Vol. 20, No. 1 Printed in U.S.A.
JOURNAL OF VIROLOGY, Oct. 1976, p. 203-210 Copyright ©D 1976 American Society for Microbiology
Semliki Forest Virus Capsid Protein Associates with the 60S Ribosomal Subunit in Infected Cells ISMO ULMANEN,* HANS SODERLUND, AND LEEVI KAARIAINEN Departments of Virology,* Zoology, and Biochemistry, University ofHelsinki, SF-00290 Helsinki 29, Finland Received for publication 1 June 1976
Semliki forest virus capsid protein cosedimented with the large ribosomal subunit at 60S in sucrose gradients after treatment of the cytoplasm from infected cells with Triton X-100 and EDTA. In CsCl gradients the capsid protein banded with the subunit at a density of 1.56 to 1.57 g/cm3. Most of the capsid protein could be detached from the 60S structure by treatment with 0.8 M KCl. The ribonucleoprotein of the 26S RNA had a sedimentation value of 53S and a density of 1.50 g/cm3 and could thus be separated from the 60S structure. The data suggest that the capsid protein binds to the large ribosomal subunit, but not to the viral 26S RNA.
Two major virus-specific RNAs are regularly found in alpha virus-infected cells: the 42S RNA genome (mol wt, 4.0 x 106 to 4.5 x 106) (33) and the 26S RNA (mol wt, 1.6 x 106) (25, 34, 44) derived from the 3'-proximal end of the 42S RNA (4, 6). Both of these RNAs are associated with polysomes (9, 18, 29, 31, 39, 46) and can stimulate protein synthesis in cell-free systems (4, 11, 14, 34, 37, 45); the 26S RNA directs the synthesis of viral structural proteins and is thus a copy of the structural cistron of 42S RNA. The in vitro products directed by 42S RNA consist mainly of nonstructural proteins. The messenger role of 42S RNA in the cells is not understood, since mainly structural proteins and their precursors are found in the infected cells during the second half of the infectious cycle (16, 19). The major proteins detected at that time are the capsid protein (C; mol wt, 33,000), envelope glycoprotein E-1 (mol wt, 49,000), and p-62 (mol wt, 62,000), which is the immediate precursor of the envelope glycoproteins E-2 and E-3 (10, 17, 36). In addition, small amounts of large structural precursor proteins with molecular weights of 130,000 (p-130 or NVP 130), 97,000 (p-97 or NVP 97), and 86,000 (p-86 or NVP 86) can be detected (3, 5, 19, 22, 32). One of the functions of the 42S RNA is to become associated with the capsid protein to form the viral nucleocapsid (15, 38). The formation of the nucleocapsid must be a rapid process, since capsid protein synthesized during a short pulse attains its maximum activity in the 130S nucleocapsid after only 5 to 7 min of chase (38). The newly formed capsid protein has also been observed associated with a structure sedimenting at about 60S together with the 26S
RNA ribonucleoprotein (RNP) (8, 40). Herein we show that the newly synthesized capsid protein sedimenting at 60S is associated with the large ribosomal subunit and can be separated from the 26S RNA RNP by sucrose or CsCl density gradient centrifugation. MATERIALS AND METHODS Virus and cells. The origin and cultivation of Semliki forest virus (SFV), prototype strain, in HeLa cells has been previously described (17, 38). Cells were grown as monolayers in 250-ml plastic bottles (Falcon). Isotope labeling. The cells were infected at a multiplicity of infection of 50. After adsorption for 1 h at 37°C, the cells were washed and the medium was replaced. The virus growth medium used was methionine-free Eagle minimum essential medium containing 2 ,ug of actinomycin D per ml and 0.2% bovine serum albumin. Virus-specific RNAs were labeled with [3H]uridine for 1 to 3 h (25 to 28 Ci/ mmol; the Radiochemical Centre, Amersham, 250 PiCi/bottle), and proteins were labeled for 3 or 15 min with [35S]methionine (160 to 280 Ci/mmol; Amersham, 250 ,uCi/bottle). To prelabel cellular components, the cells were exposed to [35S]methionine (100 uCi/bottle) or [3H]uridine (50 ,uCi/bottle) in Eagle minimum essential medium with 5% dialyzed calf serum for 13 to 16 h before infection. Cell fractionation. Cell monolayers were washed twice with ice-cold phosphate-buffered saline containing cycloheximide (100 iLg/ml), and the cells were scraped into about 1 ml of medium containing 0.15 M NaCl, 0.01 M Tris, pH 7.4, 1.5 mM MgCl2, and 0.65% Triton X-100. The suspension was kept on ice for 10 min and agitated from time to time with a Vortex mixer (21). The nuclei were removed from this lysis buffer by low-speed centrifugation, and the cytoplasmic extract was processed as described below. Isolation of RNPs. To isolate viral and cellular 203
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RNPs, the cytoplasmic extracts were treated with 25 mM EDTA and centrifuged on 15 to 30% sucrose (wt/ wt) gradients in 0.05 M Tris, pH 7.4, 0.1 M NaCl, and 0.001 M EDTA (TNE) for 2 to 6 h in a Spinco SW41 rotor at 40,000 rpm and 4°C. The gradients were collected from below and the acid-insoluble radioactivity was determined. S-values were determined according to Martin and Ames (27). The ribosomal subunits were used as markers for 60S and 40S, respectively, and encephalomyocarditis virus was used as a marker for 150S. Analysis of RNAs and proteins. RNA was released with 2% sodium dodecyl sulfate and analyzed on 15 to 30% sucrose gradients containing sodium dodecyl sulfate, as described by Tuomi et al. (44). The ribosomal 28S and 18S RNAs were used as Svalue markers. Proteins were analyzed on polyacrylamide slab gels according to the method of Laemmli (23) using 3% acrylamide in the spacer and 8% acrylamide in the separating gel. The samples (pretreated with 5 ,ug of RNase per ml if necessary) were prepared by adding sodium dodecyl sulfate to 2%, glycerol to 10%, mercaptoethanol to 5%, and bromophenol blue to 0.001% and heating at 100°C for 2 min. After electrophoresis the gels were impregnated with PPO (2,5-diphenyloxazole) as described by Bonner and Laskey (2), and the procedure of Laskey and Mills (24) was used for fluorography. As a marker for the proteins p-130 and p-86, a cytoplasmic extract from SFV mutant ts-3-infected cells was used. Determination of buoyant densities. Samples were fixed with glutaraldehyde (5 or 2%) and analyzed according to Baltimore and Huang (1).
