Vol. 24, No. 3 Printed in U.S.A.
JOURNAL OF VIROLOGY, Dec. 1977, p. 907-909 Copyright X 1977 American Society for Microbiology
Transient Association of Semliki Forest Virus Capsid Protein with Ribosomes HANS SODERLUND* AND ISMO ULMANEN Department of Virology, University of Helsinki, SF-00290 Helsinki 29, Finland Received for publication 18 May 1977
HeLa cells infected with Semliki Forest virus were exposed to [3S]methionine for 1 min and chased for various periods. The analysis of labeled ribonucleoproteins showed that the viral capsid protein associated first with the large ribosomal subunit in polysomes, from which it was chased to assembling nucleocapsids and to free monosomes. In infected cells the capsid protein of Semliki Forest virus (SFV) associates specifically with the viral genome (42S RNA) forming nucleocapsids (3, 10, 12). The protein is, however, also found with the large ribosomal subunit (13). Even capsid protein synthesized in wheat germ cell-free extract in response to viral 26S RNA binds to the large subunit (2). Binding of viral proteins to ribosomes has also been described for picornaviruses (7-9, 14) and myxoviruses (1, 5). However, in no case has a distinct function been ascribed to these complexes. In this paper, we followed the fate of newly formed SFV capsid protein by the aid of pulse-chase experiments. HeLa cell monolayers (11), infected with the prototype strain of SFV (4), were exposed for 1 min to [3S]methionine at 4.5 h postinfection. After the pulse, cells were either harvested immediately or first chased for varying periods in a medium containing an excess of unlabeled methionine. The cytoplasmic extract obtained by lysis of the cells with Triton in isotonic conditions was treated with EDTA and analyzed by sucrose gradient centrifugation. From the sample harvested immediately after the pulse, about one-third of the radioactivity sedimented at 60S, whereas two-thirds remained at the top of the gradient (Fig. 1A). After increasing chase periods, the amount of radioactivity sedimenting at 60S decreased and another peak with a sedimentation value of about 130S rose (Fig. 1B and C). During the chase, no change in the amount of radioactivity at the top fraction occurred. We have shown previously that the 130S peak consists of capsid protein and a 42S RNA, i.e., cytoplasmic viral nucleocapsid, whereas the 60S peak represents viral capsid protein associated with the large ribosomal subunit (13). The material remaining at the top of the gradient consisted of viral envelope proteins and their precursors (data not shown; see reference 13). Since no capsid protein was found in the top fractions,
the increase in the amount of capsid protein at 130S must be derived from the 60S peak, which showed a corresponding decrease in radioactivity during the chase period (Fig. 1). Quantitation of this shift (Fig. 2) showed that the transfer of capsid protein was initially very fast, about 30% being transferred during the first 2 min of chase. Thereafter, the rate of transfer became considerably slower, so that about 20% of the radioactivity was still sedimenting at 60S after 60 min of chase. A
B
60S
2
60S NC
NC
60S
Vo
u
10
20
30
10 FRACTION
20
30
10
20
30
NUMBER
FIG. 1. Pulse-chase experiment with SFV-infected HeLa cells. (A) Cells, grown as monolayers, were infected at a multiplicity of infection of 50, and virus was propagated in methionine-free Eagle minimum essential medium containing 2 pg of actinomycin D per ml and bovine serum albumin as described (13). At 4.5 h after the infection, the cells were labeled for 1 min with r5S]methionine (475 Ci/mmol; Radiochemical Centre, Amersham, England; 50 ILCi/dish). (B) Labeling as in (A) followed by a chase for 5 min and (C) for 60 min with Eagle minimum essential medium containing I mM unlabeled methionine. The cytoplasmic extracts were prepared using an isotonic buffer (0.15 M NaCI-0.01 M Tris (pH 7.4)-1.5 mM MgC42) containing 0.65% Triton X-100 (6). Ribonucleoproteins were released by adding EDTA (final concentration, 20 mM) and analyzed by centrifugation on 15 to 30% (wt/wt) sucrose gradients made in 0.05 M Tris (pH 7.4)-0.1 M NaCI-0.001 M EDTA for 2 h in a Spinco SW41 rotor at 40,000 rpm and 40C. NC, Nucleocapsid; 60S, large subunit. 907
908
J. VIROL.
NOTES
at0S
A
B os
60C
NC
75
c c *-
2?
