size of
within a mixed aggregate. Attempts to eliminate Group IV mutation from double mutants (Group IV t VIII) more often
the gene to be replaced. It would be difficult to apply this method for estimating and comparing target size of various genes because of the uncertainty as to whether different RNA species or genes are represented in an equal molar ratio in a given rescuing virus preparation or not (Palese and Schulman, 1976). The method, however, proved to be useful at least for uncovering the unknown mutation in two mutants, previously considered genotypically identical to the other Group IV mutants. A mutant of a new recombination group, ts-GOS, was obtained by segregation from a double mutant ts-60. Eight recombination groups have now been defined from ts mutants isolated from WSN (Sugiura et al., 1972, 1975). The ts mutants of the same virus collected by other groups of investigators (Simpson and Hirst, 1968; Hirst, 1973) had also been classified into eight groups by genetic characterization. Ribonucleic acid extracted from purified influenza virus particles has been resolved into eight RNA species by electrophoresis (Pons, 1976; Bean and Simpson, 1976; Content, 1976; Palese and Schulman, 1976; McGeoch et al., 1976; Scholtissek et al., 1976). Data from both genetic and biophysical studies are thus in good agreement. Which polypeptide the newly defined Group VIII gene codes for awaits a future study. Since both ts-11 and ts-60 (Group IV + VIII) synthesize virion-type RNA at the nonpermissive temperature (Sugiura et al., 1975), Group VIII gene is required presumably for a step after viral RNA synthesis. Segregation of ts-60s from ts-60 was accompanied by a decrease in the target size of the mutated gene. The attempted segregation was complicated by repeated appearances of genotypes not manifest in the parent viruses. Probably the new genotypes did not result from a spontaneous mutation during the process as had been experienced by Hirst (1973), but arose from uncovering of a preexisting mutation that had been masked by complementation
VIII) than single mutants (Group I, Group III, or Group VIII). The reason may have been that we used a criterion for the ts character too stringent for single mutants, favoring double mutants which tended to be less leaky and/or less reverting. Actually, among three clones with a mutation in Group VIII gene alone, obtained after the second cycle of segregation, only one had ts character of a sufficient stability. Velocity sedimentation analysis demonstrated that aggregated virus particles predominated in the preparation of ts-11. Preparations of other Group IV mutants (ts-3, ts-60, and ts-64) similarly contained more fast-sedimenting particles than ts+ or mutants of other recombination groups (M. Ueda, personal communication). Predominance of aggregates was caused most likely by incomplete desialylation of budding virus particles due to a low neuraminidase activity of Group IV mutants (Sugiura et al., 1972; Palese et al., 1974). The preparation of a neuraminidase-defective virus was, thus, likely to be a heterogeneous population containing, in mixed aggregates, virus particles with mutations in various other genes, in addition to the Group IV gene. The mutation in other genes might have been present from the beginning or might as well have been gained spontaneously thereafter. It may be hardly accidental, therefore, that a mutation in a hitherto unknown gene had been carried by only Group IV mutants, and by two of them.
yielded double mutants of other combinations (Group III t VIII and Group I +
REFERENCES BARRY, R. D. (1961). The multiplication of influenza virus. II. Multiplicity reactivation of ultraviolet irradiated virus. Virology 14, 398-405. BEAN, W. J. and SIMPSON, R. W. (1976). Transcriptase activity and genome composition of defective influenza virus. J. Viral. 18, 365-369. CONTENT, J. (1976). Cell-free translation of influenza virus mRNA. J. Virol. 18, 604-618. HIRST, G. K. (1973). Mechanism of influenza recom-
NAKAJIMA
AND SUGIURA
bination. I. Factors influencing recombination rates between temperature-sensitive mutants of strain WSN and the classification of mutants into complementation-recombination groups. Virology 55, 81-93. HIRST, G. K. and PONS, M. W. (1973). Mechanism of influenza recombination. II. Virus aggregation and its effect on plaque formation by so-called noninfective virus. Virology 56, 620-631. MCCAHON, D. and SCHILD, G. C. (1971). An investigation of some factors affecting cross-reactivation between influenza A viruses. J. Gen. Viral. 12, 207-219. MCGEOCH, D., FELLNER, P., and NEWTON, C. (1976). Influenza virus genome consists of eight distinct RNA species. Proc. Nat. Acad. Sci. USA 73,30453049.