RESULTS In the present study cytoplasmic RNPs from SFV-infected HeLa cells were analyzed. The viral RNAs were labeled with [3H]uridine for 1, 2, or 3 h in the presence of actinomycin D, and the proteins were labeled for 3 or 15 min with [35S]methionine. The labels are found almost exclusively in virus-specific material since both RNA and protein synthesis of the host cell are efficiently inhibited under these conditions (42, 43). The cells were lysed with Triton X-100 in isotonic buffer to avoid artefactual RNA-protein association in low salt, and the polysomes were dissociated with EDTA (13, 21, 30). The released RNPs were isolated by centrifugation on sucrose gradients. The RNA label was found in material sedimenting at about 130S (pool a) and 60S (pool b), with a shoulder at 100S (Fig. 1A). Protein label cosedimented with the 130S and 60S components, but most of the 3S activity remained on the top of the gradient. The RNA and protein composition of different fractions of the sucrose gradient were then analyzed: 42S RNA was associated with the 130S and 100S material, whereas 26S RNA was recovered from the 60S fraction (Fig. 1A, inset). This finding was inde-
J. VIROL.
pendent of the uridine-labeling period. The protein patterns on polyacrylamide gels are shown in Fig. 2. The capsid protein was found to be the dominant species associated with the 130S, 1OOS, and 60S structures after both 3- and 15min pulses of F`5S]methionine. Faint bands of envelope proteins E-1, p-62, and p-86 were sometimes seen. In the top fractions of the gradient the major proteins were E-1 and p-62, and the capsid protein could scarcely be detected. Thus, almost all the newly labeled capsid protein was found in structures sedimenting at 60S or faster, whereas the envelope proteins were in the top fractions. The 3H- and 35S-labeled material sedimenting at about 130S has the properties of the cytoplasmic nucleocapsid (38), whereas the 100S material may be the 42S messenger RNP or partially unfolded nucleocapsid (40, 41). Characterization of the 60S complex. When the resolution in the 60S region of the gradient was increased by prolonged centrifugation, the 3H and 35S labels were partly separated (Fig. 1B, pool c). Separation of virus-specific protein and RNA labels was further improved when the 60S material was resedimented in prolonged runs (Fig. 3). The sedimentation rates corresponded to about 60S for the 35S label and 53S for the 3H label. In another experiment the cellular RNA was labeled with [3H]uridine for 13 h before infection to label the rRNA's. The newly formed viral proteins were labeled 5 h postinfection with [35S]methionine. The two labels were now found to cosediment at 60S even after prolonged centrifugation (Fig. 1C, pool d). From this material the RNA label recovered was 28S rRNA (Fig. 1C, inset) and the protein label was in capsid protein (not shown). The RNA label at 40S (pool e) was in 18S rRNA as expected. Densities of the viral RNPs were determined by CsCl density gradient centrifugation after different combinations of labeling (Fig. 4). The 130S viral nucleocapsid (Fig. 4A) had a density of 1.43 g/cm3 as shown previously (38). The 60S material in which the capsid protein was labeled with [35S]methionine and the 26S RNA with [3H]uridine was split in two components with different densities. The protein peak was found at a density of 1.56 to 1.57 g/cm3, whereas the density of the RNA label was 1.50 g/cm3 (Fig. 4B). The 60S complex isolated from cells prelabeled with [35S]methionine and further labeled with [3H]uridine 4 h after infection with SFV was also split in two peaks, with densities of 1.57 g/cm3 (35S) and 1.50 g/cm3 (3H) (Fig. 4C). Part of the protein label was, however, found in the 1.45- to 1.55-g/cm3 region. Finally, when
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60S subunits were prelabeled in their 28S RNA and the capsid protein was labeled with F35S]methionine, both labels had the same density of 1.57 g/cm3 (Fig. 4D). The above results indicate that the capsid protein sedimenting at 60S is not primarily associated with the viral 26S RNA, but rather with the large ribosomal subunit, which has a density of 1.57 g/cm3 (1). The 268 RNA apparently forms a messenger RNP in combination with cellular proteins. Salt resistance of the 60S complex. From cells prelabeled with [3H]uridine and pulse labeled with P35S]methionine after the infection with SFV the 60S material was pooled as described. From this fraction the labels could be recovered in 288 RNA and capsid protein, respectively (not shown). The salt resistance of the 60S complex was determined by addition of
