Control of Protein Synthesis in Semliki Forest ... - Journal of Virology

JOURNAL OF VIROLOGY, Apr. 1977, p. 142-149 Copyright © 1977 American Society for Microbiology

Vol. 22, No. 1 Printed in U.S.A.

Control of Protein Synthesis in Semliki Forest Virus-Infected Cells BAT-EL LACHMIl* AND LEEVI KAARIAINEN Department of Virology, University of Helsinki, SF-00290 Helsinki 29, Finland Received for publication 3 August 1976

Protein synthesis in Semliki forest virus-infected chicken embryo cells was studied by labeling them with [35S]methionine for short periods at different times after infection, with or without synchronization of protein synthesis by the hypertonic block technique. The rate of host-cell protein synthesis declined almost linearly in inverse correlation to the increase in the amount of virusspecific RNA. At 5.5 h postinfection, the host-cell protein synthesis was reduced by about 70%. The viral structural proteins were detectable with certainty at 3.5 h postinfection, and their rate of synthesis increased linearly parallel to the amount of their messenger, the 26S RNA. This suggests that the rate of synthesis of the structural proteins is controlled at the level of transcription. The rate of synthesis of the virus-specific nonstructural proteins attained its maximum between 3 and 4 h postinfection and declined thereafter, whereas the amount of their messenger, the 42S RNA, continued to increase linearly in the cells. Thus, the messenger activity of the 42S RNA is reduced in the late phase of infection compared with its activity in the early phase.

The mode of expression of the genetic information of Semliki forest virus 42S RNA (molecular weight, 4.0 x 106 to 4.5 x 106) (19, 26) has recently been elucidated to a large extent. The four structural proteins (capsid protein and the glycoproteins El, E2, and E3) are translated as a polyprotein with a molecular weight of about 130,000 (16, 17). The messenger for the structural polyprotein is the intracellular 26S RNA (molecular weight, 1.6 x 106) (26), which is a replica of the 3' third of the 42S RNA (38). The translation starts from one initiation site as has been shown by several investigators (5, 7, 9). Four nonstructural proteins with molecualr weights of 70,000 (ns7O), 86,000 (ns86), 72,000 (ns72), and 60,000 (ns6O) were recently shown to be synthesized sequentially when a temperature-sensitive mutant (ts-1) of Semliki forest virus was studied (18) by the hypertonic block technique (6, 24, 25). The four nonstructural proteins were apparently synthesized as a giant polyprotein with a molecular weight of about 290,000, which is presumably cleaved to give two short-lived intermediates with molecular weights of 155,000 (nsl55) and 135,000 (ns135) (18). The messenger for the nonstructural proteins is probably the viral 42S RNA genome, which in vitro directs the synthesis of products identical to the nonstructural proteins isolated ' Present address: Department of Virology, Israel Institute for Biological Research, Tel-Aviv University Medical School, P.O.B. 19, Ness-Ziona, Israel.

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from ts-1-infected cells (N. Glanville, B. Lachmi, A. E. Smith, and L. Kaariainen, submitted for publication). Support for the idea that the nonstructural proteins are made as a polyprotein has also been obtained from in vitro studies, in which the 42S RNA-directed product was shown to yield only one formyl-[35S]methionine tryptic and pronase peptide, suggesting that there is only one active initiation site for the nonstructural proteins in this RNA (9). Here we have studied the synthesis of the structural and nonstructural proteins in Semliki forest virus wild-type-infected cells. The rate of synthesis of the nonstructural proteins was maximal early in infection and declined thereafter, despite the continuous increase in the amount of the 42S RNA. By contrast, the rate of synthesis of the structural proteins in the cells increased parallel to the amount of 26S RNA showing that the synthesis of structural and nonstructural proteins is controlled by different mechanisms. MATERIALS AND METHODS Virus and cells. Semliki forest virus prototype strain (13) and a ts-1 mutant (15, 16) were used in these studies. BHK-21 cells and secondary special pathogen-free chicken embryo fibroblasts were cultivated as before (16). All experiments were carried out at 39°C, the restrictive temperature, in the presence of 1 ug of actinomycin D per ml. Labeling of virus-specific proteins. Confluent monolayers of secondary chicken embryo fibroblasts