PALESE, P., TOBITA, K., UEDA, M., and COMPANS, R. W. (1974). Characterization of temperature-sensitive influenza virus mutants defective in neuraminidase. ViroZogy 61, 397-410. PALESE, P. and SHIJLMAN, J. L. (1976). Differences in RNA patterns of influenza A virus. J. Viral. 17, 876-884. PALESE, P. and SHULMAN, J. L. (1976). Mapping of the influenza virus genome. Proc. Nat. Acad. Sci. USA 73, 2142-2146. PONS, M. W. (1976). A reexamination of influenza single- and double-stranded RNA by gel electrophoresis. Virology 69, 789-792. SIMPSON, R. W. and HIR~T, G. K. (1961). Genetic recombination among influenza virus. I. Cross reactivation of plaque-forming capacity as a method for selecting recombinants from the prog-
eny of crosses between influenza A strains. Virology 15, 436-451. SIMPSON, R. W. and HIR~T, G. K. (1968). Temperature-sensitive mutants of influenza A virus: isolation of mutants and preliminary observation on genetic recombination and complementation. Virology 35, 41-49. SCHOLTISSEK, C., HARMS, E., ROHDE, W., ORLICH, M., and ROTT, R. (1976). Correlation between RNA fragments of fowl plague virus and their corresponding gene functions. Virology 74, 332344. SUGIURA, A. and KILBOURNE, E. D. (1966). Genetic studies of influenza virus. III. Production of plaque-type recombinants with Ao and Al strains. Virology 29, 84-91. SUGIURA, A. and UEDA, M. (1971). Marker rescue with ultraviolet-inactivated influenza virus. J. Viral.
7, 499-503.
SUGIURA, A., TOBITA, K., and KILBOURNE, E. D. (1972). Isolation and preliminary characterization of temperature-sensitive mutants of influenza virus. J. Viral. 10, 639-647. SUGIURA, A., UEDA, M., TOBITA, K., and ENOMOTO, C. (1975). Further isolation and characterization of temperature-sensitive mutants of influenza virus. Virology 65, 363-373. TUMOVA, B. and PEREIRA, H. G. (1965). Genetic interaction between influenza A viruses of human and animal origin. Virology 27, 253-261. UEDA, M. and KILBOURNE, E. D. (1976). Temperature-sensitive mutants of influenza virus: a mutation in the hemagglutinin gene. Virology 70, 425-431.
78,
365-374
(1977)
Isolation of an Influenza Virus Temperature-Sensitive Mutant of a New Recombination-Complementation Group K. NAKAJIMA Department
of Microbiology,
The Znstitute
AND
of Public Tokyo 108,
Accepted
January
A. SUGIURA Health, Japan
6-1, Shirokanedai
I-chome,
Minutoku,
3,1977
Wild-type WSN of influenza A virus which had been inactivated by ultraviolet (uv) irradiation was capable, to various extents, of rescuing temperature-sensitive (ts) mutants of the same virus, resulting in the production of wild-type recombinants at the nonpermissive temperature. The capability of wild-type virus to rescue ts mutants decreased with increasing uv dose following single-hit kinetics. Inactivation of rescue capability varied when mutants of different recombination-complementation (recombination) groups were compared, but was nearly identical to different members of one recombination group, except for Group IV mutants. The inactivation rate was significantly greater when it was determined with two mutants (ts-11 and ~9-60) than when determined with the rest of the Group IV mutants. Because the inactivation rate was expected to be greater for a double mutant than for virus strains having either one of two mutational lesions alone, it was suspected that ts-11 and ts-60 had an additional mutational lesion in a still undeiined gene. Segregation of a new mutant from ts-60 was, therefore, attempted by backcrossing it with wild-type WSN and screening progeny clones. A clone which had lost Group IV mutation during the process but still retained temperature sensitivity had been obtained. Genetic crossing of this segregant, ts-GOS, with mutants of all seven other recombination groups gave rise to recombinants of wildtype character, indicating that ts-60s was a mutant of unknown recombination group (Group VIII). Both ts-11 and ts-60 were confirmed to be double mutants (Groups IV + VIII). During the process of segregation, a number of progeny clones with a novel genotype were obtained which had not been manifest in the parent viruses, i.e., the mutation in either the group I or Group III gene, in addition ta the one in the Group VIII gene. It was found that these paradoxical phenomena were caused by mixed aggregates present in the parent viruses, b-11 and ts-60. Mixed aggregates contained, at least, triple mutants (either Groups I + IV + VIII or Groups III + IV + VIII) and the double mutant (Groups IV + VIII). The preparation of ts-11 predominantly showed aggregates which sedimented faster than wild-type virus particles and which could be dispersed by the treatment with neuraminidase. Aggregates were formed most likely as a result of incomplete desialylation due to low neuraminidase activity of Group IV mutants. INTRODUCTION
Our collection of temperature-sensitive (ts) mutants derived from the WSN strain of influenza A virus had been classified into seven recombination-complementation (recombination) groups (Sugiura et al., 1975). Since influenza A genome RNA was resolved into eight RNA species by electrophoresis (Pans, 1976; Bean and Simpson, 1976; Content, 1976; Palese and Schulman, 1976; McGeoch et al., 1976; Scholtissek et al., 19761, the eighth mutant Copyright All rights
0 197'7 by Academic Press, Inc. of reproduction in any form reserved.