different amounts of KC1 to samples of the pool. The samples were then analyzed on 15 to 30% sucrose gradients made in TNE. On top of each gradient was a layer of 5% sucrose made in TNE with additional KCl as in the sample. The salt treatment seems to remove increasing amounts of capsid protein from the 60S subunit. With 0.4 M KCl 30 to 50% was detached and with 0.8 M KCI 60 to 70% was detached, as calculated from the decreasing 35S/ 3H ratio in the peak fractions (Fig. 5). Small ribosomal subunit. The binding of capsid protein to the 60S ribosomal subunit makes it relevant to ask whether the 40S subunit also binds viral proteins. However, the viral protein on the top of the gradient always extended down to the 40S subunit despite the prolonged centrifugation (Fig. 1C). Analysis of the proteins cosedimenting with the 40S sub-
206
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unit showed that this fraction contained capsid proteins E-1, p-62, and p-86 in roughly equal amounts (Fig. 6). When the 40S fraction was resedimented on sucrose gradients with or without 0.2% Triton X-100 most of the viral protein label separated from the 3H-labeled 40S subunit (Fig. 7). The presence of Triton in the gradient increased the recovery of 35S about twofold compared to the Triton-free sample and also affected strongly its distribution in the gradient (Fig. 7). The 35Slabeled protein sedimenting at about 40S may thus consist of complexes of membrane proteins similar to those isolated from the virion envelope (35). From the 3H/35S ratio in the 60S and 40S fractions after resedimentation, it can be calculated that the binding of viral proteins to the
40S ribosomal subunit is less than one-tenth of that binding to the 60S subunit (cf. Fig. 7 and Fig. 5A).
DISCUSSION Sucrose gradient analysis of EDTA-treated cytoplasmic extracts of infected cells showed that the two major SFV-specific RNAs sedimented as RNP complexes, as revealed by their sedimentation rate and density. Most of the cytoplasmic 42S RNA was found in structures sedimenting at about 130S. Capsid protein was virtually the only virus-specific protein associated with this structure. Thus, most of the 42S RNA seems to be in the form of viral nucleocapsid (38). Capsid protein sedimented also in the 50S to
SFV CAPSID PROTEIN IN 60S RIBOSOMAL SUBUNIT
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60S region, where nearly all the cytoplasmic 26S RNA was found. The RNA and protein labels could, however, be separated by prolonged sucrose gradient centrifugation, and 3 9 analysis on CsCl density gradients separated 1.5 them almost completely. The capsid protein cosedimented with the large ribosomal subunit in I o .C sucrose gradients and it also banded at the ._ D. *lCsame density as the subunit. The 26S RNA at 53S and had a density (about O l 1.50 g/cm3) that suggested that it was associco . ft, co 'ated with some unlabeled, probably host cell, This shows that the newly syntheo? .... i y nproteins. capsid protein does not bind to the 26S x:l°sized RNA. -:lb 6 > E 1The attachment of capsid protein to the large 0J0. W1 0\f ribosomal subunit seems to be relatively stable [ 0.5 in high salt. It is also specific in the sense that Q. ?o 0 no preferential binding of capsid protein to the ° °Qa o° * 40S subunit was observed. The possibility that 0a°- \ capsid protein is artefactually bound to the 60S subunit due to the disruption of the cells cannot 20 10 but it is interesting to note that be excluded, FRACTION FRACTION NUMBER ~the binding of capsid protein to the 608 subunit cel-feprotein-synthe6 sizin FIG. 3. Resedimentation of the 60S complex. In- als ocusing f/ected cells were labeled as described in the legend to also occurs m a cell-free protein-synthesizing IFig. 1B. The 60S complex was isolated and resedi- system. The wheat germ extract programmed rnented on a 15 to 40% sucrose gradient made in 0.05 with viral 26S RNA synthesizes the viral strucAVf Tris, pH 7.4, 0.1 M NaCl for 11 h at 34,000 rpm in tural proteins (6, 11), of which the capsid protein almost quantitatively associates with the ain SW41 rotor at 4C. 60S
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FIG. 4. CsCl density gradient analysis of the 130S RNP and the 60S complex from SFV-infected HeLa cells. (A) The infected cells were labeled with [3H]uridine from 4 to 6 h postinfection (p.i.) and then pulsed for 15 min with [35S]methionine. The EDTA-treated cytoplasmic extract was centrifuged on a 15 to 30% sucrose gradient as in Fig. 1A, and the 130S material was analyzed after glutaraldehyde fixation. (B) The infected cells were labeled with [3H]uridine for 1 to 4 p.i. and pulsed with [35S]methionine for 15 min. The EDTAtreated cytoplasmic extract was centrifuged on a 15 to 30% sucrose gradient for 3 h and the 60S material was analyzed. (C) The cells were prelabeled for 13 h with [35S]methionine, infected with SFV, and labeled with [3H]uridine from 4.5 to 5.5 h p.i. The 60S complex was isolated and its density was determined. (D) The cells were labeled and the 60S complex was isolated as described for Fig. IC, and thereafter its density was determined. The preformed (1.2 to 1.6 g/cm3) CsCl gradients were centrifuged overnight at 37,500 rpm and 4'C in an SW50.1 rotor.