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PROTEIN SYNTHESIS IN ALPHAVIRUS-INFECTED CELLS

were infected at a multiplicity of 50 or 500 PFU per cell and incubated at 390C. At the times indicated, the cells were pulsed with [35S]methionine, 50 or 200 jCi per plate (210 to 250 Ci/mmol, Amersham/ Searle Corp.) in a methionine-free medium. The pulses were followed by chases in the presence of 10fold the normal concentration of unlabeled methionine. The whole cells were taken into hot 2% sodium dodecyl sulfate (SDS) as described before (16, 17). In some experiments the cells were incubated in the presence of 335 mM NaCl for 30 or 40 min before the pulse was given in isotonic medium as described (18). Protein determinations were carried out according to Lowry et al. (20) using bovine serum albumin as standard. Polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis was carried out by the discontinuous system described by Neville (23) and modified as before (17). Electrophoresis was carried out in slab gels at 10 mA for 6 to 7 h. The gels were dried and autoradiographed. In some experiments after electrophoresis the gels were impregnated with PPO (2,5-diphenyloxazole) and then dried and fluorographed (2). For quantitation, the individual lanes, taken from the slab, were cut into 1-mm slices, solubilized in NCS (Nuclear Chicago Solubilizer) and counted in a toluene-based scintillation fluid. The quantitations of the gels were done according to the method described by McAllister and Wagner (21). This method enabled us to determine the percentage of reduction in host-cell protein accumulation and the accumulation of virus-specific proteins. The region of each gel that does not contain known virus-specific proteins (fractions 5 to 15) was chosen to normalize activity. The normalization ratio (N), which indicates the degree of reduction of host-cell protein synthesis, was determined by dividing the sum of radioactivity in fractions 5 to 15 from virus-infected cells (I) by the sum of radioactivity in the same region from mock-infected cells (U), i.e., N =I/U. The virus-specific protein accumulation in each infected cell gel fraction (V) was determined by the equation i - NU, where i was the radioactivity in an infected gel fraction and U was the radioactivity in the corresponding mock-infected gel fraction, and where N is the reduction in host-cell protein synthesis and NU is the host-cell background estimated for the infected cell gel fraction. Labeling of viral RNA. Confluent monolayers were infected at a multiplicity of 500 PFU and incubated at 39°C. At the end of the adsorption period, 10 ,ACi per plate of [3H]uridine (28 Ci/mmol) was added to each plate, and the incubation was continued. At 1-h intervals, from 1.5 to 5.5 h postinfection, one plate was taken into 2% SDS, and the extracts were analyzed in 15 to 30% (wt/wt) sucrose gradients as described before (15).

RESULTS Identification of Semliki forest virus-induced proteins. The structural and nonstructural proteins synthesized in chicken cells in-

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fected with our ts-1 mutant at 39°C are shown in Fig. 1. As shown, the viral protein synthesis recovered more quickly from hypertonic block of initiation than did the host-cell protein synthesis (6, 18, 24). We tried to reduce the residual host-cell protein synthesis to the minimum in infected cells by incubating them in hypertonic medium (335 mM NaCl) for 40 min. When the hypertonic medium is replaced by an isotonic one, synchronous initiation follows (6). Therefore, infected cells were labeled immedi-

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FIG. 1. Autoradiograph of 7.5% polyacrylamide slab gel of cellular extracts from ts-1 -infected chicken embryo cells following synchronization of initiation by a high-salt block. Infected cells were incubated at 39°C for 4 h 20 min, treated with high salt (335 mM NaCl) for 40 min, and then pulsed for 15 min with [35S]methionine (100 uCi per plate) upon restoration of salt concentrations and chased for 15 min (A) or 60 min (B) in the presence of cold methionine. The slab gel was impregnated with PPO and then dried and fluorographed for 1 week. C, El, and p62 are structural proteins and 155 (ns155), 135 (ns135), 86 (ns86), and 70 (ns7O) are nonstructural proteins.