group was still to be found. Some mutants, although considered to be single at present, might possibly be double mutants involving the putative Group VIII gene. We tried, therefore, to test the singularity of mutation utilizing cross-reactivation. Multiplicity reactivation (Barry, 1961) and cross reactivation (Simpson and Hirst, 1961; Tumova and Pereira, 1965; Sugiura and Kilbourne, 1966; McCahon and Schild, 1971; Sugiura and Ueda, 1971) are the particular forms of genetic recombination
365 ISSN
0042-6822
366
NAKAJIMA
which involve parent virus inactivated by damage to the viral genome. A ts mutant would also be rescued by ultraviolet (uv)irradiated wild-type virus, when a gene in the latter that has escaped a uv hit replaces the corresponding gene in the mutant and thereby restores its reproductive capacity at the nonpermissive temperature. The ability of wild-type virus to rescue a particular mutant would be abolished by uv irradiation at the rate reflecting the target size of the mutated gene. The target size of the mutated portion of the genome in a double mutant is expected to be greater than that of constituent single mutants. The experiments described here show the validity of the assumption and revealed that two of the Group IV mutants might have an additional mutation in a still unidentified gene. This hypothesis has been verified by segregation of a mutant belonging to a new recombination group. We also describe a genetically anomalous behavior of Group IV mutants which could be attributed to the low neuraminidase activity of the mutants. MATERIALS
AND
METHODS
Cells and virus. MDBK
cells were used. The media employed for propagation and maintenance of cells have been described previously (Sugiura et al., 19721, except that the growth medium contained heatinactivated newborn calf serum, kindly provided by the Mitsubishi - Kasei Institute of Life Sciences, instead of fetal calf serum. The wild-type virus (ts+) and ts mutants of the WSN strain of influenza A virus have also been described elsewhere (Sugiura et al., 1972, 1975). Ultraviolet-irradiated virus. The ts+ was diluted tenfold in phosphate-buffered saline (PBS) containing 0.2% bovine serum albumin. The virus was distributed in 5ml amounts in lo-cm petri dishes and was irradiated by a 15-W germicidal lamp at a distance of 20 cm for various time periods, during which the open dishes were constantly rocked. Reactivation of ts mutants diated ts+ (uv-ts+) . Virus
with
uv-irra-
titration was performed by counting plaque-forming infective centers. Virus mixture containing
AND
SUGIURA
2.5 x lo4 PFU (calculated from preirradiation titer) of uv-ts+ and 5 x lo4 PF’U of a ts mutant in 0.2 ml was prepared. A suspension of 5 x lo5 freshly trypsinized cells in 0.2 ml was combined with the above mixture. Input multiplicity was 0.05 PFU/cell for uv-ts+ and 0.1 PFU/cell for a ts mutant. Virus adsorption to cells was allowed to proceed for 30 min at 37” with occasional shaking. Cells were washed and resuspended in fresh maintenance medium. Serial dilutions were prepared in fivefold steps and 0.1 ml of the cell suspension was deposited on drained monolayer cultures in 6-cm petri dishes. Inoculated cells were spread over the entire surface by shaking the dish. After incubation at 37” for 30 min for attachment of cells to the monolayer, agar overlay medium was gently added and the cultures were incubated at a nonpermissive temperature (39.5”). Plaques were counted after 4 days of incubation. Recombination test. The presence or absence of genetic recombination was determined by standard assay. Briefly, cells were infected with two mutants, both at a multiplicity of l-5 PFU/cell, and were incubated at 34”. At 3 hr, the cultures were treated with rabbit anti-WSN antiserum diluted to l:l,OOO for 10 min, washed three times with PBS containing 0.2% bovine serum albumin, and fed with fresh maintenance medium. Incubation was continued at 34”. The culture fluid was harvested at 10 hr after infection. Appropriate controls, namely, cultures singly infected with each mutant, were included. The progeny virus produced during one-step growth was titrated by plaque assay at two temperatures. Recombination frequency was calculated by the following formula (Sugiura et al., 1972):
where [AB3JS4 and [AB34]39.5are the titers of a mixed yield grown at 34” and assayed at 34” and 39.5”, respectively, and [A34139.5 and [B34139.5 are the titers of single yields grown at 34” and assayed at 39.5”. Mixed infection for simplified complementation-recombination.