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FIG. 6. Analysis of [35S]methionine-labeled virusspecific proteins cosedimenting with ribosomal subunits. The infected cells were labeled for 15 min at 4 h postinfection, and the ribosomal subunits were isolated (cf. Fig. 1C). The 60S and 40S peak fractions and the top fraction from the gradient were processed and subjected to electrophoresis as in Fig. 2. Fluorography was for 3 days. The positions of capsid (C), envelope E-1, p-62, and p-86 proteins are indicated.
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FRACTION NUMBER FIG. 7. Resedimentation of the 40S ribosomal subunit in the presence and absence of Triton X-100. Infected cells were labeled as described for Fig. 5. The cytoplasmic extract was treated with EDTA and centrifuged for 6 h on a sucrose gradient as described in Materials and Methods. The 40S subunit fraction was diluted (A) with TNE or (B) with TNE containing Triton X-100 (final concentration, 1%o). The samples were analyzed on 15 to 30% (wt/wt) sucrose gradients in TNE or TNE with 0.2% Triton. Centrifugation was for 18 h at 24,000 rpm in an SW27 rotor at 4°C.
wheat germ 60S subunit (12). This binding of capsid protein with the 60S ribosomal subunit is apparently specific also in vitro since the nonstructural products (11) of 42S RNA did not attach to the ribosomal subunits (12). Binding of viral proteins to ribosomes has been described previously for poliovirus-, encephalomyocarditis virus-, and mengovirus-infected cells. Structural and nonstructural proteins have been shown to be associated with the 40S ribosomal subunit by high-salt dissociable bonds (26, 27, 45). The influenza virus-specific nonstructural (NS) protein has been reported to bind to both 40S and 60S subunits. The viral protein is, however, released with 0.5 M salt (7, 20). Thus, the association of the SFV capsid protein with the 60S ribosomal subunit seems to be a unique phenomenon. At present we do not know whether this binding affects the translation capacity of the ribosome. Preliminary kinetic data suggest that the capsid protein is transferred from the subunit to the viral nucleocapsid relatively fast (15). Thus, one function of the 608 subunit-capsid protein complex may be connected with the virus assembly. ACKNOWLEDGMENTS The skillful technical assistance of Ritva Rajala and Mirja Salonen is gratefully acknowledged.
This investigation was supported by grants from the Finnish Academy and the Sigrid Juselius and the Alfred Kordelin Foundations. Actinomycin D was a gift from Merck Sharp & Dohme. LITERATURE CITED 1. Baltimore, D., and A. S. Huang. 1968. Isopycnic separation of subcellular components from poliovirus-infected and normal HeLa cells. Science 162:572-574. 2. Bonner, W. M., and R. A. Laskey. 1974. A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46:8388. 3. Burke, D. C. 1975. Processing of alphavirus-specific proteins in infected cells. Med. Biol. 53:352-356. 4. Clegg, C., and I. Kennedy. 1975. Synthesis of the structural proteins of Semliki Forest virus. INSERM 47:255-258. 5. Clegg, J. C. S. 1975. Sequential translation of capsid and membrane protein genes of alphaviruses. Nature (London) 254:454-455. 6. 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. 7. Compans, R. W. 1973. Influenza virus proteins. II. Association with components of the cytoplasm. Virology 51:56-70. 8. Eaton, B. T., T. P. Donaghue, and P. Faulkner. 1972. Presence of poly(A) in the polyribosome associated RNA of Sindbis-infected BHK cells. Nature (London) New Biol. 238:109-111. 9. Eaton, B. T., and R. L. Regnery. 1975. Polysomal RNA in Semliki Forest virus infected Aedes albopictus cells. J. Gen. Virol. 29:35-49. 10. Garoff, H. K., K. Simons, and 0. Renkonen. 1974. Isolation and characterization of the membrane pro-
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ribosomes of Krebs II cells infected with Encephalomyocarditis virus. FEBS Lett. 39:4-8. Mowshowitz, D. 1973. Identification of polysomal RNA in BHK cells infected by Sindbis virus. J. Virol. 11:535-543. Perry, R. P., and D. E. Kelley. 1968. Messenger RNAprotein complexes and newly synthesized ribosomal subunits: analysis of free particles and components of polyribosomes. J. Mol. Biol. 35:37-59. Rosemond, H., and T. Sreevalsan. 1973. Viral RNAs associated with ribosomes in Sindbis virus-infected cells. J. Virol. 11:399-415. Schlesinger, S., and M. J. Schlesinger. 1972. Formation of Sindbis virus proteins: identification of a precursor for one of the envelope proteins. J. Virol. 10:925-932. Simmons, D. T., and J. H. Strauss. 1972. Replication of Sindbis virus. I. Relative size and genetic content of 26 S and 49 S RNA. J. Mol. Biol. 71:599-613. Simmons, D. T., and J. H. Strauss. 