Burge & Pfefferkorn (i966a) isolated 23 temperature-sensitive mutants of a strain of. Sindbis virus which had been selected for heat resistance (the HR strain).
J. gen. Virol. (I974), 25, 371-38o
371
Printed in Great Britain
Isolation and Preliminary Characterization of Temperature-sensitive Mutants of Sindbis Virus Strain AR339 By G. J. A T K I N S , J A N I S S A M U E L S AND S. I. T. K E N N E D Y
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, England (Accepted I4 August 1974) SUMMARY
One hundred and four temperature-sensitive mutants of Sindbis virus strain AR339 have been isolated using five different mutagens. Forty-seven mutants showed no detectable RNA synthesis at the restrictive temperature (39 °C), whereas 57 showed levels ranging from 1% to over IOO% of the wild-type level. The mutants did not show complementation. The ratio of the 42S:26S RNA species in cells infected with 30 mutants showing > IO % of the wild-type level of RNA synthesis at 39 °C was measured. Of these 30 mutants, none were found which were wholly defective in production of either RNA species, although the ratio was significantly different from the wild-type for some mutants. Two mutants showed production at 39 °C of polydisperse RNA of both double-stranded and singlestranded types. INTRODUCTION
Temperature-sensitive mutants have been isolated for two alphaviruses; Semliki Forest virus (Tan, Sambrook & Bellett, I969) and Sindbis virus (Burge & Pfefferkorn, I966a). Both sets of mutants could be classified into two groups on the basis of RNA synthesis at the restrictive temperature. However, while the Sindbis virus mutants showed complementation at the restrictive temperature in certain combinations (Burge & Pfefferkorn, I966b), Semliki Forest virus mutants showed no such complementation (Tan, 1969). Burge & Pfefferkorn (i966a) isolated 23 temperature-sensitive mutants of a strain of Sindbis virus which had been selected for heat resistance (the HR strain). The present paper describes the isolation and preliminary characterization of 104 temperature-sensitive mutants of Sindbis virus strain AR339. This strain was not selected for heat resistance. It is hoped to use the mutants in screening programmes designed to find mutants defective in specific virus functions at restrictive temperature. We are particularly interested in finding mutants which are defective in various aspects of virus RNA synthesis. Thus, while previous investigators have isolated small numbers of mutants and attempted to fully characterize them, our approach is to select a much larger number of mutants and to screen these for defects in specific functions. METHODS
Materials. Actinomycin D was a gift from Merck, Sharpe and Dohme Research Laboratories, Rahway, N.J.,U.S.A. Medium 199 and minimal essential medium were obtained from the Wellcome Foundation, Beckenham, Kent. Hydroxylammonium chloride (analytical grade) was obtained from Fisons Ltd., Loughborough, Leicester, sodium nitrite (analytical grade) from Hopkin & Williams Ltd, Chadwell Heath, Essex, and 5-fluorouracil (FU), ethyl 2542
372
G.J. ATKINS, J. SAMUELS AND S. I. T. KENNEDY
methane sulphonate (EMS) and N-nitro-N-methyl-N'-nitrosoguanidine (NTG) from the Sigma Chemical Co., London. [5-aH]-uridine (24 Ci/illmOl) was purchased from the Radiochemical Centre, Amersham, Bucks. Cells and media. Primary chick cells and BHK 2i cells clone 13 were grown as described by Morser, Kennedy & Burke (1973). Virus was routinely grown in chick cell monolayers maintained in Medium I99 supplemented with 2 % calf serum. Plaque assays were performed using chick cell monolayers, with an overlay medium containing Medium 199, 7 % calf serum and 0"9 % Difco Noble Agar. Virus. The AR339 strain of Sindbis virus was obtained as 7th passage mouse brain stock from Dr J. S. Porterfield, National Institute for Medical Research, Mill Hill, London. This material was passaged in chick ceils, then cloned three times by picking large plaques. The cloned material was passaged again in mouse brain to reduce the possibility of defective interfering particles being present (Schtesinger, Schlesinger & Burge, I972), then cloned once more in chick cells. Second passage chick cell stocks of this clone were used for mutant isolation and as the wild-type in all experiments. Such stock gave uniformly large plaques, about 3 mm in diam. after 48 h incubation at 37 °C. The HR strain of Sindbis virus and a selection of temperature-sensitive mutants, originally isolated by Burge & Pfefferkorn (I966a), were obtained from Dr R. Z. Lockart, Central Research Department, E. I. du Pont de Nemours & Co., Wilmington, Delaware, U.S.A. and Dr T. Sreevalsan, Department of Microbiology, Georgetown University, Schools of Medicine and Dentistry, Washington, U.S.A. Both the wild-type and the temperaturesensitive mutants were cloned by picking single plaques, and stocks grown from these clones were used in subsequent experiments. All mutant stocks were checked for revertant frequency and yield at 39 °C as described below. The HR strain of Sindbis gives plaques which are about I mm in diam. after 48 h incubation at 37 °C. Temperature control. For all mutants, the restrictive temperature was 39 °C and the permissive 30 °C. Plaque assays were incubated in water-jacketed incubators. The 39 °C incubator was kept in a 37 °C warm room, the 30 °C incubator at room temperature. For measurement of RNA synthesis and virus yields at the restrictive temperature, infected cultures were immersed in circulating water baths set to 39 °C (+ o'o5 °C) and maintained in a 37 °C warm room.
