463
J. gen. Virol. 0978), 39, 463-473 Printed in Great Britain
Cleavage Defect in the Non-structural Polyprotein of Semliki Forest Virus has Two Separate Effects on Virus R N A Synthesis By L E E V I K , ~ R I ~ I N E N Department of Virology, University of Helsinki, Haartmaninkatu 3, SF-oo29o Helsinki 29, Finland D O R O T H E A S A W I C K I * AND P E T E R J. G O M A T O S Division of Virology, Memorial Sloan-Kettering Institute, New York, New York IOO2I, U.S.A.
(Accepted 7 December I977) SUMMARY
When Semliki Forest virus ts-4 mutant infected cultures are grown at the permissive temperature (28 °C) and shifted to the restrictive temperature (39 °C), two different defects in RNA synthesis are manifested: (i) the synthesis of 26S RNA is stopped within 60 min (Saraste et al. 1977), and (ii) the increase in RNA synthesizing activity ceases, in contrast to cultures maintained at 28 °C, indicating that no new active RNA polymerase is formed at 39 °C. Accumulation of a non-structural precursor protein with an apparent mol. wt. of about 22oooo (ns22o) was demonstrated in ts-4 infected cultures shifted to 39 °C. Ns22o was labelled during short pulses given immediately after release of protein synthesis from hypertonic initiation block, suggesting that genes coding for ns22o are located near the initiation site at the 5'-end of the 42S RNA. The viral specificity of ns22o was shown by its disappearance after a shift to 28 °C and by labelling in the presence of sucrose, when no host cell protein synthesis is detectable. The two functional defects can be explained if the polypeptides responsible for the RNA polymerizing activity and that responsible for the synthesis of 26S RNA are components of the same non-structural polyprotein. A mutation in the latter polypeptide which prevents cleavage of the polyprotein would thereby prevent the further formation of active RNA polymerase. If cleavage of the polyprotein has taken place at the permissive temperature, the RNA polymerase would remain active also at 39 °C, whereas the polypeptide responsible for 26S RNA synthesis would become inactive due to the mutation. INTRODUCTION
The alphavirus 42S RNA genome coded proteins are probably translated as two polyproteins. The messenger for the structural polyprotein pI3o (mol. wt. 13o0o0) is the 26S RNA which is a replica of the 3' third of the 42S RNA (Kennedy, I976; Wengler & Wengler, I976; Strauss & Strauss, I977). The 42S RNA seems to be the messenger for at least three to four non-structural proteins which are synthesized starting from one initiation site (Cancedda et al. 1975; Glanville et al. 1976; Lachmi & K~i/iri~iinen,1976; Brzeski & Kennedy, I977; Glanville & Lachmi, I977). In SemIiki Forest virus (SFV) infected cells four non* Present address: Department of Microbiology, Medical College of Ohio, Toledo, Ohio 43699, U.S.A.
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L. K ~ . A R I A I N E N , D. S A W I C K I
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structural proteins ns7o, ns86, ns72 and ns6o with apparent mol. wt. of 70000, 86000, 72000 and 6000o are synthesized sequentially in the above order. Ttyptic peptide mapping has recently shown that two short-lived proteins with mol. wt. of 155000 (nsI55) and 135o00 (nsI35) are precursols of the more stable non-structural proteins. The nsI55 contains the peptides of ns7 o and ns86 whereas the ns135 consists of ns72 and most probably also of ns6o (Glanville et al. 1978). An R N A negative mutant, ts-4, of Semliki Forest virus has a reversible defect in the synthesis of 26S R N A as shown by shift-up experiments (Saraste et al. I977). Here we report the detection in ts-4 infected cells of a third non-structural precursor protein, which has an apparent mol. wt. of about 22oo00, found only in cells shifted to the restrictive temperature. Similar size proteins have been described in Sindbis virus mutant (Bracha et al. I976), in Sindbis virus wild type (Brzeski & Kennedy, 1977) and in SFV wild type infected cells (Clegg et al. 1976). METHODS
Cells and virus. Secondary cultures of chicken fibroblast cells obtained from special pathogen-free animals were grown in 60 mm Petri dishes as described previously (Ker/inen & K/i/iri/iinen, I974)- SFV prototype strain was cultivated as described (Kfi/iri/iinen et al. I969), and the isolation, characterization and propagation of SFV ts-4 mutant have been described previously (Ker/inen & Kfi~rifiinen, 1974). Growth of virus and isotope labelling. Chicken cells were infected with ts-4 or wild type SFV (50 p.Lu./cell) at either 28 or 39 °C, as indicated. After I to 2 h adsorption, virus was removed and the cells were washed twice with Hanks' solution. The cells were then incubated in Eagle's minimum essential medium with or without o.2 % bovine serum albumin (BSA) and I/zg/ml of actinomycin D. In temperature shift experiments, fresh medium at either 28 or 39 °C was added to the cultures upon the temperature shift. For labelling with aH-uridine (Radiochemical Centre, Amersham, specific activity 29 to 31 Ci/mmol), 2o #Ci/dish was used. Labelling with 35S-methionine (Amersham, specific activity 92o Ci/mmol) was carried out in methionine-free Eagle's medium using lO to 5oo #Ci/dish, as indicated in individual experiments. Treatment of cells with hypertonic media. Infected chicken cells were exposed to either 335 mM-NaC1 (Saborio et al. 1974; Nuss et al. 1975) or o.I to o'5 M-sucrose for 4o to 6o rain in 3 ml of medium. The medium was removed and new medium containing 85Smethionine was added. 35S-Methionine labelling was carried out in methionine-free Eagle's medium supplemented with o-2% BSA and, as indicated in the text, also with o-I to 0"5 M-sucrose. At the end of the pulse, the medium was removed and sucrose-free Eagle's medium containing a 2o-fold excess of the normal concentration of methionine was added for variable times. The cells were washed with o.1 M-NaC1, o.oi M-tris-HC1 pH 7"4, and collected in 2 % aqueous SDS as described previously (Lachmi & K~i~iri~iinen, I976). Polyacrylamide gel electrophoresis. Polyacrylamide slab gels with lO ~o main gel and 3 to 5 % spacer gel were made according to the method of Neville (197I) and modified as described by Lachmi et al. (1975). The gels were stained with o'25 % Coomassie blue in 50 ~o trichloroacetic acid (TCA) for 45 mix and destained in 8 % acetic acid. In most cases, the stained gels were treated with dimethylsulphoxide and PPO and dried for fluorography as described by Bonner & Laskey (1974). Myosin was a generous gift from Rockefeller University, given by Dr Polly Etkind. R N A analysis. R N A was isolated from cells disrupted by 2 % sodium dodecyl sulphate (SDS) and was analysed using 15 to 30% (w/w) sucrose gradients made in o"14 M-NaC1,
RNA and protein synthesis by a ts-mutant of SFV I
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Hours Fig. I. R a t e o f R N A synthesis in ts-4 infected chicken cells m a i n t a i n e d at 28 °C or shifted to 39 °C at hourly intervals. C h i c k e n cells infected with ts-4 at 28 °C were m a i n t a i n e d at 28 °C a n d were either exposed to 3H-uridine (20 # C i / d i s h ) for 60 m i n at 28 °C or were shifted to 39 °C a n d pulsed similarly at so h post-infection. T h e cells were t a k e n into 2 % SDS a n d T C A precipitated radioactivity was determined. 0 - - 0 , Pulsed at 28 °C; (3 - - - © , shifted to 39 °C a n d pulsed between io a n d I I h post-infection.