>1
c ;
E
u
50
o0
O9
5) co
-o
25
99
91 ~~~~~90
9 o 9
9
000
0 0 u~~9999
de 20 0
5
10
15
30
60
CHASE (min)
FIG. 2. Kinetics of capsid protein transfer from a large ribosomal subunit to a viral nucleocapsid. The mS activity associated with nucleocapsids and large subunits was determined by sucrose gradient centrifugation of EDTA-treated cytoplasmic extracts. The infected cells were labeled with IpS~methionine for I min at 4.5 h after the infection and chased for different
periods. The proportion of label sedimenting
at the position of the nucleocapsid (120 to 140S) or the large subunit (60S) is compared (as percentage) to the total "sedimenting" (50 to 200S) WS activity. The latter corresponds roughly to the total amount ofcapsid protein in the gradient (see text). The circles represent data from one experiment, and the bars
indicate the range from three independent experiments. (0) -S at 120 to 140S (nucleocapsid); (@) 3MS at about 60S (the large subunit).
Similar experiments were performed in which the EDTA treatment was omitted that the polysomes remained intact. After the 1-min [35S]methionine pulse, most of the 'S activity sedimented faster than 160S and could be collected with the polysomes on a cushion of 60% sucrose layered below a 15 to 30% sucrose gradient (Fig. 3A). Essentially, no protein label sedimented with the monosomes, even if the bulk of the ribosomes was recovered at 80S (Fig. 3A). If the cytoplasmic extract was treated with EDTA, most of the sedimenting radioactivity was found at 60S (Fig. 3C) as before. When the cells were chased with unlabeled methionine so
after the pulse, the polysome-associated 35S activity was drastically reduced. The envelope proteins were now found at the top of the gradient, whereas the capsid protein sedimented both with nucleocapsids and with monosomes (Fig.
3B). When polysomes, isolated immediately after the pulse, were treated with EDTA and the resultant components were analyzed, the capsid protein sedimented with the large subunit as expected, and the nascent chains were found at the top of the gradient. These data suggest that the newly formed capsid protein first associates with those ribosomes actively engaged in protein synthesis. It
30 0
10 20 30 FRACTION NUMBER
10
20
30
FIG. 3. Isolation of polysomes from SFV-infected HeLa cells. (A) Cells were prelabeled with [3H]uridine (25 to 29 Ci/mmol; Radiochemical Centre, Amersham, England; 5 ,&Ci/dish) in Eagle minimum essential medium containing 1% fetal calf serum for 16 h before infection and pulse-labeled for I min with [3Smethionine at 4.5 h postinfection. (B) The infected cells were pulse-labeled for 1 min with rSJmethionine and chased for 15 min. Cytoplasmic extracts were prepared and centrifuged at 40,000 rpm and 40C in a Spinco SW41 rotor for 2 h and 20 min on a 15 to 30% (wt/wt) sucrose gradient made in isotonic buffer with a 0.5-ml cushion of 60% sucrose on the bottom. (C) Part of the cytoplasmic extract in (A) was treated with EDTA (20 mM) and analyzed similarly in a sucrose gradient made in 0.05 M Tris (pH 7.4)-O.1 M NaCI-0.001 M EDTA. 0, 35S; *, 3H. may then be released with the ribosomes to the monosome pool or directly transferred to assembling nucleocapsids. Thus, one function for the ribosome-capsid protein complex seems to be
protein transport in the nucleocapsid assembly. We thank L Ki .en for fruitful discussions and M. Lehtonen and R. Rajala for skillful technical assistance. Actinomyci D was a kind gift from Merck Sharp & Dohme. This work was supported by grants from the Finnish Academy and from the Leo & Regina Weinstein Foundation. LITERATURE CITED 1. Compans, R. W. 1973. Influenza vimr proteins. II. Association with components of the cytoplasm. Virology 51:56-70. 2. Glanville, N., and I. Ulnen 1976. Biological activity ofin vitro synthesized protein: binding of Semiiki Forest virus capsid protein to the large ribosomal subunit. Biochem. Biophys. Res. Commun. 71:393-399. 3. Kaie, L, 8. Ker ]en,B.ehi S8d rhund, 1975. Replication of SemK. T1oml, and L liki Forest virus. Med. BioL 53:342-362. 4. Ksarlam~en, L, K. Simons, and C.-H. von Bonsdorff. 1969. Studies in subviral components of Semliki Forest virus Ann. Med. Exp. Biol. Fenn. 47:235-248. 5. Krug, R. AL, and P. R. Etkind. 1973. Cytoplasmic and nuclear virus-specific proteins in influenza virus-infected MDCK-cells. Virology 5:334-348. 6. Kumar, A., and U. Lindberg. 1972. Characterization of messenger ribonucleoprotein and messenger RNA from KB cells. Proc. Natl. Acad. Sci. U.S.A. 60:681-85. 7. Manak, X. I., S. L Abreu, and J. Lucas-Lenard. 1975. Association of mengo-virus proteins with host cell native 408 ribosomal subunit. INSERM 47:387-396. 8. Matthewsa, B., E. Butterworth, L Chaffin, and R. R.