Chemical mutagenesis FU. Monolayers were infected at an input multiplicity of IO at 4 °C. Minimal essential medium lacking calf serum but containing 50 #g/ml FU was then added. A control was set up at the same time with no mutagen present. The cultures were incubated at 3o °C until maximum c.p.e, was observed in the control (about 24 h), when the fluids from both cultures were harvested. NTG. Virus was treated with lOO/zg/ml N T G in phosphate-buffered saline (PBS) for 15 min at room temperature, then dialysed overnight at 4 °C against PBS. EMS. Virus was treated with 1% EMS in PBS for 4 h at room temperature, then dialysed overnight at 4 °C against PBS. Nitrous acid (NA). Virus was treated with 2 M-sodium nitrite in o.25 M-phosphate buffer, pH 6"7, for a period of 2 h at room temperature. The pH was then adjusted to 8-0, and the sample dialysed overnight at 4 °C against PBS. Hydroxylamine (HA). Virus was treated with o-I M-hydroxylammonium chloride in PBS (pH 7"4) for a period of2o min at 37 °C, followed immediately by Ioo-fold dilution with cold maintenance medium.
Sindbis virus ts mutants
373
Selection of mutants. A temperature shift technique similar to that described by Lake & Mackenzie (I973) for foot-and-mouth disease virus, was used in the isolation of mutants. Mutagenized virus suspensions were plated to give between 5 and ~o plaques/plate. The plates were incubated at 3o °C for 48 h, followed by incubation at 39 °C for a further 24 h. Small plaques were then picked, and suspensions made. These were plated at 3o °C and 39 °C, and isolates showing plaques at 39 °C were discarded. Those isolates showing plaques at 30 °C but not at 39 °C were recloned and replated at both temperatures. Plaque suspensions from the second cloning were used to grow virus stocks at 3o °C .These stocks were plaque assayed at 39 °C and 3o °C. The ratio of the plaque titre at 39 °C to that at 3o °C was taken as the revertant frequency. Any stocks showing a revertant frequency greater than 5 × Io-~ or a titre at 30 °C of less than 2 × ~o7 p.f.u./ml were discarded. The total yield at 39 °C was also measured for each mutant. Virus collected 4 to 6 h after infection at an initial input multiplicity of Io was plaque assayed at 3o °C (for method see Complementation tests). The yield ratio is expressed as the titre of virus produced by the mutant at 39 °C divided by that produced by the wild-type at 39 °C. Any mutants giving a yield ratio greater than 5 × I o -2 compared to the wild-type were discarded. Complementation tests. These were carried out essentially as described by Burge & Pfefferkorn (1966 b). Chick cell monolayers in 3o ml (I oz) screw-capped bottles were washed with cold PBS, immersed in 5 ml cold maintenance medium, and left for I h at 4 °C. One mutant at an input multiplicity of IO, or two mutants each at an input multiplicity of 5, were added to the monolayer and adsorption allowed to proceed for I h at 4 °C. The monolayers were then drained, washed with 5 ml warm (39 °C) PBS, 5 ml warm maintenance medium added, and the bottles transferred to a water-bath held at 39 °C. After 4 h incubation, the monolayers were drained, washed twice with warm PBS, immersed in 5 ml warm maintenance medium, and replaced in the 39 °C water-bath. Two hours later, the fluids were harvested and stored at - 7 o °C before being plaque assayed at 30 °C. The complementation level is defined as the yield at 39 °C on mixed infection divided by the sum of the yields of the two mutants on single infection at 39 °C, levels above two being regarded as indicating complementation (Burge & Pfefferkorn, I966b). Total RNA synthesis. Chick cell monolayers in 3o ml screw-capped bottles were