o.oI ~-tris-HC1 pH 7"4, and o.ooI M-EDTA (TNE) and o.1% SDS. Centrifugation was for I4 h at 24ooo rev/min in the SW27~ rotor at 23 °C. The gradients were collected from the bottom by a collecting device connected to a peristaltic pump. Determination of radioactivity. For determination of 35S-methionine-labelled proteins, samples from SDS-solubilized cells were precipitated with Io % TCA, heated for 2o min at 9o °C, and the precipitates were collected on C F / C glass-fibre filters, sH-Uridinelabelled R N A was directly precipitated with Io % TCA. RESULTS
RNA synthesis The rate of R N A synthesis in ts-4 infected chicken cell cultures was studied at the permissive temperature (28 °C) as well as in cultures which had been shifted to the restrictive temperature (39 °C). Two cultures were taken at hourly intervals: one was labelled with 3H-uridine for 6o min at 18 °C and the other shifted to 39 °C. All the shifted cultures were labelled simultaneously for 6o min with 3H-uridine at IOh post-infection. After 4 h incubation at 28 °C the rate of R N A synthesis increased rapidly (Fig. 0 , similar to that of the wild type SFV. The R N A synthesis continued in all cultures which had
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Fig. 2. Sucrose gradient analysis of ts-4 specific RNAs synthesized between 6 and 7 h postinfection at 28 °C (a) or between IO and TT h at 39 °C (b) after shift up from 28 °C to 39 °C at 6 h after infection. Bottom of the gradient is to the left. The fast sedimenting peak is 42S RNA.
been shifted to the restrictive temperature. Interestingly, there was no increase in the rate of R N A synthesis in any of the cultures shifted to 39 °C. There was rather a decrease, maximally of about 5o%, compared to the R N A synthesizing activity observed at z8 °C at the time of shift (Fig. 0 . These results suggest that after the shift to the restrictive temperature no new active polymerase is formed, and that the R N A synthesizing activity detected at 39 °C as long as 8 h after the shift was due to R N A polymerase which was formed during the incubation at z8 °C. The same result was obtained when wild type SFV infected cultures were shifted to 39 °C in the presence of cycloheximide (IOO/~g/ml). Analysis of the RNAs formed at 28 °C and after the shift to 39 °C confirmed our previous findings that virtually no 26S R N A is synthesized at the restrictive temperature (Fig. 2; Saraste et al. I977; Sawicki et al. I978). Thus, two different steps are affected when ts-4 infected cultures are shifted to the restrictive temperature: (i) the synthesis of 26S R N A is shut off rapidly, and (ii) the formation of new polymerase is prevented. Protein synthesis
Duplicate ts-4 infected cultures maintained for 6 h at 28 °C were either shifted to 39 °C or maintained at 28 °C and labelled with 35S-methionine for I5 min with or without pretreatment with 335 mM-NaC1 or 0"5 M-sucrose. The hypertonic media were used to reduce the host protein synthesis which, during the early period of infection, makes the detection of virus proteins difficult (Lachmi & Kft~iri~iinen, T977). The labelled proteins resolved on a discontinuous polyacrylamide slab gel are shown in Fig. 3At 39 °C two large proteins can clearly be seen in ts-4 infected cultures treated with either
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Fig. 3. Fluorogram of IO % polyacrylamideslab gel of ts-4 induced, 85S-methionine-labelled proteins at 39 °C (A, B, C) and at 28 °C (E, F, (3). Ts-4 infected cells maintained for 6 h at 18 °C were either shifted to 39 °C or kept at 28 °C and were exposed to o'5 M-sucrose (B, F) or to 335 mM-NaC1 (C, G), or were kept as controls (A, E) for 4o rain and were thereafter labelled with 35S-methionine for I5 min at Ioo/zCi/dish and subsequently chased for 45 rain before harvest. Ts-I marker containing the previously identified non-structural proteins is shown in lane D, and the proteins of ~sS-methionine-labelled SFV is shown in lane H. Position of capsid (C) and envelope proteins EI and Ez as well as the precursor of E2 and E3, p6z, and the non-structural proteins ns7o (70), ns72 (72), ns86 (86), nsI35 (I35) and nsI55 (I55) are indicated, as is the new ts-4 specific protein (22o). salt or sucrose. T h e smaller o f these p r o t e i n s m i g r a t e d with the p r e v i o u s l y identified nonstructural p r e c u r s o r p r o t e i n nsI55 (mol. wt. 15500o) (Lachmi & Kfi/iri/iinen, I976, I977). T h e larger protein, which is n o t detected in cultures m a i n t a i n e d a n d labelled at 28 °C, m i g r a t e d with m y o s i n a n d thus has a mol. wt. close to 2200oo. T h e possibility t h a t t h e 22ooo0 mol. wt. p r o t e i n is virus-specific, a n d p e r h a p s a new nonstructural p r e c u r s o r p r o t e i n like the large p r o t e i n f o u n d in Sindbis virus infected cells ( B r a c h a et al. ~976; Brzeski & K e n n e d y , I977), was investigated as follows: ts-4 infected cultures i n c u b a t e d at 28 °C for 6 h were shifted to 39 °C a n d t r e a t e d with 335 mM-NaC1 for 4o min. This t r e a t m e n t specifically b l o c k s the initiation o f p r o t e i n synthesis b u t allows e l o n g a t i o n to t a k e place ( S a b o r i o et al. I974). W h e n the h y p e r t o n i c b l o c k o f initiation is r e m o v e d b y restoring the isotonicity o f the m e d i u m , s y n c h r o n o u s i n i t i a t i o n o f p r o t e i n
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Fig. 4. Fluorogram of IO ~oo polyacrylamide gel showing the sequential labelling of ts-4 induced proteins at 39 °C followed by a chase at 28 °C (A, C, E) or 39 °C (B, D, F). Ts-4 infected cultures maintained at 28 °C for 6 h were shifted to 39 °C and exposed to 335 mM-NaC1 for 4o min at 39 °C. They were labelled thereafter with 35S-methionine for 2 min (A, B) with 3oo/zCi/dish, for 6 min (C, D) with Ioo #Ci/dish, or for 28 min (E, F) with 33 #Ci/dish. The cultures were chased after the pulse for 6o min. The designation of the proteins is the same as in Fig. 3.