1974. Translation of Sindbis virus 26 S RNA and 49 S RNA in lysates of rabbit reticulocytes. J. Mol. Biol. 86:397-409. Simons, K., A. Helenius, and H. Garoff. 1973. Solubilization of the membrane proteins from Semliki Forest virus with Triton X-100. J. Mol. Biol. 80:119-133. Simons, K., S. Keranen, and L. Kaariainen. 1973. Identification of a precursor for one of the Semliki Forest virus membrane proteins. FEBS Lett. 29:87-91. Smith, A. E., T. Wheeler, N. Glanville, and L. Kaariainen. 1974. Translation of Semliki-Forest-virus 42 S RNA in a mouse cell-free system to give virus-coat protein. Eur. J. Biochem. 49:101-110. S6derlund, H. 1973. Kinetics of formation of Semliki Forest virus nucleocapsid. Intervirology 1:354-361. S6derlund, H., N. Glanville, and L. Kaari.inen. 1973/ 74. Polysomal RNAs in Semliki Forest virus-infected cells. Intervirology 2:100-113. S6derlund, H., and L. Kaariminen. 1974. Association of capsid protein with Semliki Forest virus messenger RNAs. Acta Pathol. Microbiol. Scand. Sect. B 82:3340. Sederlund, H., L. Kaariainen, and C.-H. von Bonsdorff. 1975. Properties of Semliki Forest virus nucleocapsid. Med. Biol. 53:412-417. Strauss, J. H., B. W. Burge, and J. E. Darnell. 1969. Sindbis virus infection of chick and hamster cells: synthesis of virus-specific proteins. Virology 37:367-
376. 43. Taylor, J. 1965. Studies on the mechanism of action of interferon I. Interferon action and RNA synthesis in chick embryo fibroblasts infected with Semliki Forest virus. Virology 25:340-349. 44. Tuomi, K., L. Kaariainen, and H. S6derlund. 1975. Quantitation of Semliki Forest virus RNAs in infected cells using 32p equilibrium labelling. Nucleic Acids Res. 2:555-565. 45. Wengler, G., M. Beato, and B.-A. Hackemack. 1974. Translation of 26 S virus specific RNA from Semliki Forest virus-infected cells in vitro. Virology 61:120128. 46. Wengler, G., and G. Wengler. 1975. Studies on the polyribosome-associated RNA in BHK21 cells infected with Semliki Forest virus. Virology 59:21-35. 47. Wright, P. J., and P. D. Cooper. 1974. Poliovirus proteins associated with ribosomal structures in infected cells. Virology 59:1-20.
JOURNAL OF VIROLOGY, Oct. 1976, p. 203-210 Copyright ©D 1976 American Society for Microbiology
Semliki Forest Virus Capsid Protein Associates with the 60S Ribosomal Subunit in Infected Cells ISMO ULMANEN,* HANS SODERLUND, AND LEEVI KAARIAINEN Departments of Virology,* Zoology, and Biochemistry, University ofHelsinki, SF-00290 Helsinki 29, Finland Received for publication 1 June 1976
Semliki forest virus capsid protein cosedimented with the large ribosomal subunit at 60S in sucrose gradients after treatment of the cytoplasm from infected cells with Triton X-100 and EDTA. In CsCl gradients the capsid protein banded with the subunit at a density of 1.56 to 1.57 g/cm3. Most of the capsid protein could be detached from the 60S structure by treatment with 0.8 M KCl. The ribonucleoprotein of the 26S RNA had a sedimentation value of 53S and a density of 1.50 g/cm3 and could thus be separated from the 60S structure. The data suggest that the capsid protein binds to the large ribosomal subunit, but not to the viral 26S RNA.
Two major virus-specific RNAs are regularly found in alpha virus-infected cells: the 42S RNA genome (mol wt, 4.0 x 106 to 4.5 x 106) (33) and the 26S RNA (mol wt, 1.6 x 106) (25, 34, 44) derived from the 3'-proximal end of the 42S RNA (4, 6). Both of these RNAs are associated with polysomes (9, 18, 29, 31, 39, 46) and can stimulate protein synthesis in cell-free systems (4, 11, 14, 34, 37, 45); the 26S RNA directs the synthesis of viral structural proteins and is thus a copy of the structural cistron of 42S RNA. The in vitro products directed by 42S RNA consist mainly of nonstructural proteins. The messenger role of 42S RNA in the cells is not understood, since mainly structural proteins and their precursors are found in the infected cells during the second half of the infectious cycle (16, 19). The major proteins detected at that time are the capsid protein (C; mol wt, 33,000), envelope glycoprotein E-1 (mol wt, 49,000), and p-62 (mol wt, 62,000), which is the immediate precursor of the envelope glycoproteins E-2 and E-3 (10, 17, 36). In addition, small amounts of large structural precursor proteins with molecular weights of 130,000 (p-130 or NVP 130), 97,000 (p-97 or NVP 97), and 86,000 (p-86 or NVP 86) can be detected (3, 5, 19, 22, 32). One of the functions of the 42S RNA is to become associated with the capsid protein to form the viral nucleocapsid (15, 38). The formation of the nucleocapsid must be a rapid process, since capsid protein synthesized during a short pulse attains its maximum activity in the 130S nucleocapsid after only 5 to 7 min of chase (38). The newly formed capsid protein has also been observed associated with a structure sedimenting at about 60S together with the 26S