cells were infected with mutants
test of
MDBK individ-
A NEW
INFLUENZA
ually or in combination. The multiplicity was 0.2 PFU/cell for each mutant. After virus adsorption for 30 min at 37” cultures received maintenance medium and were immersed in the water bath maintained at 39.5” (*O.lO). At 22-24 hr, culture fluids were harvested and titrated for hemagglutination (HA) activity (Sugiura et al., 1975). The criterion of positive complementation-recombination was an HA titer at least eightfold higher in the mixedly infected culture than the sum of the HA titers in singly infected cultures. Activity of HA and neuraminidase was measured as previously described (Sugiura et al., 1972). Velocity sedimentation of virus. MDBK cells in 6-cm petri dishes were infected with either ts+ or ts-11 at a multiplicity of 1 PFU/cell at 34”. At 3 hr, 10 $i of L3Hluridine (42.5 Ci/mmole, New England Nuclear) was added per dish, and incubation was continued at 34” overnight. After the culture fluid was clarified at 6000 rpm for 20 min at 4”, the supernatant was treated with neuraminidase. The supernatant was layered onto the lo-40% (w/v) linear sucrose gradient in 0.1 M NaCl, 0.001 it4 EDTA, 0.01 M Tris-HCI (pH 7.21, and was centrifuged at 24,000 rpm for 50 min at 4” in a Spinco SW 27 rotor. Fractions of 1.4 ml each were collected from the bottom. One-milliliter aliquots were used for determination of acid-insoluble radioactivity in an Aloka Model LSC-601 scintillation spectrometer. The remaining portion was tested for infectivity by plaque assay at the permissive temperature. Neuraminidase treatment of ts-11. One milliliter of virus preparation was incubated with 50 units of V. cholerae neuraminidase (Behringwerke, Marburg, Germany) for 30 min at 37”. RESULTS
Rescue of ts Mutants and uv-Irradiated
When
with ts +
Unirradiated
cells infected with unirradiated for infective centers, the number of plaques formed was 57% of input PFU (5 x lo5 cells infected with ts+ at a multiplicity of 0.05 PFU/cell should have given 25,000 infective centers; Table 1). ts+ were plated
367
ts MUTANT
Since cultures for infective center assay were incubated at the nonpermissive temperature, at which temperature the plaquing efficiency of ts+ was between 25 and 40% of that at the permissive temperature (Sugiura et al., 19721, adsorption of input virus to cells in suspension was nearly complete. Coinfection with ts-3 resulted in a 3.4-fold increase in the number of infective centers, although the single infection with this mutant at the same multiplicity yielded only a small number of infective centers at the nonpermissive temperature. Most mutants caused a variable degree of increment, up to fivefold. Additional infective centers must .have been initiated by ts+-type recombinants resulting from interaction between non-plaque-forming, but genetically competent virus particles contained by ts+ and those contained by a mutant virus preparation (Hirst, 1973). Throughout this study, cells were infected with ts mutants at the multiplicity of 0.1 PFU/cell. With many mutants, a higher multiplicity led to the reduction in infective centers among mixedly infected cells. A few mutants (ts-52 and ts-57) depressed the number of infective centers by 20 to 40% even at a multiplicity of 0.1 PFU/cell. These results indicated that the multiplicity employed was a saturating dose of nonTABLE NUMBER OR MIXEDLY
Time bed
OF INFECTIVE
CENTERS
INFECTED CELLS W-IRRADIATION
Number
FORMED
BY SINGLY
AT VARIOUS OF t.S+ a
of infective centers lo5 cells ts+ b
None 10 20 30 60
1
14,300 (100) 11 (0.77) -
DOSES
OF
per 5 x
ts+ b + ts-3’ 49,400 24,000 8,892 4,400 450
(1001 (48.6) (18.0) (8.9) (0.9)
(1 Singly or mixedly infected cells were plated on MDBK monolayers as described in Materials and Methods. Plaque-forming infective centers were counted after I-day incubation at the nonpermissive temperature (39.5”). b Input multiplicity:0.05 PFU/cell (calculated from preirradiation titer). c Input multiplicity:O. 1 PFU/cell. Single infection with ts-3 at this multiplicity yielded 50 plaqueforming infective centers per 5 x lo5 cells plated.