synthesis takes place. Using different pulse lengths, it is possible to determine the gene order in polyproteins (Saborio et al. 1974; Clegg, 1975)- We have used this method to determine the gene order in the non-structural polyproteins of Semliki Forest virus to be ns7o, ns86, ns72 and ns6o (Lachmi & K~i~iri~iinen, i976 ). Duplicate cultures of ts-4 infected cells were released from the hypertonic block of initiation and labelled for a, 6 and I8 min at 39 °C to label the n~7o, ns7o plus ns86, and finally all the non-structural proteins (Lachmi & K~i~iri~tinen, I976). After the pulse, unlabelled methionine was added and incubation was continued either at the restrictive or permissive temperature for a further 6o min. The fluorogram of proteins separated in a polyacrylamide slab gel is shown in Fig. 4. After a 2 min pulse at 39 °C followed by a 6o min chase at 28 °C only ns7o of the non-structural proteins is labelled (Fig. 4, lane A). It seems to consist of a double band under these conditions. Of the structural proteins, the Nterminal capsid protein is labelled but in this polyacrylamide gel it has been run out from the gel to increase the resolution of the large proteins. Lane B in Fig. 4 shows the proteins labelled in cultures pulsed for 2 min at 39 °C followed by a 6o min chase at 39 °C. Essentially
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Fig. 5. Effect of hypertonic media on protein synthesis in SFV and mock infected cells. (a) SFV wild type and mock infected cells were incubated at 39 °C for 3 h and exposed to different concentrations of sucrose between 3 and 4 h post-infection. They were labelled thereafter in the same media with 35S-methionine at 2o #Ci/dish for I5 rain and chased for 15 rain in the presence of unlabelled methionine. (b) SFV wild type and mock infected cells maintained for 4 h at 39 °C were exposed to 335 mM-NaCl for 4o min and were labelled for 30 min thereafter in the presence of various amounts of sucrose with 2o #Ci/dish of asS-methionine. 0 - - 0 , Wild type; • . . . . . • , mock infected. o n l y two p r o t e i n s are labelled, n a m e l y nsI55 and the 220000 d a l t o n p r o t e i n shown in Fig. 3- This result w o u l d indicate t h a t b o t h these p r o t e i n s are derived f r o m the N - t e r m i n a l p a r t o f the n o n - s t r u c t u r a l p o l y p r o t e i n a n d thus c o n t a i n ns7o. Labelling for 6 min at 39 °C followed b y a 60 min chase at 28 °C shows the presence o f ns7o, n o w clearly as a d o u b l e b a n d , a n d also o f ns86 (Fig. 4, lane C). This was as expected f r o m o u r previous results using the ts-I m u t a n t o f S F V ( L a c h m i & Kii~iri~iinen, I976). T h e p a t t e r n o f labelled p r o t e i n s becomes, however, m o r e c o m p l i c a t e d due to the start o f host p r o t e i n synthesis. A g a i n , cultures labelled at 39 °C a n d chased at 39 °C show the nsI55 a n d the 22oooo d a l t o n p r o t e i n b u t a l m o s t no ns86 o r ns7o, suggesting t h a t b o t h ns7o a n d ns86 a r e constituents o f the two large proteins. W h e n the labelling p e r i o d is extended to 18 min, the ns72 p r o t e i n also b e c o m e s labelled in cultures shifted to 28 °C for the chase (Fig. 4, lane E). O n l y small a m o u n t s o f nsI55 can be seen a n d the presence o f the 22oooo d a l t o n 3I
V I R 39
470
L. KAAR1AINEN~ D. SAW1CKI AND P. J. GOMATOS A
B
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Fig. 6. Fluorogram of a Io ~ polyacrylamide slab gel showing large tool. wt. proteins from ts-4 (B, D, F, H) and mock infected cells (C, E, G, I). The ts-4 and mock infected cultures maintained at 28 °C for 8 h were shifted to 39 °C and exposed to 335 mM-NaC1 for 40 min and were labelled thereafter for 3o min with 50 #Ci/dish of 35S-methionine in methionine-free Eagle's medium (B, C), or in methionine-free Eagle's medium containing o'I M-sucrose (D, E), o'I5 M-sucrose (F, G) or 0.2 M-sucrose (H, I). All the cultures were chased for ~o5 min in the presence of unlabelled methionine. Designation of the ts-I marker protein (A) is as in Fig. 3protein is difficult to exclude due to the heavy labelling o f host proteins in this region. The cultures labelled and chased at 39 °C show nsI55 as heavily labelled, as is the ns~35, which appears only after IO min or longer labelling periods (Fig. 4, lane F). There seems to be some processing of the non-structural polyprotein even at 39 °C, since ns86 and ns72 can be seen when large amounts o f radioactivity are applied to the gel. These results suggest that the new protein with mol. wt. o f about 22oooo f o u n d in ts-4 infected cells pulsed at 39 °C is a virus-specific non-structural precursor protein derived f r o m the N-terminal part o f the non-structural polyprotein.
Shut-off of host protein synthesis by hypertonie media Specific labelling o f virus proteins after the hypertonic block o f initiation is limited to only a short period o f time, since host cell protein synthesis starts to recover within a few minutes after the release o f the block (Saborio et al. I974) as can clearly be seen also f r o m Fig, 4. To overcome this difficulty we determined conditions to specifically label the virus proteins in the presence o f hypertonic media. SFV and m o c k infected cells were grown for 3 h at 39 °C and then incubation was continued in the presence o f different concentrations o f sucrose for 6o min, followed by a 15 min pulse with 35S-methionine in the same media (Fig. 5 a). The protein synthesis in the m o c k infected cultures is almost completely shut off when incubated and labelled in the presence o f o.2 M-sucrose. U n d e r these conditions protein synthesis in SFV infected cells was reduced to about 2o % of the level in the presence of isotonic medium. We t o o k advantage o f these results to label the cells after hypertonic block o f initiation induced by 335 mM-NaCI. As can be seen, there is some recovery o f host cell protein synthesis during the 3o min pulse with 35S-methionine when m o c k infected cells are transferred to isotonic medium (Fig. 5 b). I f the m o c k infected cells are labelled in o. I M or higher sucrose, no recovery o f protein synthesis is seen. However, SFV infected cultures showed reduced but detectable protein synthesis in media containing up to o'3 M-sucrose.
R N A and protein synthesis by a ts-mutant o f S F V
47I
The polyacrylamide slab gel in Fig. 6 shows the effect of different concentrations of sucrose on the labelling of the large proteins in ts-4 and mock infected cells after release from the hypertonic block of initiation. The cells were labelled for 3o rain and chased for IO5 rain before analysis of the proteins. In the mock infected cells, a large protein migrating more slowly than the 22o ooo dalton ts-4 protein is clearly seen if the labelling is carried out in the absence of sucrose (Fig. 6, lane C), but is not detectable when o- I M or higher sucrose concentrations are used during the labelling (Fig. 6, lanes E, G and I). The same protein is also seen in the ts-4 infected cells labelled without sucrose but not in those labelled in the presence of sucrose. The most heavily labelled protein is the nsI55, but the 22oooo dalton protein is seen with all the sucrose concentrations used during the labelling. These results support the idea that the 22oooo dalton protein in ts-4 infected cells is clearly a virus-specific one. The pulse-chase experiments suggested that it is most probably a precursor of the non-structural proteins, and therefore we designate it as ns22o. The results also show that unequal labelling of some host proteins after the release of hypertonic block of initiation can cause severe difficulties in the interpretation of the results.
DISCUSSION
We have studied the R N A and protein synthesis of a tempeiature sensitive mutant, ts-4, of Semliki Forest virus. Ts- 4 is phenotypically an R N A negative mutant, which cannot synthesize significant amounts of virus R N A when infection is initiated at the restrictive temperature (KerSnen & K~ri~inen, 1974). When infection is started at the permissive temperature, it is able to continue RNA synthesis for long periods of time, showing that the R N A polymerase function of this mutant is intact. As we have previously reported, the relative amounts of the RNAs synthesized are affected by the shift to the restrictive temperature: synthesis of 26S R N A stops rapidly whereas that of 42S RNA continues (Saraste et a[. 1977; Sawicki et aL 1978 ). Thus the mutation apparently affects a protein which regulates the synthesis of 26S R N A (Sawicki et al. 1978). The functional defect is reversible since 26S RNA synthesis starts again when the cultures are shifted back to the permissive temperature in the presence of cycloheximide. The fact that there is no increase in the R N A synthesizing activity after the ts-4 infected cultures were shifted to 39 °C suggests that no new active RNA polymerase was formed at this temperature. The same result was obtained for SFV wild type infected cultures exposed to cycloheximide (see also Wengler & Wengler, I975; Clegg et al. I976). These results can be simply explained by the accumulation of a large 22oooo dalton protein in the ts-4 infected cultures labelled after shift to 39 °C. This protein seems to be virus-specific and a non-structural protein precursor since it is labelled under conditions when practically no host proteins are synthesized and is processed in cultures labelled at 39 °C and chased at 28 °C, to yield previously identified non-structural proteins. The labelling of ns22o and ns155 at 39 °C during a 2 min pulse after release of the cells from hypertonic block of initiation would suggest that both these proteins are translated from the 5'-end of the 42S RNA, which is the messenger R N A for the non-structural proteins of Semliki Forest virus (Glanville & Lachmi, I977). The functional defects of ts-4 can be understood if we assume that the mutated protein regulating the synthesis of 26S RNA is one of the non-structural proteins, which are apparently cleaved from a giant polyprotein (Cancedda et al. 1975; Lachmi & K~i/iri/iinen, 1976; Glanville et al. 1976 ). A mutation in one of the non-structural proteins apparently causes a cleavage defect in the polyprotein and results in accumulation of ns22o and nsI55. 31-2
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L. KAARI.AINEN, D. SAW1CKI A N D P. J. GOMATOS
The resultant large proteins contain polypeptides of the RNA polymerase which cannot function in the uncleaved form. This would explain why no increase in RNA synthesis takes place after the ts-4 infected cultures have been shifted to the restrictive temperature. In wild type SFV infected cells a short-lived protein of comparable size to our ns22o has been described by Clegg et al. (I976). Pulse-chase experiments suggested that this protein (p2oo) is a precursor of the RNA polymerase components pol 63 and pol 9o (Clewley & Kennedy, 1976). Two non-structural precursor proteins with apparent mol. wt. of 2300o0 and 215 ooo accumulate in Sindbis virus wild type infected cells in the presence of zinc ions (Brzeski & Kennedy, 1977). Taken together, these results suggest that our 22o may represent accumulation of an intermediate in the cleavage of the large non-structural polyprotein detectable in the wild type infected ceils as well. More than one mutation in the non-structural proteins of ts- 4 cannot be ruIed out at present. Since the synthesis of 42S RNA continues after the shift to 39 °C this mutation is not manifested at 39 °C when the protein has been synthesized at the permissive temperature. A second mutation would presumably only affect the cleavage defect(s) resulting in the accumulation of ns22o and nsI55. It is of interest that in the structural polyprotein of alphaviruses, mutation of the capsid protein is known to cause accumulation of the whole polyprotein in uncleaved form indicating that several cleavages can be inhibited by a single mutation (Burge & Pfefferkorn, I966; Scheele & Pfefferkorn, I97O; Schlesinger & Schlesinger, I973; Keriinen & K/i~iri/iinen, 1975 ; Lachmi et al. I975)This investigation was supported by the Finnish Academy, Sigrid Juselius Foundation and Finnish Science Society. We are grateful for the technical assistance of Ms Raija Lahdensivu. Actinomycin D was a kind gift from Merck, Sharp & Dohme.