RNA ribonucleoprotein (RNP) (8, 40). Herein we show that the newly synthesized capsid protein sedimenting at 60S is associated with the large ribosomal subunit and can be separated from the 26S RNA RNP by sucrose or CsCl density gradient centrifugation. MATERIALS AND METHODS Virus and cells. The origin and cultivation of Semliki forest virus (SFV), prototype strain, in HeLa cells has been previously described (17, 38). Cells were grown as monolayers in 250-ml plastic bottles (Falcon). Isotope labeling. The cells were infected at a multiplicity of infection of 50. After adsorption for 1 h at 37°C, the cells were washed and the medium was replaced. The virus growth medium used was methionine-free Eagle minimum essential medium containing 2 ,ug of actinomycin D per ml and 0.2% bovine serum albumin. Virus-specific RNAs were labeled with [3H]uridine for 1 to 3 h (25 to 28 Ci/ mmol; the Radiochemical Centre, Amersham, 250 PiCi/bottle), and proteins were labeled for 3 or 15 min with [35S]methionine (160 to 280 Ci/mmol; Amersham, 250 ,uCi/bottle). To prelabel cellular components, the cells were exposed to [35S]methionine (100 uCi/bottle) or [3H]uridine (50 ,uCi/bottle) in Eagle minimum essential medium with 5% dialyzed calf serum for 13 to 16 h before infection. Cell fractionation. Cell monolayers were washed twice with ice-cold phosphate-buffered saline containing cycloheximide (100 iLg/ml), and the cells were scraped into about 1 ml of medium containing 0.15 M NaCl, 0.01 M Tris, pH 7.4, 1.5 mM MgCl2, and 0.65% Triton X-100. The suspension was kept on ice for 10 min and agitated from time to time with a Vortex mixer (21). The nuclei were removed from this lysis buffer by low-speed centrifugation, and the cytoplasmic extract was processed as described below. Isolation of RNPs. To isolate viral and cellular 203
204
ULMANEN, SODERLUND, AND KAARIAINEN
RNPs, the cytoplasmic extracts were treated with 25 mM EDTA and centrifuged on 15 to 30% sucrose (wt/ wt) gradients in 0.05 M Tris, pH 7.4, 0.1 M NaCl, and 0.001 M EDTA (TNE) for 2 to 6 h in a Spinco SW41 rotor at 40,000 rpm and 4°C. The gradients were collected from below and the acid-insoluble radioactivity was determined. S-values were determined according to Martin and Ames (27). The ribosomal subunits were used as markers for 60S and 40S, respectively, and encephalomyocarditis virus was used as a marker for 150S. Analysis of RNAs and proteins. RNA was released with 2% sodium dodecyl sulfate and analyzed on 15 to 30% sucrose gradients containing sodium dodecyl sulfate, as described by Tuomi et al. (44). The ribosomal 28S and 18S RNAs were used as Svalue markers. Proteins were analyzed on polyacrylamide slab gels according to the method of Laemmli (23) using 3% acrylamide in the spacer and 8% acrylamide in the separating gel. The samples (pretreated with 5 ,ug of RNase per ml if necessary) were prepared by adding sodium dodecyl sulfate to 2%, glycerol to 10%, mercaptoethanol to 5%, and bromophenol blue to 0.001% and heating at 100°C for 2 min. After electrophoresis the gels were impregnated with PPO (2,5-diphenyloxazole) as described by Bonner and Laskey (2), and the procedure of Laskey and Mills (24) was used for fluorography. As a marker for the proteins p-130 and p-86, a cytoplasmic extract from SFV mutant ts-3-infected cells was used. Determination of buoyant densities. Samples were fixed with glutaraldehyde (5 or 2%) and analyzed according to Baltimore and Huang (1).
RESULTS In the present study cytoplasmic RNPs from SFV-infected HeLa cells were analyzed. The viral RNAs were labeled with [3H]uridine for 1, 2, or 3 h in the presence of actinomycin D, and the proteins were labeled for 3 or 15 min with [35S]methionine. The labels are found almost exclusively in virus-specific material since both RNA and protein synthesis of the host cell are efficiently inhibited under these conditions (42, 43). The cells were lysed with Triton X-100 in isotonic buffer to avoid artefactual RNA-protein association in low salt, and the polysomes were dissociated with EDTA (13, 21, 30). The released RNPs were isolated by centrifugation on sucrose gradients. The RNA label was found in material sedimenting at about 130S (pool a) and 60S (pool b), with a shoulder at 100S (Fig. 1A). Protein label cosedimented with the 130S and 60S components, but most of the 3S activity remained on the top of the gradient. The RNA and protein composition of different fractions of the sucrose gradient were then analyzed: 42S RNA was associated with the 130S and 100S material, whereas 26S RNA was recovered from the 60S fraction (Fig. 1A, inset). This finding was inde-