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ately with [35S]methionine for 15 min and only been demonstrated by C-terminal labeling chased in the presence of excess unlabeled me- after treatment with pactamycin (18). thionine for 15 min (lane A) and 60 min (lane We have recently shown by tryptic peptide B). The fastest migrating band in the autoradi- analysis that ns155 and ns135 are different proogram of a discontinuous SDS slab gel was the teins, the former being the precursor for ns7O viral capsid protein (C) followed by the enve- and ns86 and the latter for ns72 and probably lope protein (El) and the precursor of envelope ns6O (Glanville et al., submitted for publicaproteins E2 and E3 (p62). The bands migrating tion). more slowly than p62 were the nonstructural Nonstructural proteins in Semliki forest viproteins with apparent molecular weights of rus wild-type-infected cells. The function of 70,000, 72,000 (previously determined 78,000 the more stable nonstructural proteins is not from its migration in 11% gels [16]), and 86,000. yet known. Since Sindbis virus RNA- temperaThese proteins were designated ns7O, ns72, and ture-sensitive mutants fall into four complens86 according to the recommendation of an mentation groups (3, 33), one would expect at international group of virologists (1). In lane A, least some of the nonstructural proteins to be which represents the 15-min chase, two addi- involved in the replication of the viral RNA. tional bands with apparent molecular weights Since RNA synthesis is an early event in the of 155,000 (nsl55) and 135,000 (nsl35) can be infectious cycle, the presence of the nonstrucseen. These proteins were scarcely detectable tural proteins in wild-type-infected cells was after the 60-min chase suggesting that they studied mainly during the early phase of the were precursors for the nonstructural proteins. growth cycle. The fourth nonstructural protein, ns6O, could Wild-type-infected cells were labeled at difnot be separated from p62 using the usual poly- ferent times postinfection both with and withacrylamide gel electrophoresis system. It has out hypertonic salt treatment. The autoradio-

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FIG. 2. Autoradiograph of 7.5% discontinuous SDS-polyacrylamide gel of cellular extracts from Semliki forest virus wild-type-infected and mock-infected cells. Infected chicken embryo cells were incubated at 39°C and at 2.5 h (A), 3.5 h (C), 4.5 h (D), and 5.5 h (E); one plate was treated with high salt (335 mM NaCl) for 30 min. The cells were pulsed immediately with [35S]methionine in isotonic medium for 20 min and chased for 5 min. Mock-infected cells (B) were not treated with high salt but were pulsed and chased similarly at 5 h postinfection. Each sample applied to the gel contained about 180,000 cpm. C and El are the structural proteins. p62 is a precursor of envelope proteins E2 and E3. 72, 86, 135, and 155 are the nonstructural proteins.


VOL. 22, 1977

graph of a polyacrylamide slab gel in Fig. 2 shows the [35S]methionine-labeled proteins of Semliki forest virus at 2.5, 3.5, 4.5, and 5.5 h postinfection. The infected cells were treated with the hypertonic medium for 30 min prior to the 20-min pulse given in isotonic medium followed by a 5-min chase. In addition to the above described structural proteins, the envelope protein E2 is seen between p62 and El (Fig. 3). The nonstructural proteins ns155 and ns135 are clearly identifiable, as are ns86, Ms72, and ns7O. Similar labeling carried out with cells not exposed to the high salt treatment is shown in Fig. 3. The presence of the above described nonstructural proteins among host-cell proteins can be seen clearly at 3.5 h postinfection (lane C) with the exception of ns7O, which is not properly resolved from ns72. Individual lanes of the dried slab gels were cut into 1-mm slices and then solubilized and counted in liquid scintillator (Fig. 4). To estimate the proportions of viral structural and

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nonstructural proteins, the difference analysis method of McAllister and Wagner was used (21). A region of the gel from virus-infected and mock-infected cells (fractions 5 to 15 in Fig. 4) was chosen for evaluation of the reduction in host-cell-specific protein synthesis. The total radioactivity in this region of gels from infected cells was divided by the radioactivity in the same region of mock-infected cells. This normalization ratio expresses the degree of inhibition of host-cell protein synthesis (Fig. 5A). The multiplication of the radioactivity in each of the mock-infected gel fractions by this ratio gave the value of residual host-cell protein synthesis. This value was then subtracted from the radioactivity in the corresponding fractions of the virus-infected gels to give the virus-specific radioactivity. The amount of virus-specific radioactivity at the positions of capsid, envelope proteins El, E2, and p62 were summed up to yield the total radioactivity of structural proteins synthesized at different times after infec-

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FIG. 3. Autoradiograph of 7.5% discontinuous SDS-polyacrylamide gel of cellular extracts from Semliki forest virus-infected and mock-infected cells. Infected chicken embryo cells were incubated at 39°C and at 2 h 10 min (A), 3 h 10 min (C), 4 h 10 min (D), and 5 h 10 min (E) postinfection; they were pulsed for 20 min with 50 ,Ci of [35S]methionine per plate and then chased for an additional 5 min. Mock-infected cells (B) were pulsed and chased similarly at 5 h 10 min postinfection. The slab gel was dried and exposed for 2 days. Symbols and amounts of radioactivity are as for Fig. 2. The main host proteins are marked with H.