368
NAKAJIMA
AND
plaque-forming but genetically competent virus particles, but the exact mechanism for the reduction of infective centers was not known. Because some mutants exerted a strong interfering effect upon the growth of ts+ virus at the nonpermissive temperature (Sugiura et al., 1975), the interference might be partially responsible for the observed inhibition. When cells were infected with the mixture of ts-3 and ts+ irradiated for various periods, the number of infective centers decreased with increasing uv dose, but at a much slower rate than the loss of infectivity of ts+. Deviation of the former from the latter is accounted for by the cross-reactivation of uv-ts+ by the portion of ts-3 viral genome that was functional at the nonpermissive temperature, or stated otherwise, by the replacement of the mutated gene of ts-3 genome by the corresponding gene of ts+ that had escaped a uv hit. Thus the decrease in I
I
SUGIURA
infective centers among mixedly infected cells represented the inactivation of the apability of ts+ to rescue ts-3 at the nonpermissive temperature. When the survival rate is plotted on a logarithmic scale against uv dose, it can be seen that the inactivation of both infectivity and rescue capability proceeds with single-hit kinetics (Fig. 1). The capability of ts+ to rescue ts-4 (Group II) and ts-15 (Group III) also declined as a single-hit curve, but the slope of the inactivation curve was steeper than for ts-3 (Group IV). It was examined, therefore, whether the difference in the inactivation rate was not accidental, but was based upon the genotypic difference among three recombination groups. Two to five mutant strains were chosen from each of seven recombination groups defined previously (Sugiura et al., 1975) and similar rescue experiments were repeated. The results presented in Fig. 2 show that the slopes of inactivation curves were reasonably close together for mutants of the same group, and that the inactivation rate was characteristic of each group of mutants. The only exception to the former conclusion was Group IV mutants. Comparison of Znactivation Kinetics for the Rescue of Single and Double Mutan ts
1 \ ,t
10
/
20
I
30
UV IRRADIATION
40 TIME.
50
60
SECONDS
FIG. 1. Kinetics of uv inactivation of the capability of wild-type WSN (ts+) to rescue ts mutants. The UV-ts+ was mixed with each ts mutant as described in the legend to Table 1. The number of plaqueforming infective centers on plating cells mixedly infected with unirradiated ts+ and a ts mutant was taken as 100% (see Table 11. Values for mixtures consisting of uv-ts+ and mutants were plotted on a logarithmic scale on the ordinate against uv dose on the abscissa. The dotted line marked as ts+ shows the inactivation of ts+ infectivity. Throughout all figures in this paper G is the abbreviation for Group. G-II (ts-4), x; G-III (ts-15), 0; G-IV (ts-3), 0.
The finding that the capability of ts+ to rescue all mutants belonging to a particular recombination group was lost at the same rate suggested that the inactivation rate reflected the target size of the mutated gene to UV irradiation. If this is the case, the inactivation curve should be significantly steeper when a double mutant is rescued than when mutants containing either one of the mutated genes alone are tested. The ts-2 which possessed mutation in both Group II and Group III genes and single mutants, ts-4 (Group II) and ts-15 (Group III), were rescued with uv-ts+. The results presented in Fig. 3 show that the capability of ts+ to rescue the double mutant was more vulnerable to uv irradiation than that for either of the constituent single mutants, as expected. Group IV mutants were delineated into two classes showing different slopes. One class com-
A NEW 100
INFLUENZA
369
ts MUTANT
a G-I
c G-III
d G-N
f G-VI
g G-W
a,
/
-i?&-&
i0 UV IRRADIATION
20 30 10 TIME, SECONDS
20
30
FIG. 2. Kinetics of uv inactivation of the capability of wild-type WSN to rescue ts mutants of seven recombination groups. See the legend to Fig. 1. (a) G-I: ts-6, 0; ts-8, 0; h-54, x. (b) G-II: ts-4, 0; ts-7, 0; ts52, X; ts-57, A; ts-59, 0. (c) G-III: ts-5, 0; ts-15, 0; ts-101, x. (d) G-IV: ts-3, 0; ts-11, 0; ts-60, x; ts-64, A. (e) G-V: ts-12, 0; ts-56, 0. (f) G-VI: ts-GlS, 0; ts-104, 0. (g) G-VII: ts-51, 0; ts-62, 0.