REFERENCES BONNER, M. W. & LASKEY, R. (1974). A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. EuropeanJournal of Biochemistry 46, 83-88. BRACHA, i . , LEONE, A. & SCHLESINGER, M. J. (1976). F o r m a t i o n o f a Sindbis virus n o n s t r u c t u r a l protein and its relation to 42S m R N A function. Journal of Virology 7,0, 612-62o. BRZESKI, H. & KENNEDY, S. I. T. (1977). Synthesis o f Sindbis virus nonstructural polypeptides in chicken e m b r y o fibroblasts. Journal of Virology 22, 42o-429. BURGE, B.W. & PFEFFERKORN, E. R. (1966). Complementation between temperature-sensitive mutants o f Sindbis virus. Virology 3 o, 214-223. CANCEDDA, R., VILLA-KOMAROFF,L., LODISH, H. & SCHLESINGER, M. (I975). Initiation sites for translation o f Sindbis virus 42S a n d 26S m e s s e n g e r R N A s . Cell 6, 215-222. CLEGG, J. C. S. (1975). Sequential translation of capsid and membrane proteins in alphaviruses. Nature, London 254, 454-455. CLEGG, J. C. S., BRZESKI, H. & KENNEDY, S. I. T. (I976). R N A polymerase components in Semliki F o r e s t virus infected cells: synthesis from large precursors. Journal of General Virology 32, 413-43o. CLEWLEY, J. P. & KENNEDY, S. I. T. (I976). Purification and polypeptide c o m p o s i t i o n o f Semliki F o r e s t virus R N A polymerase. Journal of General Virology 32, 395-41 I. GLANVILLE, N. & LACHMI, B. (1977)- T r a n s l a t i o n o f proteins accounting for the full coding capacity of the Semliki F o r e s t virus 42S R N A g e n o m e . Federation of the European Biochemical Societies Letters 8 i , 399-402. GLANVILLE, N., LACHMI, B., SMITH, A. E. & KAARI~,INEN,L. 0978)- Tryptic peptide mapping of the nonstructural proteins o f Semliki F o r e s t virus. Bioehimica et Biophysiea Acta (in the press). GLANVILLE, N., RANKI, M., MORSER, J., K~RI~.INEN, L. & SMlXH, A. E. (I 976). Initiation o f translation directed by 42S a n d 26S R N A s f r o m Semliki F o r e s t virus in vitro. Proceedings of the National Academy of Sciences of the United States of America 73, 3o59-3o63. KXXV.I~.~NEN,L., SIMONS,K. & VON BONSDOKFF,C.-n. (1969). Studies on subviral components of Semliki F o r e s t virus. Annales Medieinae Experimentalis et Biologiae Fenniae 47, 235-248. KENNEDY, S. I. T. (1976). Sequence relationships between the genome and the intracellular R N A species o f standard and defective interfering Semliki F o r e s t virus. Journal of Molecular Biology 1o8, 491-51 I.
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KER~,NEN, S. & K.K~;I~I~INEN,L. (I974). Isolation and basic characterization of temperature-sensitive mutants from Semliki Forest virus. Aeta Pathologica et Microbiologica Seandinavica B82, 8Io-82o. KEg~_NEN, S. & K~,~,RIXINE~, L. (I975)- Proteins synthesized by Semliki Forest virus and its I6 temperaturesensitive mutants. Journal of Virology x6, 388-396. LACrIMI, B., GLANVILLE, N., KER~NEN, S. ~ K~RI~INEN, L. (I975). Tryptic peptide analysis of nonstructural and structural precursor proteins from Semliki Forest virus mutant-infected cells. Journal of Virology x6, I615-I629. LACHMI, B. & K~.~RI~INEN, L. (I976). Sequential translation of nonstructural proteins in cells infected with Semliki Forest virus mutant. Proceedings of the National Academy of Sciences of the United States of America 73, t936-I94O. LACHMI, B. & K~J,Rt~INEN,~L 0977). Control of protein synthesis in Semliki Forest virus infected cells. Journal of Virology 2z, I42-149. NEWLLB, D. r~. (I97I). Molecular weight determination of protein dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer. Journal of Biological Chemistry 246, 6328-6334. NtrSS, D. L., OPPERMEN, rL & I(OCH, G. (I975). Selective blockage of initiation of host cell protein synthesis in RNA-virus-infected cells. Proceedings o f the National Academy of Sciences o f the United States o f America 72, t258-I262. SABORIO, J. L., PONG, S.S. & KOCrIL G. (I974). Selective and reversible inhibition of initiation of protein synthesis in mammalian cells. Journal of Molecular Biology 85, I95-212. SARASTE, J., I~,gt~INErq, L., S6DERLUNO, H. & KER~NEN, S. 0977)- R N A synthesis directed by a temperaturesensitive mutant of Semliki Forest virus. Journal of General Virology 37, 399-4o6. SAWICKI, D. L,, K.~.~RI,~INEN, L., LAMBECK, C. & GOMATOS, P. J. (t978). Mechanism for control of synthesis of Semliki Forest virus z6S and 42S RNA. Journal of Virology 25, I9-27. SCnE~LE, C. M. 8, Pr~FrERKORN, E. g. 097O). Virus-specific proteins synthesized in cells infected with R N A + temperature-sensitive mutants of Sindbis virus. Journal of Virology 5, 329-337. SCaLESINGER, M. s. & SCI~LESING~R, S. (I973). Large-molecular-weight precursors of Sindbis virus proteins. Journal of Virology xx, IO13-ioi6. STRAUSS, J. H. & S'rrtAUSS, Z. (t977). Togaviruses. In The Molecular Biology of Animal Viruses pp. ~I~-I66. Edited by D. P. Nayak. New York: Marcel Dekker. WENGLER, G. & WV~LER, 6. (I975). Studies on the synthesis of viral RNA-polymerase-template complexes in B H K z~ cells infected with Semliki Forest virus. Virology 66, 322-326. WENGL~R, a. & WENGLEa, G. 0976). Localization of the 26S R N A sequence on the viral genome type 42S R N A isolated from Semtiki Forest virus infected cells. Virology 73, ~9o-~99.