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FRACTION NUMBER FIG. 4. Quantitation ofpolyacrylamide gels representing wild-type virus-infected cells at 3.5 h (A) and 5.5 h (B) postinfection, representing lanes C and E in Fig. 3, respectively. The profile of the quantitation of mockinfected cells (lane B in Fig. 3) is drawn for reference. The arrows show the positions of nonstructural 155 (nsl155), 135 (ns135), 86 (ns86), 72 (ns 72), and structural proteins El, E2 and C. Symbols: 0, virus infected;

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tion. The result is expressed as the percentage of total radioactivity recovered from the gels in Fig. 5C. Similarly, the virus-specific radioactivities at the positions of ns155, ns135, ns86, and ns72 plus ns7O were summed up to yield the total

amount of nonstructural proteins synthesized during the 20-min pulse. In this quantitation the fourth nonstructural protein ns6O, which cannot be distinguished from p62 by the gel electrophoresis method used, was included in the structural proteins. Thus the amount of


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FIG. 5. Synthesis rate of host-cell proteins (A), virus-specific nonstructural proteins (B), and structural proteins (C) compared with the amount of 42S RNA (B) and 26S RNA (C) at different times after infection. Lanes of the slab gel shown in Fig. 3 were cut into 1-mm slices and then solubilized and counted in a liquid scintillator. The radioactivity profile from mock-infected cells, shown in Fig. 4, was used to calculate the degree of inhibition of host-cell protein synthesis and for the estimation of virusspecific nonstructural and structural proteins according to the difference analysis method of McAllister and Wagner (21). The radioactivities of hostcell proteins and viral nonstructural and structural proteins are presented as percentage of the total radioactivity recovered from the gels. The cumulative amount of 42S RNA (B) and 26S RNA (C) at different times of infection are shown for comparison. Labeling of cells with [3Hiuridine was carried out as described in the legend to Fig. 6. Symbols: 0, rate of protein synthesis by the difference analysis method; A, residual host protein synthesis determined by dividing the sum of radioactivity in fractions 5 to 15 (see Fig. 4) of infected cells by respective sum from uninfected cells x 100; 0, percent radioactivity in 42S RNA and in 26S RNA of total at 5.5 h postinfection.

radioactivity in the nonstructural proteins represents an underestimate (Fig. 5B). The rest of the radioactivity in the gels was taken as host proteins giving another estimate for the residual host protein synthesis (Fig. 5A). There is a progressive decrease in the rate of host-cell protein synthesis and a simultaneous increase in the rate of synthesis of the nonstructural proteins. The maximum rate of synthesis of the nonstructural proteins is reached at 3.5 h postinfection, at a time when host-cell protein synthesis is inhibited only by about 20%. At this time the synthesis rates of structural and nonstructural proteins are almost equal. At 5.5 h postinfection, the synthesis rate of the nonstructural proteins is about half of the maximum. The difference analysis revealed that 1.4% of the total radioactivity was in nonstructural proteins at 2.5 h postinfection, whereas no structural proteins could be detected at this time.

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Amount of virus-specific RNAs. Cumulative labeling of wild-type-infected cells with [3H]uridine starting from 1 h postinfection was carried out in the presence of actinomycin D. Part of the cultures were removed at hourly intervals, and the RNAs were analyzed, after treatment with SDS, by sucrose gradient centrifugation as shown in Fig. 6. The radioactivity in the viral RNAs was taken to represent the total amount of these RNAs in the cell (35). The main RNA species are the 42S RNA genome and 26S RNA, which were synthesized in a molar ratio of close to 1 at all times after infection. The increase in the radioactivity in both RNA species is almost linear beginning from 2.5 h postinfection (Fig. 5). The increase in the rate of synthesis of the structural proteins correlates well with the increase in the amount of the 26S RNA; however, there does not seem to be a simple correlation between the rate of synthesis of the nonstructural proteins and the amount of the 42S RNA, which is regarded as the messenger of these proteins (5, 8, 9). 15

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FRACTION NUMBER FIG. 6. Sucrose gradient analysis of RNAs from Semliki forest virus-infected cells. Secondary chicken embryo fibroblasts were infected with 500 PFU per cell and maintained in minimal essential medium supplemented with 0.2% bovine serum albumin in the presence of 1 Mg of actinomycin D per ml. The cells were labeled with [3H]uridine (10 1iCi per plate) from 1.0 to 5.5 h postinfection. The whole cells were dissolved in 2% SDS in a buffer containing 0.15 M NaCl, 0.001 M EDTA, and 0.01 M Tris (TSE; pH 7.4). The extract was analyzed in 15 to 30% (wtlwt) sucrose made in TSE containing 0.1% SDS. Centrifugation was for 12 h at 24,000 rpm in an SW27 Spinco rotor at 230C.