pretreated with 5 ml maintenance medium containing I/zg/ml actinomycin D for I h at 37 °C. The monolayers were placed at 4 °C for 15 min, before being infected with virus at an input multiplicity of io. Adsorption was allowed to proceed in the presence of I #g/ml actinomycin D for I h at 4 °C. The monolayers were drained, and 5 ml of pre-warmed maintenance medium containing I #g/ml actinomycin D added. The bottles were then immediately placed in a 39 °C water-bath for 2½ h. Next, the medium was replaced by 5 ml pre-warmed Earle's medium containing 2 ~o dialysed calf serum, I/zg/ml actinomycin D and 2/zCi/ml of [3H]-uridine. The monolayers were then returned to 39 "C for a further 2 h before being drained, washed 3 times with ice-cold PBS and dissolved in 4 ml o'5 ~o ammonia solution. One ml of5o ~o trichoroacetic acid (TCA) in o. • M-sodium pyrophosphate was added, and the mixture allowed to stand on ice for 3o min. The resulting precipitate was collected and washed on glass fibre discs before being counted in a Packard scintillation counter (Clegg & Kennedy, 1974). R N A synthesis was measured for three cultures of each mutant, the quoted figure being the mean. For each experiment, wild-type infected and uninfected control cultures were set up at the same time. R N A synthesis by mutants at the restrictive temperature is expressed as a percentage of the wild-type level, the lower limit of detection of the method being about 1%. Extraction and sedimentation analysis of virus-specified RNA. Monolayer cultures of B H K
374
G . J . A T K I N S , J. S A M U E L S A N D S. I. T. K E N N E D Y
Table I. Isolation o f temperature-sensitive mutants
Mutagen FUr EMS NA HA NTG NTG:~
Series F E A H N R
Proportion of
Number of
Percentage of
survivors after treatment* I IO-2
mutants isolated 45 23
mutants among plaques tested 3'z 7"9
io -g 10.2 Io -1 Io -J
20 27 x6 3
IO'O 12'4 I4"5 ~-z
* Measured by comparing p.f.u, present after mutagen treatment to p.f.u, present in samples subjected to the same treatment without mutagen. For N A treatment, sodium chloride was substituted for sodium nitrite in the control. t No lethality was detected after F U treatment. ;~ Mutants isolated after NTG treatment but without employing the temperature shift technique (see
Methods). cells in I 1 bottles (2 × IO7 cells/monolayer) were infected with virus at 4 °C as described by Tan et al. (1969), 25 ml of pre-warmed (42 °C) maintenance medium containing I/zg/ml actinomycin D added to the cultures, and the bottles placed in a 39 °C water-bath. After I h the monolayers were drained and Io ml of pre-warmed Earle's medium containing I #g/ml actinomycin D, 2o0 #Ci pH]-uridine, and 2 % dialysed calf serum added. The monolayers were replaced in the 39 °C water-bath and incubated for a further 5 h before the nucleic acid was extracted by the method of Clegg & Kennedy (I974) , and precipitated with 2½ vol. of ethanol at - 2 o °C. Labelled R N A from infected cells was dissolved in T N E buffer (50 mM-tris containing o- 1 x~NaC1 and I mM-EDTA, p H 7"4) and analysed by sedimentation at 135ooo g for 2 h 5o rain at lO °C through 4"8 ml linear 6 to 30 % (w/v) sucrose gradients prepared in T N E buffer containing o.1% SDS. Gradients were loaded with approx. IOOOOOct/min of R N A and fractionated by upward displacement. Nuclease treatment. (i) Labelled virus-specified RNA from infected cells in 50 mM-tris containing o'5 M-NaC1, I mM-EDTA and Io mM-MgCI~, (pH 7"4), was incubated for 3° min at 37 °C with 4 #g/ml pancreatic ribonuclease and Io units/ml ribonuclease T1. Digestion was stopped by adding naphthalene-i,5-disulphonate to o.1% (Clegg & Kennedy, I974) and the samples analysed on sucrose gradients as described above. (ii) Fractions collected after analysis of virus-specified R N A through sucrose gradients prepared in T N E buffer were treated with pancreatic and T1 ribonucleases as above, then precipitated at 4 °C for 3o rain with cold 5 % TCA in the presence of 250/zg carrier yeast RNA. Precipitates were collected on glass fibre discs, washed and counted as previously described (Clegg & Kennedy, 1974). RESULTS