(Received I I October I 9 7 7 )
J. gen. Virol. 0978), 39, 463-473 Printed in Great Britain
Cleavage Defect in the Non-structural Polyprotein of Semliki Forest Virus has Two Separate Effects on Virus R N A Synthesis By L E E V I K , ~ R I ~ I N E N Department of Virology, University of Helsinki, Haartmaninkatu 3, SF-oo29o Helsinki 29, Finland D O R O T H E A S A W I C K I * AND P E T E R J. G O M A T O S Division of Virology, Memorial Sloan-Kettering Institute, New York, New York IOO2I, U.S.A.
(Accepted 7 December I977) SUMMARY
When Semliki Forest virus ts-4 mutant infected cultures are grown at the permissive temperature (28 °C) and shifted to the restrictive temperature (39 °C), two different defects in RNA synthesis are manifested: (i) the synthesis of 26S RNA is stopped within 60 min (Saraste et al. 1977), and (ii) the increase in RNA synthesizing activity ceases, in contrast to cultures maintained at 28 °C, indicating that no new active RNA polymerase is formed at 39 °C. Accumulation of a non-structural precursor protein with an apparent mol. wt. of about 22oooo (ns22o) was demonstrated in ts-4 infected cultures shifted to 39 °C. Ns22o was labelled during short pulses given immediately after release of protein synthesis from hypertonic initiation block, suggesting that genes coding for ns22o are located near the initiation site at the 5'-end of the 42S RNA. The viral specificity of ns22o was shown by its disappearance after a shift to 28 °C and by labelling in the presence of sucrose, when no host cell protein synthesis is detectable. The two functional defects can be explained if the polypeptides responsible for the RNA polymerizing activity and that responsible for the synthesis of 26S RNA are components of the same non-structural polyprotein. A mutation in the latter polypeptide which prevents cleavage of the polyprotein would thereby prevent the further formation of active RNA polymerase. If cleavage of the polyprotein has taken place at the permissive temperature, the RNA polymerase would remain active also at 39 °C, whereas the polypeptide responsible for 26S RNA synthesis would become inactive due to the mutation. INTRODUCTION
The alphavirus 42S RNA genome coded proteins are probably translated as two polyproteins. The messenger for the structural polyprotein pI3o (mol. wt. 13o0o0) is the 26S RNA which is a replica of the 3' third of the 42S RNA (Kennedy, I976; Wengler & Wengler, I976; Strauss & Strauss, I977). The 42S RNA seems to be the messenger for at least three to four non-structural proteins which are synthesized starting from one initiation site (Cancedda et al. 1975; Glanville et al. 1976; Lachmi & K~i/iri~iinen,1976; Brzeski & Kennedy, I977; Glanville & Lachmi, I977). In SemIiki Forest virus (SFV) infected cells four non* Present address: Department of Microbiology, Medical College of Ohio, Toledo, Ohio 43699, U.S.A.
464
L. K ~ . A R I A I N E N , D. S A W I C K I
A N D P. J. G O M A T O S
structural proteins ns7o, ns86, ns72 and ns6o with apparent mol. wt. of 70000, 86000, 72000 and 6000o are synthesized sequentially in the above order. Ttyptic peptide mapping has recently shown that two short-lived proteins with mol. wt. of 155000 (nsI55) and 135o00 (nsI35) are precursols of the more stable non-structural proteins. The nsI55 contains the peptides of ns7 o and ns86 whereas the ns135 consists of ns72 and most probably also of ns6o (Glanville et al. 1978). An R N A negative mutant, ts-4, of Semliki Forest virus has a reversible defect in the synthesis of 26S R N A as shown by shift-up experiments (Saraste et al. I977). Here we report the detection in ts-4 infected cells of a third non-structural precursor protein, which has an apparent mol. wt. of about 22oo00, found only in cells shifted to the restrictive temperature. Similar size proteins have been described in Sindbis virus mutant (Bracha et al. I976), in Sindbis virus wild type (Brzeski & Kennedy, 1977) and in SFV wild type infected cells (Clegg et al. 1976). METHODS
Cells and virus. Secondary cultures of chicken fibroblast cells obtained from special pathogen-free animals were grown in 60 mm Petri dishes as described previously (Ker/inen & K/i/iri/iinen, I974)- SFV prototype strain was cultivated as described (Kfi/iri/iinen et al. I969), and the isolation, characterization and propagation of SFV ts-4 mutant have been described previously (Ker/inen & Kfi~rifiinen, 1974). Growth of virus and isotope labelling. Chicken cells were infected with ts-4 or wild type SFV (50 p.Lu./cell) at either 28 or 39 °C, as indicated. After I to 2 h adsorption, virus was removed and the cells were washed twice with Hanks' solution. The cells were then incubated in Eagle's minimum essential medium with or without o.2 % bovine serum albumin (BSA) and I/zg/ml of actinomycin D. In temperature shift experiments, fresh medium at either 28 or 39 °C was added to the cultures upon the temperature shift. For labelling with aH-uridine (Radiochemical Centre, Amersham, specific activity 29 to 31 Ci/mmol), 2o #Ci/dish was used. Labelling with 35S-methionine (Amersham, specific activity 92o Ci/mmol) was carried out in methionine-free Eagle's medium using lO to 5oo #Ci/dish, as indicated in individual experiments. Treatment of cells with hypertonic media. Infected chicken cells were exposed to either 335 mM-NaC1 (Saborio et al. 1974; Nuss et al. 1975) or o.I to o'5 M-sucrose for 4o to 6o rain in 3 ml of medium. The medium was removed and new medium containing 85Smethionine was added. 35S-Methionine labelling was carried out in methionine-free Eagle's medium supplemented with o-2% BSA and, as indicated in the text, also with o-I to 0"5 M-sucrose. At the end of the pulse, the medium was removed and sucrose-free Eagle's medium containing a 2o-fold excess of the normal concentration of methionine was added for variable times. The cells were washed with o.1 M-NaC1, o.oi M-tris-HC1 pH 7"4, and collected in 2 % aqueous SDS as described previously (Lachmi & K~i~iri~iinen, I976). Polyacrylamide gel electrophoresis. Polyacrylamide slab gels with lO ~o main gel and 3 to 5 % spacer gel were made according to the method of Neville (197I) and modified as described by Lachmi et al. (1975). The gels were stained with o'25 % Coomassie blue in 50 ~o trichloroacetic acid (TCA) for 45 mix and destained in 8 % acetic acid. In most cases, the stained gels were treated with dimethylsulphoxide and PPO and dried for fluorography as described by Bonner & Laskey (1974). Myosin was a generous gift from Rockefeller University, given by Dr Polly Etkind. R N A analysis. R N A was isolated from cells disrupted by 2 % sodium dodecyl sulphate (SDS) and was analysed using 15 to 30% (w/w) sucrose gradients made in o"14 M-NaC1,
RNA and protein synthesis by a ts-mutant of SFV I
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Hours Fig. I. R a t e o f R N A synthesis in ts-4 infected chicken cells m a i n t a i n e d at 28 °C or shifted to 39 °C at hourly intervals. C h i c k e n cells infected with ts-4 at 28 °C were m a i n t a i n e d at 28 °C a n d were either exposed to 3H-uridine (20 # C i / d i s h ) for 60 m i n at 28 °C or were shifted to 39 °C a n d pulsed similarly at so h post-infection. T h e cells were t a k e n into 2 % SDS a n d T C A precipitated radioactivity was determined. 0 - - 0 , Pulsed at 28 °C; (3 - - - © , shifted to 39 °C a n d pulsed between io a n d I I h post-infection.