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DISCUSSION The translation of Semliki forest virus-coded proteins consists of two different processes. (i) The structural proteins are translated conceptually as a polyprotein. Due to the nascent polysomal cleavage of capsid protein and the rapid cleavage between envelope proteins El and p62 (the precursor of E2 and E3), these three proteins are the earliest detectable products of translation (6, 30). (ii) The nonstructural proteins are probably also translated as a polyprotein, which should have a molecular weight to 300,000 (9, 18). Such a protein has not been found, which may indicate that a nascent cleavage, similar to that of the capsid protein, takes place, giving rise to the short-lived precursors nsl55 and nsl35. The former gives rise to two more stable polypeptides, ns7O and ns86, and the latter gives rise to polypeptides ns72 and ns6O (18; Glanville et al., submitted for publication). These nonstructural proteins have thus far been demonstrated only in cells infected with our temperature-sensitive mutant ts-1 (15-17) where they accumulate in easily detectable quantities (11, 12). In this study we have demonstrated the presence of the nonstructural proteins also in the wild-type-infected cells. Their synthesis was followed during the early hours of infection and reached its maximum rate at 3.5 h postinfection. At this time, they were synthesized at a rate similar to that of the structural proteins (Fig. 5). Estimates at 2.5 h postinfection, although less reliable because of the ongoing host-cell protein synthesis, would suggest that more nonstructural than structural proteins were made at this time. All our attempts to demonstrate the synthesis of either nonstructural or structural proteins earlier than 2.5 h postinfection have failed. It seems that at least 10% of the amount of viral RNA found at 5.5 h postinfection must be presented before the proteins become detectable by the methods used here. This already represents several thousands of newly synthesized viral RNA molecules (35). The fact that a given RNA can direct the synthesis of a known product in a cell-free protein-synthesizing system, together with the finding that the same RNA is associated with polysomes, has been regarded as sufficient evidence of its role as a messenger in the cell. The alphavirus 26S RNA fulfills both these criteria and is regarded as the cellular messenger for the structural proteins (4, 6, 7, 8, 14, 22, 27, 31, 32, 37). According to the same criteria, the 42S RNA should be regarded as the messenger for the nonstructural proteins; it has been found associated with the polysomes in infected cells

J. VIROL.

(22, 27, 31), and it stimulates the synthesis of nonstructural proteins in vitro (5, 8, 9,. 28). It was therefore interesting to correlate the rate of synthesis of the structural and nonstructural proteins with the amount of their respective messenger RNAs. The rate of synthesis of the structural proteins and the amount of 26S RNA rose with almost identical kinetics throughout the infection (Fig. 5C), suggesting that the amount of structural proteins synthesized is dependent on the amount of their messenger RNA. This situation is similar to that found for the synthesis of a- and ,3-globin chains in the reticulocyte lysate (34). The rate of synthesis of the nonstructural proteins correlated poorly with the amount of their 42S RNA messenger. The maximum rate of synthesis of these proteins was observed at 3.5 h postinfection when only about half of the 42S RNA found at 5.5 h postinfection had been synthesized (Fig. 5B). A rough estimate of the efficiency of 42S RNA at 3.5 and 5.5 h postinfection can be obtained if the amount ofradioactivity incorporated into the nonstructural proteins during the 20-min pulse is divided by the amount of radioactivity accumulated in 42S RNA from 1 h postinfection. There is about a sixfold drop in the "messenger efficiency" of this RNA between 3.5 and 5.5 h postinfection. One apparent possibility to explain the reduced messenger activity of the 42S RNA is the consumption of this RNA in the formation of viral nucleocapsids (11, 29, 32), which would make the 42S RNA inactive as messenger. The maximum rate of synthesis of the nonstructural proteins taking place at 3.5 h postinfection would then be the result of two processes: the synthesis of enough 42S RNA and the lack of structural proteins, i.e., capsid protein to encapsidate this RNA. Later, when the rate of synthesis of the structural proteins increased, all or almost all the newly synthesized 42S RNA would become encapsidated, and only small amounts of nonstructural proteins could be translated due to "leakiness" of the encapsidation process. This would explain why the bulk of 42S RNA synthesized between 4 and 5 h postinfection is found in the 140S nucleocapsid (27, 29, 32, 36). The process of association of the capsid protein with the 42S RNA is seemingly fast and, at 5 to 6 h, postinfection takes place within 5 to 8 min after the protein has been synthesized (29). The mechanism of nucleocapsid assembly is poorly understood at present. It has been demonstrated recently that the SFV capsid protein binds rapidly the 60S ribosomal subunit in both infected cells (36) and in vitro (10). The significance of the capsid protein-60S complex in the nucleocapsid assembly (11) and in the control of