Isolation of mutants A total of lO4 mutants was isolated. The mutants are labelled according to the mutagen employed in their isolation, i.e. F series-FU, N series-NTG, E series-EMS, A series-NA and H series-HA. The total number of mutants in each series and their frequency of isolation are shown in Table I. In order to test whether the shift procedure gave a significant increase in the number of mutants isolated per plaque tested, 256 randomly isolated plaques from NTG-treated virus were tested for temperature sensitivity without employing the shift. Of these plaques three were found to be suitable temperature-sensitive mutants,
Sindbis virus ts mutants
375
Table 2. Total RNA synthesis by temperature-sensitive mutants at 39 °C RNA synthesis (% of wild-type at 39 °C)
Undetectable
I-I0 IO-20 20-30 30-40 4o--50 50-60 60-70 7o-80 80--90 9o-Ioo -'> I00
Mutants F85, FIo4, F212, F295, F294, F367, NI, N2, N26, N24, N3o, N75, Nloo, RI64, RI9I, R22o, E39, E27, E84, E268, E285, E25o, E233, E248, EI9I, E2o6, E22I, A7, A49, Aio8, AIo7, A47, A8I, A65, AI, AI5I, A2ol, H5, H2t, H83, H92, HII9, HI45, HI61, H175, H2o7, H2o8 F182, F249, F346, N22, N7, NI7, N28, N33, N74, N98, E48, Ei8o, E286, EI93, E28o, E249, E234, E228, E279, A5, A82, AI9I, AI83, AI85, H75, I-I98,H132, H5 I A7o, A158, HIS, H76 AII, H38, HI5o, A93, AI2o, N32 F448, N4, H37, H133 F127, HIO7, HI57 HI94 HI87, F3o9 F296, HI78 E9o, H134 Elo2 F36, F214, EIo4, H191
Total 47
28 4 6 4 3 I 2 2 2 I 4 IO4
i.e. 1.2 %. These mutants constitute the R series (Table I). As the frequency of isolation of NTG-induced mutants using the temperature shift was I4"5 % (Table I), the temperature shift increases the probability of isolation of mutants approx, tenfold. Since no lethality was detected after growth of the virus in the presence of FU, and the frequency of isolation was lowest with this mutagen, it is possible that the F series of mutants are spontaneous. However, since the frequency of isolation of mutants was higher for the other mutagens, mutants produced using these agents are likely to be induced. RNA synthesis at the restrictive temperature Forty-seven of the mutants isolated showed no detectable RNA synthesis at 39 °C. The rest showed levels of RNA synthesis which varied from barely detectable to above the wild-type level (Table 2). Eight mutants, with undetectable RNA synthesis at 39 °C in chick cells were also tested for RNA synthesis at 39 °C in BHK cells; no RNA synthesis was detectable for any mutant. Complemen tation No complementation could be detected in crosses involving the temperature-sensitive mutants of Sindbis strain AR339, even between mutants which differed greatly in RNA synthesis at the restrictive temperature. Representative data (from a total of 57 different complementation tests performed) are shown in Table 3. This lack of complementation was observed even when complementation between mutants of the HR strain could be easily detected (Table 4). Cross-complementation between the HR and AR339 mutants could be demonstrated in some crosses, although at relatively low levels (< 20). As found for both Semliki Forest virus (Tan, I969) and the HR strain of Sindbis virus (Burge & Pfefferkorn, I966b), no recombination could be detected in crosses involving AR339 mutants.