o.oI ~-tris-HC1 pH 7"4, and o.ooI M-EDTA (TNE) and o.1% SDS. Centrifugation was for I4 h at 24ooo rev/min in the SW27~ rotor at 23 °C. The gradients were collected from the bottom by a collecting device connected to a peristaltic pump. Determination of radioactivity. For determination of 35S-methionine-labelled proteins, samples from SDS-solubilized cells were precipitated with Io % TCA, heated for 2o min at 9o °C, and the precipitates were collected on C F / C glass-fibre filters, sH-Uridinelabelled R N A was directly precipitated with Io % TCA. RESULTS
RNA synthesis The rate of R N A synthesis in ts-4 infected chicken cell cultures was studied at the permissive temperature (28 °C) as well as in cultures which had been shifted to the restrictive temperature (39 °C). Two cultures were taken at hourly intervals: one was labelled with 3H-uridine for 6o min at 18 °C and the other shifted to 39 °C. All the shifted cultures were labelled simultaneously for 6o min with 3H-uridine at IOh post-infection. After 4 h incubation at 28 °C the rate of R N A synthesis increased rapidly (Fig. 0 , similar to that of the wild type SFV. The R N A synthesis continued in all cultures which had
466
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Fig. 2. Sucrose gradient analysis of ts-4 specific RNAs synthesized between 6 and 7 h postinfection at 28 °C (a) or between IO and TT h at 39 °C (b) after shift up from 28 °C to 39 °C at 6 h after infection. Bottom of the gradient is to the left. The fast sedimenting peak is 42S RNA.
been shifted to the restrictive temperature. Interestingly, there was no increase in the rate of R N A synthesis in any of the cultures shifted to 39 °C. There was rather a decrease, maximally of about 5o%, compared to the R N A synthesizing activity observed at z8 °C at the time of shift (Fig. 0 . These results suggest that after the shift to the restrictive temperature no new active polymerase is formed, and that the R N A synthesizing activity detected at 39 °C as long as 8 h after the shift was due to R N A polymerase which was formed during the incubation at z8 °C. The same result was obtained when wild type SFV infected cultures were shifted to 39 °C in the presence of cycloheximide (IOO/~g/ml). Analysis of the RNAs formed at 28 °C and after the shift to 39 °C confirmed our previous findings that virtually no 26S R N A is synthesized at the restrictive temperature (Fig. 2; Saraste et al. I977; Sawicki et al. I978). Thus, two different steps are affected when ts-4 infected cultures are shifted to the restrictive temperature: (i) the synthesis of 26S R N A is shut off rapidly, and (ii) the formation of new polymerase is prevented. Protein synthesis
Duplicate ts-4 infected cultures maintained for 6 h at 28 °C were either shifted to 39 °C or maintained at 28 °C and labelled with 35S-methionine for I5 min with or without pretreatment with 335 mM-NaC1 or 0"5 M-sucrose. The hypertonic media were used to reduce the host protein synthesis which, during the early period of infection, makes the detection of virus proteins difficult (Lachmi & Kft~iri~iinen, T977). The labelled proteins resolved on a discontinuous polyacrylamide slab gel are shown in Fig. 3At 39 °C two large proteins can clearly be seen in ts-4 infected cultures treated with either
R N A and protein synthesis by a ts-mutant o f S F V
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Fig. 3. Fluorogram of IO % polyacrylamideslab gel of ts-4 induced, 85S-methionine-labelled proteins at 39 °C (A, B, C) and at 28 °C (E, F, (3). Ts-4 infected cells maintained for 6 h at 18 °C were either shifted to 39 °C or kept at 28 °C and were exposed to o'5 M-sucrose (B, F) or to 335 mM-NaC1 (C, G), or were kept as controls (A, E) for 4o rain and were thereafter labelled with 35S-methionine for I5 min at Ioo/zCi/dish and subsequently chased for 45 rain before harvest. Ts-I marker containing the previously identified non-structural proteins is shown in lane D, and the proteins of ~sS-methionine-labelled SFV is shown in lane H. Position of capsid (C) and envelope proteins EI and Ez as well as the precursor of E2 and E3, p6z, and the non-structural proteins ns7o (70), ns72 (72), ns86 (86), nsI35 (I35) and nsI55 (I55) are indicated, as is the new ts-4 specific protein (22o). salt or sucrose. T h e smaller o f these p r o t e i n s m i g r a t e d with the p r e v i o u s l y identified nonstructural p r e c u r s o r p r o t e i n nsI55 (mol. wt. 15500o) (Lachmi & Kfi/iri/iinen, I976, I977). T h e larger protein, which is n o t detected in cultures m a i n t a i n e d a n d labelled at 28 °C, m i g r a t e d with m y o s i n a n d thus has a mol. wt. close to 2200oo. T h e possibility t h a t t h e 22ooo0 mol. wt. p r o t e i n is virus-specific, a n d p e r h a p s a new nonstructural p r e c u r s o r p r o t e i n like the large p r o t e i n f o u n d in Sindbis virus infected cells ( B r a c h a et al. ~976; Brzeski & K e n n e d y , I977), was investigated as follows: ts-4 infected cultures i n c u b a t e d at 28 °C for 6 h were shifted to 39 °C a n d t r e a t e d with 335 mM-NaC1 for 4o min. This t r e a t m e n t specifically b l o c k s the initiation o f p r o t e i n synthesis b u t allows e l o n g a t i o n to t a k e place ( S a b o r i o et al. I974). W h e n the h y p e r t o n i c b l o c k o f initiation is r e m o v e d b y restoring the isotonicity o f the m e d i u m , s y n c h r o n o u s i n i t i a t i o n o f p r o t e i n
468
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Fig. 4. Fluorogram of IO ~oo polyacrylamide gel showing the sequential labelling of ts-4 induced proteins at 39 °C followed by a chase at 28 °C (A, C, E) or 39 °C (B, D, F). Ts-4 infected cultures maintained at 28 °C for 6 h were shifted to 39 °C and exposed to 335 mM-NaC1 for 4o min at 39 °C. They were labelled thereafter with 35S-methionine for 2 min (A, B) with 3oo/zCi/dish, for 6 min (C, D) with Ioo #Ci/dish, or for 28 min (E, F) with 33 #Ci/dish. The cultures were chased after the pulse for 6o min. The designation of the proteins is the same as in Fig. 3.