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the translation of the nonstructural proteins is under investigation. ACKNOWLEDGMENTS We thank Ritva Rajala and Mizja Salonen for excellent technical assistance. Actinomycin D was a gift from Merck, Sharp and Dohme. This work was supported by grants from the Sigrid Juselius Foundation and the Finnish Academy. B.L. is a recipient of a scholarship from the Finnish Ministry of Education. LITERATURE CITED 1. Baltimore, D., D. C. Burke, M. C. Horzinek, A. S. Huang, L. Kiiriainen, E. M. Pfefferkorn, M. J. Schlesinger, S. Schlesinger, W. R. Schlesinger, and C. Scholtissek. 1976. Proposed nomenclature for alphavirus polypeptides. J. Gen. Virol. 30:273. 2. Bonner, M. W., and R. Laskey. 1974. A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46:83-88. 3. Burge, B. W., and E. M. Pfefferkorn. 1966. Complementation between temperature-sensitive mutants of Sindbis virus. Virology 30:214-223. 4. Cancedda, R., and M. J. Schlesinger. 1974. Formation of Sindbis virus capsid protein in mammalian cellfree extracts programmed with viral messenger RNA. Proc. Natl. Acad. Sci. U.S.A. 71:1843-1847. 5. Cancedda, R., L. Villa-Komaroff, H. Lodish, and M. Schlesinger. 1975. Initiation sites for translation of Sindbis virus 42S and 26S messenger RNAs. Cell 6:215-222. 6. Clegg, J. C. S. 1975. Sequential translation of capsid and membrane proteins in alphaviruses. Nature (London) 254:454-455. 7. Clegg, J. C. S., and S. I. T. Kennedy. 1975. Initiation of the synthesis of the structural proteins of Semliki forest virus. J. Mol. Biol. 97:401-411. 8. Glanville, N., J. Morser, P. Uomala, and L. Kkiriainen. 1976. Simultaneous translation of structural and nonstructural proteins from Semliki forest virus RNA in two eukaryotic systems in vitro. Eur. J. Biochem. 64:167-175. 9. Glanville, N., M. Ranki, J. Morser, L.Kaariainen, and A. E. Smith. 1976. Initiation of translation directed by 42S and 26S RNAs from Semliki forest virus in vitro. Proc. Natl. Acad. Sci. U.S.A. 73:3059-3063. 10. Glanville, N., and I. Ulmanen. 1976. Biological activity of in vitro synthesized protein: binding of Semliki forest virus capsid protein to the large ribosomal subunit. Biochem. Biophys. Res. Commun. 71:393399. 11. Kaariainen, L., S. Keranen, B. Lachmi, H. Soderlund, K. Tuomi, and I. Ulmanen. 1975. Replication of Semliki forest virus. Med. Biol. 53:342-352. 12. Kaariainen, L., B. Lachmi, and N. Glanville. 1976. Translational control in Semliki forest virus infected cells. Ann. Microbiol. (Paris) 127A:197-203. 13. Kaariainen, L., K. Simons, and C.-H. von Bonsdorff. 1969. Studies on subviral components of Semliki forest virus. Ann. Med. Exp. Biol. Fenn. 47:235-248. 14. Kennedy, S. I. T. 1972. Isolation and identification of the virus-specific RNA species found on membranebound polyribosomes of chick embryo cells infected with Semliki forest virus. Biochem. Biophys. Res. Commun. 48:1254-1258. 15. Keranen, S., and L. Kairiainen. 1974. Isolation and basic characterization of temperature-sensitive mutants from Semliki forest virus. Acta Pathol. Microbiol. Scand. Sect. B 82:810-820. 16. Keranen, S., and L. Kaariminen. 1975. Proteins synthesized by Semliki forest virus and its 16 temperaturesensitive mutants. J. Virol. 16:388-396. 17. Lachmi, B., N. Glanville, S. Keranen, and L. Kasri-

18.