376
G . J . ATKINS, J. SAMUELS AND S. I. T. KENNEDY Table 3- Complementation yields for the first six mutants isolated
RNA synthesis at 39 °C (% of Mutant wild-type)*
Yield on single infection at 39 °C : (p.f.u./ml) F36
F36
6"2 X I 0 a
156
NI
u~
5'3 x lo 3
F85
u
6"I x lo a
N22
2
5"8 X I03
N2
U
5"3 x 102
N26
u
9"4 x 102
Wild-type
I00
2"7 x I 0 s
--
Yield on double infection at 39 °C (p.f.u./ml)l" -I NI
F85
N22
N2
N26
I ' 0 X 104
5 ' 5 x 1o 3
7 . 8 X IO a
2 ' 9 x 102
(0.8)
(0"5) 4 " 3 x I0~
(o'7) 3'6 x Io3
(0'4) 9"8 x lO3 (o'2) 6'5 x I o ~ (0"I)
--
(0"4) --
(0'3) 4'0
to z (0"3) x
--
8-8
x
1o 2
(o-,)
3'o x I o3
(0'4) 7.2 × l O 2
(o.i) 3"9 x I 0 2
(0.6) 9 " 7 × 103
(I "4) 6-8 x l O 2
(o'5)
* Data from Table 2. ~ The complementation level is indicated in parentheses. ~: Undetectable. Table 4- Complementation between HR mutants and AR339 mutants Input virus
Yield at 39 °C (p.f.u./ml)
Complementation level
N26
I ' 2 × IO 3
--
F36 ts 5 ts 7
9"4 x t o 3 2"6 × I 0 a 3"7 × lO2
----
N26/F36 ts 5/ts 7 N26/ts 5 F36/ts 7
4"7 × lO3 2'0 × to5 4"I x IO4 l.t x lO4
0"4 66 IO-8 I-2
HR +
6"6 × I 0 t;
--
AR339+
5'4 x Ios
--
All yields were measured in the same experiment from monolayers set up at the same time. N26 shows no RNA synthesis at 39 °C, while F36 shows more RNA synthesis a t 39 °C than the wild-type (Table 2). ts 5 is an RNA + mutant of the HR strain, ts 7 an RNA- mutant (Burge & Pfefferkom, I966a).
Sucrose gradient analysis of virus-specified RNA Labelled virus-specified R N A analysed o n sucrose gradients gave 2 m a j o r peaks o f radioactivity sedimenting at z6S a n d 42S, as observed previously (Burge & Pfefferkorn, 1968). The ratio of 42 S: 26 S R N A was measured by plotting a graph of these radioactive R N A species a n d cutting out a n d weighing the corresponding peak areas. This ratio was measured for the wild-type virus in a series of 19 experiments, a n d gave a value of o'73 with a s t a n d a r d error of o'o14. A total of 30 temperature-sensitive m u t a n t s synthesizing more t h a n I o ~o of the wild-type level of R N A was also analysed o n sucrose gradients. All these m u t a n t s m a d e b o t h 26 S a n d 42S R N A . However, a n F-test (variance ratio) analysis conducted o n m e a s u r e m e n t s m a d e o n the ratio o f 42 S:26 S R N A isolated from m u t a n t infected cells, a n d repeated measurements of the ratio of these R N A species from wild-type infected cells (samples (a) a n d (b) of
Sindbis virus ts mutants 9 8 7 6 5 4
377
(a)
~3 E 2
(b) El04 F127 Al~nH194.1-1~/ ~. ^aa H51 . . . .
6 5
F214 HI07 H150 A158
l~.~,
4
3 2
HN
1 -
I I t- 0
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I 1 F309
-
0.1
0.2
0-3
0.4
0.5
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I I I I
IAII I I I I HI8 F296
0.6 0-7 0.8 0.9 Ratio of 42 S: 26 S peaks
1.0
1.1
1.2
1.3
Fig. I. Distribution of the ratio of 42 S:26S RNA species formed at 39 °C from I to 6 h post-infection in the presence of I #g/ml actinomycin D. Sedimentation was for 2 h 50 min on 6 to 30 % (w/v) sucrose density gradients. (a) wild-type; (b) mutants showing more than IO % of the wild-type level of RNA synthesis at 39 °C (excluding H76 and F448; see text). Fig. I), gave a value o f 9, corresponding to a probability o f < 0.02. Thus the variance o f measurements made on R N A f r o m m u t a n t infected cells was significantly greater than that o f repeated measurements on R N A f r o m wild-type infected cells. A representative experiment indicating two extremes is shown in Fig. 2. The sedimentation pattern of the two mutants H76 and F448 was anomalous (see below). Eleven o f the mutants, selected randomly, were tested for the ability to synthesize doublestranded R N A at 39 °C. Preparations o f labelled R N A were treated with ribonuclease before sedimentation on sucrose gradients. All these mutants synthesized approximately the same a m o u n t o f ribonuclease-resistant R N A as the wild-type. M u t a n t s H76 and F448
The sedimentation pattern o f the R N A made by these mutants showed dispersion o f the 26 S and 42 S peaks, as well as the presence of a large a m o u n t of low tool. wt. material (Fig. 3)- The R N A produced by these mutants was also examined by ribonuclease treatment o f fractions collected f r o m sucrose gradients (Fig. 4). Whereas a sharp peak o f doublestranded R N A was obtained for the wild-type both mutants showed the presence o f ribonuclease-resistant R N A dispersed t h r o u g h the b o t t o m portion o f the gradient. The low tool. wt. material observed at the top o f the gradient in Fig. 3 is therefore single-stranded. However, it is not k n o w n if this material is virus-specified, since control gradients o f mock-infected cells contain appreciable amounts o f radioactivity in this region.