synthesis takes place. Using different pulse lengths, it is possible to determine the gene order in polyproteins (Saborio et al. 1974; Clegg, 1975)- We have used this method to determine the gene order in the non-structural polyproteins of Semliki Forest virus to be ns7o, ns86, ns72 and ns6o (Lachmi & K~i~iri~iinen, i976 ). Duplicate cultures of ts-4 infected cells were released from the hypertonic block of initiation and labelled for a, 6 and I8 min at 39 °C to label the n~7o, ns7o plus ns86, and finally all the non-structural proteins (Lachmi & K~i~iri~tinen, I976). After the pulse, unlabelled methionine was added and incubation was continued either at the restrictive or permissive temperature for a further 6o min. The fluorogram of proteins separated in a polyacrylamide slab gel is shown in Fig. 4. After a 2 min pulse at 39 °C followed by a 6o min chase at 28 °C only ns7o of the non-structural proteins is labelled (Fig. 4, lane A). It seems to consist of a double band under these conditions. Of the structural proteins, the Nterminal capsid protein is labelled but in this polyacrylamide gel it has been run out from the gel to increase the resolution of the large proteins. Lane B in Fig. 4 shows the proteins labelled in cultures pulsed for 2 min at 39 °C followed by a 6o min chase at 39 °C. Essentially
RNA and protein synthesis by a ts-mutant of SFV i
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Fig. 5. Effect of hypertonic media on protein synthesis in SFV and mock infected cells. (a) SFV wild type and mock infected cells were incubated at 39 °C for 3 h and exposed to different concentrations of sucrose between 3 and 4 h post-infection. They were labelled thereafter in the same media with 35S-methionine at 2o #Ci/dish for I5 rain and chased for 15 rain in the presence of unlabelled methionine. (b) SFV wild type and mock infected cells maintained for 4 h at 39 °C were exposed to 335 mM-NaCl for 4o min and were labelled for 30 min thereafter in the presence of various amounts of sucrose with 2o #Ci/dish of asS-methionine. 0 - - 0 , Wild type; • . . . . . • , mock infected. o n l y two p r o t e i n s are labelled, n a m e l y nsI55 and the 220000 d a l t o n p r o t e i n shown in Fig. 3- This result w o u l d indicate t h a t b o t h these p r o t e i n s are derived f r o m the N - t e r m i n a l p a r t o f the n o n - s t r u c t u r a l p o l y p r o t e i n a n d thus c o n t a i n ns7o. Labelling for 6 min at 39 °C followed b y a 60 min chase at 28 °C shows the presence o f ns7o, n o w clearly as a d o u b l e b a n d , a n d also o f ns86 (Fig. 4, lane C). This was as expected f r o m o u r previous results using the ts-I m u t a n t o f S F V ( L a c h m i & Kii~iri~iinen, I976). T h e p a t t e r n o f labelled p r o t e i n s becomes, however, m o r e c o m p l i c a t e d due to the start o f host p r o t e i n synthesis. A g a i n , cultures labelled at 39 °C a n d chased at 39 °C show the nsI55 a n d the 22oooo d a l t o n p r o t e i n b u t a l m o s t no ns86 o r ns7o, suggesting t h a t b o t h ns7o a n d ns86 a r e constituents o f the two large proteins. W h e n the labelling p e r i o d is extended to 18 min, the ns72 p r o t e i n also b e c o m e s labelled in cultures shifted to 28 °C for the chase (Fig. 4, lane E). O n l y small a m o u n t s o f nsI55 can be seen a n d the presence o f the 22oooo d a l t o n 3I
V I R 39
470
L. KAAR1AINEN~ D. SAW1CKI AND P. J. GOMATOS A
B
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I
Fig. 6. Fluorogram of a Io ~ polyacrylamide slab gel showing large tool. wt. proteins from ts-4 (B, D, F, H) and mock infected cells (C, E, G, I). The ts-4 and mock infected cultures maintained at 28 °C for 8 h were shifted to 39 °C and exposed to 335 mM-NaC1 for 40 min and were labelled thereafter for 3o min with 50 #Ci/dish of 35S-methionine in methionine-free Eagle's medium (B, C), or in methionine-free Eagle's medium containing o'I M-sucrose (D, E), o'I5 M-sucrose (F, G) or 0.2 M-sucrose (H, I). All the cultures were chased for ~o5 min in the presence of unlabelled methionine. Designation of the ts-I marker protein (A) is as in Fig. 3protein is difficult to exclude due to the heavy labelling o f host proteins in this region. The cultures labelled and chased at 39 °C show nsI55 as heavily labelled, as is the ns~35, which appears only after IO min or longer labelling periods (Fig. 4, lane F). There seems to be some processing of the non-structural polyprotein even at 39 °C, since ns86 and ns72 can be seen when large amounts o f radioactivity are applied to the gel. These results suggest that the new protein with mol. wt. o f about 22oooo f o u n d in ts-4 infected cells pulsed at 39 °C is a virus-specific non-structural precursor protein derived f r o m the N-terminal part o f the non-structural polyprotein.
Shut-off of host protein synthesis by hypertonie media Specific labelling o f virus proteins after the hypertonic block o f initiation is limited to only a short period o f time, since host cell protein synthesis starts to recover within a few minutes after the release o f the block (Saborio et al. I974) as can clearly be seen also f r o m Fig, 4. To overcome this difficulty we determined conditions to specifically label the virus proteins in the presence o f hypertonic media. SFV and m o c k infected cells were grown for 3 h at 39 °C and then incubation was continued in the presence o f different concentrations o f sucrose for 6o min, followed by a 15 min pulse with 35S-methionine in the same media (Fig. 5 a). The protein synthesis in the m o c k infected cultures is almost completely shut off when incubated and labelled in the presence o f o.2 M-sucrose. U n d e r these conditions protein synthesis in SFV infected cells was reduced to about 2o % of the level in the presence of isotonic medium. We t o o k advantage o f these results to label the cells after hypertonic block o f initiation induced by 335 mM-NaCI. As can be seen, there is some recovery o f host cell protein synthesis during the 3o min pulse with 35S-methionine when m o c k infected cells are transferred to isotonic medium (Fig. 5 b). I f the m o c k infected cells are labelled in o. I M or higher sucrose, no recovery o f protein synthesis is seen. However, SFV infected cultures showed reduced but detectable protein synthesis in media containing up to o'3 M-sucrose.
R N A and protein synthesis by a ts-mutant o f S F V
47I
The polyacrylamide slab gel in Fig. 6 shows the effect of different concentrations of sucrose on the labelling of the large proteins in ts-4 and mock infected cells after release from the hypertonic block of initiation. The cells were labelled for 3o rain and chased for IO5 rain before analysis of the proteins. In the mock infected cells, a large protein migrating more slowly than the 22o ooo dalton ts-4 protein is clearly seen if the labelling is carried out in the absence of sucrose (Fig. 6, lane C), but is not detectable when o- I M or higher sucrose concentrations are used during the labelling (Fig. 6, lanes E, G and I). The same protein is also seen in the ts-4 infected cells labelled without sucrose but not in those labelled in the presence of sucrose. The most heavily labelled protein is the nsI55, but the 22oooo dalton protein is seen with all the sucrose concentrations used during the labelling. These results support the idea that the 22oooo dalton protein in ts-4 infected cells is clearly a virus-specific one. The pulse-chase experiments suggested that it is most probably a precursor of the non-structural proteins, and therefore we designate it as ns22o. The results also show that unequal labelling of some host proteins after the release of hypertonic block of initiation can cause severe difficulties in the interpretation of the results.
DISCUSSION
We have studied the R N A and protein synthesis of a tempeiature sensitive mutant, ts-4, of Semliki Forest virus. Ts- 4 is phenotypically an R N A negative mutant, which cannot synthesize significant amounts of virus R N A when infection is initiated at the restrictive temperature (KerSnen & K~ri~inen, 1974). When infection is started at the permissive temperature, it is able to continue RNA synthesis for long periods of time, showing that the R N A polymerase function of this mutant is intact. As we have previously reported, the relative amounts of the RNAs synthesized are affected by the shift to the restrictive temperature: synthesis of 26S R N A stops rapidly whereas that of 42S RNA continues (Saraste et a[. 1977; Sawicki et aL 1978 ). Thus the mutation apparently affects a protein which regulates the synthesis of 26S R N A (Sawicki et al. 1978). The functional defect is reversible since 26S RNA synthesis starts again when the cultures are shifted back to the permissive temperature in the presence of cycloheximide. The fact that there is no increase in the R N A synthesizing activity after the ts-4 infected cultures were shifted to 39 °C suggests that no new active RNA polymerase was formed at this temperature. The same result was obtained for SFV wild type infected cultures exposed to cycloheximide (see also Wengler & Wengler, I975; Clegg et al. I976). These results can be simply explained by the accumulation of a large 22oooo dalton protein in the ts-4 infected cultures labelled after shift to 39 °C. This protein seems to be virus-specific and a non-structural protein precursor since it is labelled under conditions when practically no host proteins are synthesized and is processed in cultures labelled at 39 °C and chased at 28 °C, to yield previously identified non-structural proteins. The labelling of ns22o and ns155 at 39 °C during a 2 min pulse after release of the cells from hypertonic block of initiation would suggest that both these proteins are translated from the 5'-end of the 42S RNA, which is the messenger R N A for the non-structural proteins of Semliki Forest virus (Glanville & Lachmi, I977). The functional defects of ts-4 can be understood if we assume that the mutated protein regulating the synthesis of 26S RNA is one of the non-structural proteins, which are apparently cleaved from a giant polyprotein (Cancedda et al. 1975; Lachmi & K~i/iri/iinen, 1976; Glanville et al. 1976 ). A mutation in one of the non-structural proteins apparently causes a cleavage defect in the polyprotein and results in accumulation of ns22o and nsI55. 31-2
472
L. KAARI.AINEN, D. SAW1CKI A N D P. J. GOMATOS
The resultant large proteins contain polypeptides of the RNA polymerase which cannot function in the uncleaved form. This would explain why no increase in RNA synthesis takes place after the ts-4 infected cultures have been shifted to the restrictive temperature. In wild type SFV infected cells a short-lived protein of comparable size to our ns22o has been described by Clegg et al. (I976). Pulse-chase experiments suggested that this protein (p2oo) is a precursor of the RNA polymerase components pol 63 and pol 9o (Clewley & Kennedy, 1976). Two non-structural precursor proteins with apparent mol. wt. of 2300o0 and 215 ooo accumulate in Sindbis virus wild type infected cells in the presence of zinc ions (Brzeski & Kennedy, 1977). Taken together, these results suggest that our 22o may represent accumulation of an intermediate in the cleavage of the large non-structural polyprotein detectable in the wild type infected ceils as well. More than one mutation in the non-structural proteins of ts- 4 cannot be ruIed out at present. Since the synthesis of 42S RNA continues after the shift to 39 °C this mutation is not manifested at 39 °C when the protein has been synthesized at the permissive temperature. A second mutation would presumably only affect the cleavage defect(s) resulting in the accumulation of ns22o and nsI55. It is of interest that in the structural polyprotein of alphaviruses, mutation of the capsid protein is known to cause accumulation of the whole polyprotein in uncleaved form indicating that several cleavages can be inhibited by a single mutation (Burge & Pfefferkorn, I966; Scheele & Pfefferkorn, I97O; Schlesinger & Schlesinger, I973; Keriinen & K/i~iri/iinen, 1975 ; Lachmi et al. I975)This investigation was supported by the Finnish Academy, Sigrid Juselius Foundation and Finnish Science Society. We are grateful for the technical assistance of Ms Raija Lahdensivu. Actinomycin D was a kind gift from Merck, Sharp & Dohme.