19.

20. 21.

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ainen. 1975. Tryptic peptide analysis of nonstructural and structural precursor proteins from Semliki forest virus mutant-infected cells. J. Virol. 16:1615-1629. Lachmi, B., and L. Kaariinen. 1976. Sequential translation of nonstructural proteins in cells infected with Semliki forest virus mutant. Proc. Natl. Acad. Sci. U.S.A. 73:1936-1940. Levin, J. G., and R. M. Friedman. 1971. Analysis of arbovirus ribonucleic acid forms by polyacrylamide gel electrophoresis. J. Virol. 7:504-514. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. McAllister, P. E., and R. R. Wagner. 1976. Differential inhibition of host protein synthesis in L cells infected with RNA- temperature-sensitive mutants of vesicular stomatitis virus. J. Virol. 18:550-558.

22. Mowshowitz, D. 1973. Identification of polysomal RNA in BHK cells infected by Sindbis virus. J. Virol. 11:535-543. 23. Neville, D. M. 1971. Molecular weight determination of

24.

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33.

34. 35.

36.

37.

38.

protein dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer. J. Biol. Chem. 246:63286334. Nuss, D. L., H. Oppermann, and G. Koch. 1975. Selective blockage of initiation of host cell protein synthesis in RNA-virus-infected cells. Proc. Natl. Acad. Sci. U.S.A. 72:1258-1262. Saborio, J. L., S. S. Pong, and G. Koch. 1974. Selective and reversible inhibition of initiation of protein synthesis in mammalian cells. J. Mol. Biol. 85:195-211. Simmons, D. T., and J. H. Strauss. 1972. Replication of Sindbis virus. I. Relative size and genetic content of 26S and 49S RNA. J. Mol. Biol. 71:599-613. Simmons, D. T., and J. H. Strauss. 1974. Replication of Sindbis virus. V. Polyribosomes and mRNA in infected cells. J. Virol. 14:552-559. Simmons, D. T., and J. H. Strauss. 1974. Translation of Sindbis virus 26S RNA and 49S RNA in lysates of rabbit reticulocytes. J. Mol. Biol. 86:397-409. Sdderlund, H. 1973. Kinetics of formation of the Semliki forest virus nucleocapsid. Intervirology 1:354361. Soderlund, H. 1976. The post-translational processing of Semliki forest virus structural proteins in puromycin treated cells. FEBS Lett. 63:56-58. Soderlund, H., N. Glanville, and L. Kiiridinen. 1973/ 74. Polysomal RNAs in Semliki forest virus-infected cells. Intervirology 2:100-113. Soderlund, H., and L. Kiiriainen. 1974. Association of capsid protein with Semliki forest virus messenger RNAs. Acta Pathol. Microbiol. Scand. Sect. B 82:3340. Strauss, E. G., M. Lenches, and J. H. Strauss. 1976. Mutants of Sindbis virus. I. Isolation and partial characterization of 89 new temperature-sensitive mutants. Virology 74:154-168. Temple, G., and H. F. Lodish. 1975. Competititon between alpha-globin and beta-globin messenger-RNA. Biochem. Biophys. Res. Commun. 63:971-979. Tuomi, K., L. K11riainen, and H. Sdderlund. 1975. Quantitation of Semliki forest virus RNAs in infected cells using 32P equilibrium labeling. Nucleic Acids Research 2:555-565. Ulmanen, I., H. S6derlund, and L. Kaariainen. 1976. Semliki forest virus capsid protein associates with the 608 ribosomal subunit in infected cells. J. Virol. 20:203-210. Wengler, G., M. Beato, and B. A. Hackemack. 1974. Translation of 26S virus-specific RNA from Semliki forest virus-infected cells in vitro. Virology 61:120128. Wengler, G., and G. Wengler. 1976. Localization of the 26S RNA sequence on the viral genome type 42S RNA isolated from SFV infected cells. Virology 73:190-199.