prising h-3 and ts-64 was characterized by a significantly slower inactivation rate than the other consisting of La-11 and ts-60. From the relationship between single mutants and a double mutant (Fig. 31, it was suggested that ts-11 and ts-60 contained at least a second mutational lesion, in addition to the one in the Group IV gene, and that, since both ts-11 and ts-60 crossed readily with any mutants of the other six recombination groups, the second mutation resided in a still undefined gene (Group VIII). Segregation of a Single Mutant of a New Recombination-Complementation (Recombination) Group from Double Mutants We attempted, therefore, to segregate a single mutant of putative Group VIII from these double mutants, essentially follow-
ing the method employed by Ueda and Kilbourne (1976). However, a clone was considered to be a Group VIII single mutant if recombination and complementation occurred in its cross with Group IV single mutants (ts-3 and ts-64) and were absent when it was crossed with the parent double mutants (ts-11 and ts-60). MDBK cells were infected with the mixture of ts+ (1-3 PFU/cell) and either ts-11 or ts-60 (3 PFU/cell). The culture fluid harvested after 20 hr of incubation at the permissive temperature was plated on monolayer cultures of MDBK cells. Well-isolated plaques formed at the permissive temperature were picked. Eighty-one clones were collected from a cross between ts-11 and ts+, and 77 clones from two separate crosses between ts-60 and ts+. These clones were inoculated into MDBK cells in duplicate, one set of cultures being incu-
370
NAKAJIMA
AND
bated at the permissive temperature (34”) and the other at the nonpermissive temperature (39.5”) for 22 hr. Culture fluids were tested for HA activity. A clone was regarded as possessing ts character when 34” fluid titered 1:128 or higher, while 39.5 fluid titered 1:8 or less. Twenty out of 158 clones turned out to be ts. The 20 ts clones were then screened against the mutation in the Group IV gene in a simplified test of
UV IRRADIATION
TIME,
SECONDS
FIG. 3. Comparison of inactivation kinetics of ts+ rescue capability for single and double mutants. k-4 (G-II), x; ts-15 (G-III), 0; h-2 (G-II + III), 0.
GENETIC
CHARACTERIZATION
OF
CLONES
SUGIURA
complementation-recombination with ts64 (Group IV). Seven clones (A to G) complemented ts-64 and were, therefore, free from Group IV mutation. They were further plaque-purified and their genotypes were determined by pairwise crosses with mutants of known recombination groups. Surprisingly, all clones were found to have a mutation that had never been detected in their parents (Table 2). Four clones, B, C, D, and G, were double mutants of Group III and Group VIII. Clone E was a single mutant of Group III, and clone F was a double mutant of Group I and Group VIII. The reason for the unexpected appearance of Group I or Group III mutation was investigated in the experiment described in the following section. By whatever mechanism these additional mutations had arisen, they had to be eliminated by further segregation in order to obtain a Group VIII single mutant. Backcrossing of clone C (Group III + VIII) with ts+ and screening of progeny clones were conducted essentially in the same way as had been described. Nineteen clones were of ts character among 49 clones examined. They were screened by a simplified complementation-recombination test with each of ts-15 (Group III), ts-64 (Group IV), and ts-60 (Group IV + VIII). Three clones complemented ts-15 and ts-64, but not ts60. They belonged, therefore, to neither Group III nor Group IV, but still retained
TABLE 2 BY RECOMBINATION
Clone@
WITH PROTOTYPE
STRAINS”
Groups
A B C D E F G
I h-6
II h-4
III ts-15
IV k-64
V ts-12
fC + + + + -
+ + + + + + +
-d -
+ + + + + + +
+ + + + + + +
+
+ -
a The presence or absence of genetic recgmbination Materials and Methods. b Clones A and B were derived from ts-11; the other c Recombination frequency of 1% or higher. d Recombination frequency of less than 0.1%. e Not tested.
was clones
tested from
VI ts-61s
VII h-51
+ + + + + + + by ts-60.