G. J. A T K I N S , J. S A M U E L S A N D S. I. T. K E N N E D Y
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From the above data it is apparent that the temperature-sensitive mutants of Sindbis AR339 differ from those of the HR strain in that complementation is observed between some HR mutants but not between AR339 mutants in any combination tested. In this respect, the AR339 mutants are similar to those of Semliki Forest virus (Tan, I969). At least four possibilities, not necessarily mutually exclusive, may account for this lack of complementation. First, the AR339 mutants may bear multiple ts lesions, as has been suggested for the Sindbis mutants isolated from persistently infected mosquito cells (Schenk, Koshelnyk, & Stollar, I974). However since the AR339 mutants were produced using lower levels of exposure to mutagens than used for the production of the HR mutants, resulting in lower lethality, this possibility seems unlikely. Secondly, the different isolation procedures used in selecting the mutants may have produced mutants differing in their ability to complement. It may be that the selection for heat resistance which Burge & Pfefferkorn (I966a) employed, affected the virus in other ways. Also, theAR339 mutants were selected using a temperature shift technique, whereas the HR mutants were randomly selected. Thirdly, the AR339 mutants may interfere with each other on multiple infection, whereas the
Sindbis virus ts mutants t
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Fig. 3. Sucrose density gradient analysis of R N A species from B H K cells infected with Sindbis virus and incubated at 39 °C in the presence of I #g/ml actinomycin D. R N A was labelled with 20 #Ci/ml [SH]-uridine from I to 6 h post-infection. © . . . . O, wild-type; A ~ , H76; • @, F448. Arrows show the position of [a4C]-uridine-labelled B H K marker RNA. Fig. 4. Ribonuclease treatment of R N A isolated from B H K cells infected with Sindbis virus and incubated at 39 °C in the presence of i #g/ml actinomycin D. R N A was labelled with 2o #Ci/ml [aH]-uridine from I to 6 h post-infection. Fractions were collected after sedimentation of R N A for 3 h in a 6 to 30 % linear sucrose gradient, treated with a mixture of pancreatic A and I"1 ribonuclease and TCA precipitated. © . . . . O, wild-type; ~ A, H76; • "-, F448. Arrows show the position of [uC]-uridine-labelled B H K marker RNA.
H R mutants do not. Lastly, it is apparent that, since the original isolation by Taylor et al. ([955), the two Sindbis strains have been cultivated for many passages in different laboratories. During this time, one of the strains may have lost or gained the ability to complement whereas the other has not. These possibilities are currently under investigation. The two sets of mutants also differ in the level of R N A synthesis at the restrictive temperature. No two distinct groups of R N A + and R N A - mutants could be distinguished for the AR339 mutants. Although a large number of mutants showed undetectable R N A synthesis at 39 °C, levels of R N A synthesis ranging from barely detectable to above the wild-type level could be detected among the other mutants. This could merely be a consequence of the fact that many more AR339 mutants have been examined than H R mutants. The analysis of virus-specified R N A species on sucrose gradients has shown that none of the 3o mutants examined is wholly defective in the production of either the 42 S or the 26 S
380
G . J . ATKINS, J. SAMUELS AND S. I, T. KENNEDY
R N A species at the restrictive temperature. T a n et aL (1969) came to a similar conclusion after e x a m i n i n g the R N A species formed at the restrictive temperature by I3 R N A + m u t a n t s o f Semliki Forest virus. Thus, if separate enzymes are involved in the synthesis of 26 S a n d 4 2 S R N A , m u t a n t s of the genes coding for these enzymes have n o t yet been detected. It is clear from the present data, however, that m a n y m u t a n t s do n o t synthesize the two singlestranded R N A species in the same p r o p o r t i o n as the wild-type, a n d this m a y indicate a defect in a f u n c t i o n which n o r m a l l y regulates the p r o p o r t i o n of 26 S to 42 S R N A . The work was supported by grants from the Medical Research Council a n d the Cancer Research Campaign. We t h a n k Mrs Christine Lancashire for excellent technical assistance.
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844-862. (Received 20 May I974)