J. VIROL.
pendent of the uridine-labeling period. The protein patterns on polyacrylamide gels are shown in Fig. 2. The capsid protein was found to be the dominant species associated with the 130S, 1OOS, and 60S structures after both 3- and 15min pulses of F`5S]methionine. Faint bands of envelope proteins E-1, p-62, and p-86 were sometimes seen. In the top fractions of the gradient the major proteins were E-1 and p-62, and the capsid protein could scarcely be detected. Thus, almost all the newly labeled capsid protein was found in structures sedimenting at 60S or faster, whereas the envelope proteins were in the top fractions. The 3H- and 35S-labeled material sedimenting at about 130S has the properties of the cytoplasmic nucleocapsid (38), whereas the 100S material may be the 42S messenger RNP or partially unfolded nucleocapsid (40, 41). Characterization of the 60S complex. When the resolution in the 60S region of the gradient was increased by prolonged centrifugation, the 3H and 35S labels were partly separated (Fig. 1B, pool c). Separation of virus-specific protein and RNA labels was further improved when the 60S material was resedimented in prolonged runs (Fig. 3). The sedimentation rates corresponded to about 60S for the 35S label and 53S for the 3H label. In another experiment the cellular RNA was labeled with [3H]uridine for 13 h before infection to label the rRNA's. The newly formed viral proteins were labeled 5 h postinfection with [35S]methionine. The two labels were now found to cosediment at 60S even after prolonged centrifugation (Fig. 1C, pool d). From this material the RNA label recovered was 28S rRNA (Fig. 1C, inset) and the protein label was in capsid protein (not shown). The RNA label at 40S (pool e) was in 18S rRNA as expected. Densities of the viral RNPs were determined by CsCl density gradient centrifugation after different combinations of labeling (Fig. 4). The 130S viral nucleocapsid (Fig. 4A) had a density of 1.43 g/cm3 as shown previously (38). The 60S material in which the capsid protein was labeled with [35S]methionine and the 26S RNA with [3H]uridine was split in two components with different densities. The protein peak was found at a density of 1.56 to 1.57 g/cm3, whereas the density of the RNA label was 1.50 g/cm3 (Fig. 4B). The 60S complex isolated from cells prelabeled with [35S]methionine and further labeled with [3H]uridine 4 h after infection with SFV was also split in two peaks, with densities of 1.57 g/cm3 (35S) and 1.50 g/cm3 (3H) (Fig. 4C). Part of the protein label was, however, found in the 1.45- to 1.55-g/cm3 region. Finally, when
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60S subunits were prelabeled in their 28S RNA and the capsid protein was labeled with F35S]methionine, both labels had the same density of 1.57 g/cm3 (Fig. 4D). The above results indicate that the capsid protein sedimenting at 60S is not primarily associated with the viral 26S RNA, but rather with the large ribosomal subunit, which has a density of 1.57 g/cm3 (1). The 268 RNA apparently forms a messenger RNP in combination with cellular proteins. Salt resistance of the 60S complex. From cells prelabeled with [3H]uridine and pulse labeled with P35S]methionine after the infection with SFV the 60S material was pooled as described. From this fraction the labels could be recovered in 288 RNA and capsid protein, respectively (not shown). The salt resistance of the 60S complex was determined by addition of
different amounts of KC1 to samples of the pool. The samples were then analyzed on 15 to 30% sucrose gradients made in TNE. On top of each gradient was a layer of 5% sucrose made in TNE with additional KCl as in the sample. The salt treatment seems to remove increasing amounts of capsid protein from the 60S subunit. With 0.4 M KCl 30 to 50% was detached and with 0.8 M KCI 60 to 70% was detached, as calculated from the decreasing 35S/ 3H ratio in the peak fractions (Fig. 5). Small ribosomal subunit. The binding of capsid protein to the 60S ribosomal subunit makes it relevant to ask whether the 40S subunit also binds viral proteins. However, the viral protein on the top of the gradient always extended down to the 40S subunit despite the prolonged centrifugation (Fig. 1C). Analysis of the proteins cosedimenting with the 40S sub-
206
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unit showed that this fraction contained capsid proteins E-1, p-62, and p-86 in roughly equal amounts (Fig. 6). When the 40S fraction was resedimented on sucrose gradients with or without 0.2% Triton X-100 most of the viral protein label separated from the 3H-labeled 40S subunit (Fig. 7). The presence of Triton in the gradient increased the recovery of 35S about twofold compared to the Triton-free sample and also affected strongly its distribution in the gradient (Fig. 7). The 35Slabeled protein sedimenting at about 40S may thus consist of complexes of membrane proteins similar to those isolated from the virion envelope (35). From the 3H/35S ratio in the 60S and 40S fractions after resedimentation, it can be calculated that the binding of viral proteins to the
40S ribosomal subunit is less than one-tenth of that binding to the 60S subunit (cf. Fig. 7 and Fig. 5A).