REFERENCES BONNER, M. W. & LASKEY, R. (1974). A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. EuropeanJournal of Biochemistry 46, 83-88. BRACHA, i . , LEONE, A. & SCHLESINGER, M. J. (1976). F o r m a t i o n o f a Sindbis virus n o n s t r u c t u r a l protein and its relation to 42S m R N A function. Journal of Virology 7,0, 612-62o. BRZESKI, H. & KENNEDY, S. I. T. (1977). Synthesis o f Sindbis virus nonstructural polypeptides in chicken e m b r y o fibroblasts. Journal of Virology 22, 42o-429. BURGE, B.W. & PFEFFERKORN, E. R. (1966). Complementation between temperature-sensitive mutants o f Sindbis virus. Virology 3 o, 214-223. CANCEDDA, R., VILLA-KOMAROFF,L., LODISH, H. & SCHLESINGER, M. (I975). Initiation sites for translation o f Sindbis virus 42S a n d 26S m e s s e n g e r R N A s . Cell 6, 215-222. CLEGG, J. C. S. (1975). Sequential translation of capsid and membrane proteins in alphaviruses. Nature, London 254, 454-455. CLEGG, J. C. S., BRZESKI, H. & KENNEDY, S. I. T. (I976). R N A polymerase components in Semliki F o r e s t virus infected cells: synthesis from large precursors. Journal of General Virology 32, 413-43o. CLEWLEY, J. P. & KENNEDY, S. I. T. (I976). Purification and polypeptide c o m p o s i t i o n o f Semliki F o r e s t virus R N A polymerase. Journal of General Virology 32, 395-41 I. GLANVILLE, N. & LACHMI, B. (1977)- T r a n s l a t i o n o f proteins accounting for the full coding capacity of the Semliki F o r e s t virus 42S R N A g e n o m e . Federation of the European Biochemical Societies Letters 8 i , 399-402. GLANVILLE, N., LACHMI, B., SMITH, A. E. & KAARI~,INEN,L. 0978)- Tryptic peptide mapping of the nonstructural proteins o f Semliki F o r e s t virus. Bioehimica et Biophysiea Acta (in the press). GLANVILLE, N., RANKI, M., MORSER, J., K~RI~.INEN, L. & SMlXH, A. E. (I 976). Initiation o f translation directed by 42S a n d 26S R N A s f r o m Semliki F o r e s t virus in vitro. Proceedings of the National Academy of Sciences of the United States of America 73, 3o59-3o63. KXXV.I~.~NEN,L., SIMONS,K. & VON BONSDOKFF,C.-n. (1969). Studies on subviral components of Semliki F o r e s t virus. Annales Medieinae Experimentalis et Biologiae Fenniae 47, 235-248. KENNEDY, S. I. T. (1976). Sequence relationships between the genome and the intracellular R N A species o f standard and defective interfering Semliki F o r e s t virus. Journal of Molecular Biology 1o8, 491-51 I.
RNA and protein synthesis by a ts-mutant of SFV
473
KER~,NEN, S. & K.K~;I~I~INEN,L. (I974). Isolation and basic characterization of temperature-sensitive mutants from Semliki Forest virus. Aeta Pathologica et Microbiologica Seandinavica B82, 8Io-82o. KEg~_NEN, S. & K~,~,RIXINE~, L. (I975)- Proteins synthesized by Semliki Forest virus and its I6 temperaturesensitive mutants. Journal of Virology x6, 388-396. LACrIMI, B., GLANVILLE, N., KER~NEN, S. ~ K~RI~INEN, L. (I975). Tryptic peptide analysis of nonstructural and structural precursor proteins from Semliki Forest virus mutant-infected cells. Journal of Virology x6, I615-I629. LACHMI, B. & K~.~RI~INEN, L. (I976). Sequential translation of nonstructural proteins in cells infected with Semliki Forest virus mutant. Proceedings of the National Academy of Sciences of the United States of America 73, t936-I94O. LACHMI, B. & K~J,Rt~INEN,~L 0977). Control of protein synthesis in Semliki Forest virus infected cells. Journal of Virology 2z, I42-149. NEWLLB, D. r~. (I97I). Molecular weight determination of protein dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer. Journal of Biological Chemistry 246, 6328-6334. NtrSS, D. L., OPPERMEN, rL & I(OCH, G. (I975). Selective blockage of initiation of host cell protein synthesis in RNA-virus-infected cells. Proceedings o f the National Academy of Sciences o f the United States o f America 72, t258-I262. SABORIO, J. L., PONG, S.S. & KOCrIL G. (I974). Selective and reversible inhibition of initiation of protein synthesis in mammalian cells. Journal of Molecular Biology 85, I95-212. SARASTE, J., I~,gt~INErq, L., S6DERLUNO, H. & KER~NEN, S. 0977)- R N A synthesis directed by a temperaturesensitive mutant of Semliki Forest virus. Journal of General Virology 37, 399-4o6. SAWICKI, D. L,, K.~.~RI,~INEN, L., LAMBECK, C. & GOMATOS, P. J. (t978). Mechanism for control of synthesis of Semliki Forest virus z6S and 42S RNA. Journal of Virology 25, I9-27. SCnE~LE, C. M. 8, Pr~FrERKORN, E. g. 097O). Virus-specific proteins synthesized in cells infected with R N A + temperature-sensitive mutants of Sindbis virus. Journal of Virology 5, 329-337. SCaLESINGER, M. s. & SCI~LESING~R, S. (I973). Large-molecular-weight precursors of Sindbis virus proteins. Journal of Virology xx, IO13-ioi6. STRAUSS, J. H. & S'rrtAUSS, Z. (t977). Togaviruses. In The Molecular Biology of Animal Viruses pp. ~I~-I66. Edited by D. P. Nayak. New York: Marcel Dekker. WENGLER, G. & WV~LER, 6. (I975). Studies on the synthesis of viral RNA-polymerase-template complexes in B H K z~ cells infected with Semliki Forest virus. Virology 66, 322-326. WENGL~R, a. & WENGLEa, G. 0976). Localization of the 26S R N A sequence on the viral genome type 42S R N A isolated from Semtiki Forest virus infected cells. Virology 73, ~9o-~99.
(Received I I October I 9 7 7 )