standard
+ + + + + + + assay
IV + VIII b-60 n.d.’ + as described
in
A NEW
INFLUENZA
Group VIII mutation and were candidates for Group VIII single mutants. One of these clones with the least leakiness was plaque-purified, grown up into virus stocks, and designated as ts-GOS, because it had been derived from ts-60 by two cycles of segregation. Properties
of ts-60s
Plaquing efficiency of ts-60s at 39.5” was less than 5 x 10e6 of that at 34”. The genetic character was examined either by recombination test with mutants of other recombination groups or by simplified complementation-recombination test with other mutants of Group IV. Recombination occurred between ts-6OS and mutants of all other groups except ts-60 (Table 3); ts-6OS complemented ts-3 and ts-64 (Group IV) but not ts-11 and ts-60 (data not shown). From these results, we concluded that ts60s did not belong to any of preexisting seven recombination groups, but shared a mutational lesion only with ts-11 and ts60. The presence of a new recombination group, Group VIII, has now been confirmed. Both ts-11 and ts-60 were double mutants of Group IV and Group VIII and ts-60s was a segregant from ts-60 which had inherited only the latter mutation. The ultraviolet (uv) irradiation study has also confirmed the above conclusion. Target size of Group IV, VIII, and Group IV + VIII genes was compared by cross reactivaor uv-irradition with ts+, unirradiated ated. A more gradual decline of the capability of ts+ to rescue ts-60s (Group VIII) than ts-60 (Group IV + VIII) indicated the decreased target size of the mutated gene in the former (data not shown). The ts-60s also differed from its parent ts-60 in physiological characteristics. Unlike ts-60, TABLE RECOMBINATION
3
FREQUENCY AND
OTHER
(%)
BETWEEN
ts-60s
MUTANTS’
Groups I
II ts-4
III ts-15
IV ts-64
ts-6
,s:2
1.8
3.0
6.0
2.5
8.0
tsk
,::i
‘;I; ts-60
n Recombination scribed in Materials
10.0
16.0
frequency was calculated and Methods.
0.02
as de-
ts MUTANT
371
which failed to induce a detectable level of HA and neuraminidase activities at the nonpermissive temperature (Sugiura et al., 1975), infection of ts-60s resulted in the appearance of both activities at 39.5”, about tenfold lower than at 34”. Presence of Mixed Aggregates minidase-Defective Mutants
in Neura-
The backcross of either ts-11 or ts-60 with ts+ gave rise to progeny clones of novel genotype, Group I or Group III mutation, unrecognized in the parents (Table 2). In order to explain this puzzling finding, we made the following postulate. Group IV mutants were defective in neuraminidase, and virus particles budding from infected cells formed large aggregates at the nonpermissive temperature (Palese et al., 1974). Since these mutant vu-ions had a very low neuraminidase activity even when grown at the permissive temperature (Sugiura et al., 19721, it was conceivable that virus preparations consisted predominantly of aggregates of various sizes. If an additional mutation had been present or had occurred during subsequent passages in some virions, it would have passed unrecognized as long as it was complemented by virions of original genotype present in an aggregate. Although ts11 and ts-60 had both undergone several plaque-to-plaque passages, the predominance of aggregates would have virtually precluded effective cloning, thereby masking and perpetuating the presence of the additional mutation. The validity of this postulate was then tested, first by examining whether and to what extent the preparation of a neuraminidase-defective mutant contained aggregates by the method employed by Hirst (1973) and Hirst and Pons (1973). Mutant ts-11 was grown in the presence of [3Hluridine at the permissive temperature. Virus-containing culture fluid, either before or after the neuraminidase treatment, was immediately layered onto and centrifuged through a sucrose gradient. As shown in Fig. 4, both 3H label and the infectivity of ts-11 sedimented faster and in a broader peak than those of ts +, indicating the predominance of aggregates in the population. The treatment of
372
NAKAJIMA
b
AND
~-11
SUGIURA
(NAI
c
ts’
‘5-
-7
O-
-5
5-
-2
c 4 1 FRACTION
NO
IV + VIII) viruses labeled with FIG. 4. Velocity sedimentation patterns of ts+ and ts-11 (Group PHluridine. Both ts-11, either before (a) or after (b) the treatment with neuraminidase, and ts+ (c) viruses were centrifuged through sucrose gradients. Infectivity and radioactivity were determined for each fraction. Experimental details were described in Materials and Methods. TABLE ANALYSIS OF CLONES OR UNTREATED Neuraminidase treatment
+
4
DERIVED FROM b-11, WITH NEURAMINIDASE”
Number of clones isolated
24 23
TREATED
Number of clones not complementing ts-15” 0 3
a k-11 was treated with neuraminidase as described in Materials and Methods. b Examined by simplified complementation-recombination test as described in Materials and Methods.