DISCUSSION Sucrose gradient analysis of EDTA-treated cytoplasmic extracts of infected cells showed that the two major SFV-specific RNAs sedimented as RNP complexes, as revealed by their sedimentation rate and density. Most of the cytoplasmic 42S RNA was found in structures sedimenting at about 130S. Capsid protein was virtually the only virus-specific protein associated with this structure. Thus, most of the 42S RNA seems to be in the form of viral nucleocapsid (38). Capsid protein sedimented also in the 50S to
SFV CAPSID PROTEIN IN 60S RIBOSOMAL SUBUNIT
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207
60S region, where nearly all the cytoplasmic 26S RNA was found. The RNA and protein labels could, however, be separated by prolonged sucrose gradient centrifugation, and 3 9 analysis on CsCl density gradients separated 1.5 them almost completely. The capsid protein cosedimented with the large ribosomal subunit in I o .C sucrose gradients and it also banded at the ._ D. *lCsame density as the subunit. The 26S RNA at 53S and had a density (about O l 1.50 g/cm3) that suggested that it was associco . ft, co 'ated with some unlabeled, probably host cell, This shows that the newly syntheo? .... i y nproteins. capsid protein does not bind to the 26S x:l°sized RNA. -:lb 6 > E 1The attachment of capsid protein to the large 0J0. W1 0\f ribosomal subunit seems to be relatively stable [ 0.5 in high salt. It is also specific in the sense that Q. ?o 0 no preferential binding of capsid protein to the ° °Qa o° * 40S subunit was observed. The possibility that 0a°- \ capsid protein is artefactually bound to the 60S subunit due to the disruption of the cells cannot 20 10 but it is interesting to note that be excluded, FRACTION FRACTION NUMBER ~the binding of capsid protein to the 608 subunit cel-feprotein-synthe6 sizin FIG. 3. Resedimentation of the 60S complex. In- als ocusing f/ected cells were labeled as described in the legend to also occurs m a cell-free protein-synthesizing IFig. 1B. The 60S complex was isolated and resedi- system. The wheat germ extract programmed rnented on a 15 to 40% sucrose gradient made in 0.05 with viral 26S RNA synthesizes the viral strucAVf Tris, pH 7.4, 0.1 M NaCl for 11 h at 34,000 rpm in tural proteins (6, 11), of which the capsid protein almost quantitatively associates with the ain SW41 rotor at 4C. 60S
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FIG. 4. CsCl density gradient analysis of the 130S RNP and the 60S complex from SFV-infected HeLa cells. (A) The infected cells were labeled with [3H]uridine from 4 to 6 h postinfection (p.i.) and then pulsed for 15 min with [35S]methionine. The EDTA-treated cytoplasmic extract was centrifuged on a 15 to 30% sucrose gradient as in Fig. 1A, and the 130S material was analyzed after glutaraldehyde fixation. (B) The infected cells were labeled with [3H]uridine for 1 to 4 p.i. and pulsed with [35S]methionine for 15 min. The EDTAtreated cytoplasmic extract was centrifuged on a 15 to 30% sucrose gradient for 3 h and the 60S material was analyzed. (C) The cells were prelabeled for 13 h with [35S]methionine, infected with SFV, and labeled with [3H]uridine from 4.5 to 5.5 h p.i. The 60S complex was isolated and its density was determined. (D) The cells were labeled and the 60S complex was isolated as described for Fig. IC, and thereafter its density was determined. The preformed (1.2 to 1.6 g/cm3) CsCl gradients were centrifuged overnight at 37,500 rpm and 4'C in an SW50.1 rotor.
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208
SFV CAPSID PROTEIN IN 60S RIBOSOMAL SUBUNIT
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wheat germ 60S subunit (12). This binding of capsid protein with the 60S ribosomal subunit is apparently specific also in vitro since the nonstructural products (11) of 42S RNA did not attach to the ribosomal subunits (12). Binding of viral proteins to ribosomes has been described previously for poliovirus-, encephalomyocarditis virus-, and mengovirus-infected cells. Structural and nonstructural proteins have been shown to be associated with the 40S ribosomal subunit by high-salt dissociable bonds (26, 27, 45). The influenza virus-specific nonstructural (NS) protein has been reported to bind to both 40S and 60S subunits. The viral protein is, however, released with 0.5 M salt (7, 20). Thus, the association of the SFV capsid protein with the 60S ribosomal subunit seems to be a unique phenomenon. At present we do not know whether this binding affects the translation capacity of the ribosome. Preliminary kinetic data suggest that the capsid protein is transferred from the subunit to the viral nucleocapsid relatively fast (15). Thus, one function of the 608 subunit-capsid protein complex may be connected with the virus assembly. ACKNOWLEDGMENTS The skillful technical assistance of Ritva Rajala and Mirja Salonen is gratefully acknowledged.
This investigation was supported by grants from the Finnish Academy and the Sigrid Juselius and the Alfred Kordelin Foundations. Actinomycin D was a gift from Merck Sharp & Dohme. LITERATURE CITED 1. Baltimore, D., and A. S. Huang. 1968. Isopycnic separation of subcellular components from poliovirus-infected and normal HeLa cells. Science 162:572-574. 2. Bonner, W. M., and R. A. Laskey. 1974. A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46:8388. 3. Burke, D. C. 1975. Processing of alphavirus-specific proteins in infected cells. Med. Biol. 53:352-356. 4. Clegg, C., and I. Kennedy. 1975. Synthesis of the structural proteins of Semliki Forest virus. INSERM 47:255-258. 5. Clegg, J. C. S. 1975. Sequential translation of capsid and membrane protein genes of alphaviruses. Nature (London) 254:454-455. 6. 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. 7. Compans, R. W. 1973. Influenza virus proteins. II. Association with components of the cytoplasm. Virology 51:56-70. 8. Eaton, B. T., T. P. Donaghue, and P. Faulkner. 1972. Presence of poly(A) in the polyribosome associated RNA of Sindbis-infected BHK cells. Nature (London) New Biol. 238:109-111. 9. Eaton, B. T., and R. L. Regnery. 1975. Polysomal RNA in Semliki Forest virus infected Aedes albopictus cells. J. Gen. Virol. 29:35-49. 10. Garoff, H. K., K. Simons, and 0. Renkonen. 1974. Isolation and characterization of the membrane pro-
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