ts-11 with bacterial neuraminidase restored the sedimentation pattern to the one similar to that of ts+, demonstrating the effectiveness of neuraminidase in dispersing aggregates. We examined, next, whether some virions in ts-I1 population contained a hidden mutational lesion which might be unmasked through dispersal with neuraminidase treatment. Stock virus of ts-11 was treated with either bacterial neuraminidase or PBS and plated for plaque isolation. Clones were grown once in the fluid medium and tested for the presence of
Group III mutation by a complementationrecombination test with ts-15 (Group III) (Table 4). All clones descended from untreated ts-11 complemented ts-15, while three out of 23 clones derived from neuraminidase-treated sample failed to complement ts-15. Such clones as these three must have been responsible for the results in Table 2 showing that the backcross of ts-11 with ts+ gave rise to clones containing Group III mutation. The above experiments have shown the presence of mixed aggregates composed at least of double (Group IV + VIII) and triple (Group III + IV + VIII) mutants in ts-11 population. DISCUSSION
Ultraviolet-irradiated ts+ was capable of replacing mutated genes of ts mutants and of giving rise to progeny virus of wild-type character. The inactivation of this ability by uv irradiation followed a single-hit kinetics, and was fairly constant when different virus strains of the same recombination group were rescued, whereas it varied when mutants of different groups were tested. Furthermore, the inactivation occurred more rapidly when a double mutant
A NEW
INFLUENZA
373
ts MUTANT
was rescued than when either of its constituent mutants was rescued. The inactivation rate is, thus, a measure of radiation sensitivity of a rescuing gene, and, as a consequence, indirectly reflects the size of
within a mixed aggregate. Attempts to eliminate Group IV mutation from double mutants (Group IV t VIII) more often
the gene to be replaced. It would be difficult to apply this method for estimating and comparing target size of various genes because of the uncertainty as to whether different RNA species or genes are represented in an equal molar ratio in a given rescuing virus preparation or not (Palese and Schulman, 1976). The method, however, proved to be useful at least for uncovering the unknown mutation in two mutants, previously considered genotypically identical to the other Group IV mutants. A mutant of a new recombination group, ts-GOS, was obtained by segregation from a double mutant ts-60. Eight recombination groups have now been defined from ts mutants isolated from WSN (Sugiura et al., 1972, 1975). The ts mutants of the same virus collected by other groups of investigators (Simpson and Hirst, 1968; Hirst, 1973) had also been classified into eight groups by genetic characterization. Ribonucleic acid extracted from purified influenza virus particles has been resolved into eight RNA species by electrophoresis (Pons, 1976; Bean and Simpson, 1976; Content, 1976; Palese and Schulman, 1976; McGeoch et al., 1976; Scholtissek et al., 1976). Data from both genetic and biophysical studies are thus in good agreement. Which polypeptide the newly defined Group VIII gene codes for awaits a future study. Since both ts-11 and ts-60 (Group IV + VIII) synthesize virion-type RNA at the nonpermissive temperature (Sugiura et al., 1975), Group VIII gene is required presumably for a step after viral RNA synthesis. Segregation of ts-60s from ts-60 was accompanied by a decrease in the target size of the mutated gene. The attempted segregation was complicated by repeated appearances of genotypes not manifest in the parent viruses. Probably the new genotypes did not result from a spontaneous mutation during the process as had been experienced by Hirst (1973), but arose from uncovering of a preexisting mutation that had been masked by complementation
VIII) than single mutants (Group I, Group III, or Group VIII). The reason may have been that we used a criterion for the ts character too stringent for single mutants, favoring double mutants which tended to be less leaky and/or less reverting. Actually, among three clones with a mutation in Group VIII gene alone, obtained after the second cycle of segregation, only one had ts character of a sufficient stability. Velocity sedimentation analysis demonstrated that aggregated virus particles predominated in the preparation of ts-11. Preparations of other Group IV mutants (ts-3, ts-60, and ts-64) similarly contained more fast-sedimenting particles than ts+ or mutants of other recombination groups (M. Ueda, personal communication). Predominance of aggregates was caused most likely by incomplete desialylation of budding virus particles due to a low neuraminidase activity of Group IV mutants (Sugiura et al., 1972; Palese et al., 1974). The preparation of a neuraminidase-defective virus was, thus, likely to be a heterogeneous population containing, in mixed aggregates, virus particles with mutations in various other genes, in addition to the Group IV gene. The mutation in other genes might have been present from the beginning or might as well have been gained spontaneously thereafter. It may be hardly accidental, therefore, that a mutation in a hitherto unknown gene had been carried by only Group IV mutants, and by two of them.
yielded double mutants of other combinations (Group III t VIII and Group I +
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