VOL. 24, 1977 Rueckert. 1973. Encephalomyocarditis (EMC) virus and rhinovirus 1A (HRV-1A) peptides associated with the infected cell ribosomes. Fed. Proc. Fed. Am. Soc. Exp. Biol. 32:461. 9. Medvedkina, 0. A., L V. Scarlat, N. 0. Kalina, and V. I. Agol. 1974. Virus specific proteins associated with ribosomes of Krebs cells infected with Encephalomyocarditis virus& FEBS Lett. 39:4-8. 10. Pfefferkorn, E. R., and D. Shapiro. 1974. Reproduction of togaviruses, p. 171-230. In H. Fraenkel-Conrat and R. R Wagner (ed.), Comprehensive virology, vol. 2. Plenum Publishing Corp., New York.
NOTES 11. Sdderlund,
909
H. 1973. Kinetics of formation of Semliki Forest virus nucleocapsid. Intervirology 1:354-361. 12. Strauss, J. IL, and E. G. Strauss. 1977. Togaviruses, p. 111-166. In D. Nayak (ed.), The molecular biology of animal viruses. Marcel Dekker, Inc., New York. 13. Ulnen, L, H. S6derlund, and L Kiiriinen. 1976. Semliki Forest virus capsid protein associates with the 608 ribosomal subunit in infected cells J. Virol. 20:203-210. 14. Wright, P. J., and P. D. Cooper. 1974. Poliovirus proteins associated with ribosomal structures in infected cells. Virology 59:1-20.
JOURNAL OF VIROLOGY, Dec. 1977, p. 907-909 Copyright X 1977 American Society for Microbiology
Transient Association of Semliki Forest Virus Capsid Protein with Ribosomes HANS SODERLUND* AND ISMO ULMANEN Department of Virology, University of Helsinki, SF-00290 Helsinki 29, Finland Received for publication 18 May 1977
HeLa cells infected with Semliki Forest virus were exposed to [3S]methionine for 1 min and chased for various periods. The analysis of labeled ribonucleoproteins showed that the viral capsid protein associated first with the large ribosomal subunit in polysomes, from which it was chased to assembling nucleocapsids and to free monosomes. In infected cells the capsid protein of Semliki Forest virus (SFV) associates specifically with the viral genome (42S RNA) forming nucleocapsids (3, 10, 12). The protein is, however, also found with the large ribosomal subunit (13). Even capsid protein synthesized in wheat germ cell-free extract in response to viral 26S RNA binds to the large subunit (2). Binding of viral proteins to ribosomes has also been described for picornaviruses (7-9, 14) and myxoviruses (1, 5). However, in no case has a distinct function been ascribed to these complexes. In this paper, we followed the fate of newly formed SFV capsid protein by the aid of pulse-chase experiments. HeLa cell monolayers (11), infected with the prototype strain of SFV (4), were exposed for 1 min to [3S]methionine at 4.5 h postinfection. After the pulse, cells were either harvested immediately or first chased for varying periods in a medium containing an excess of unlabeled methionine. The cytoplasmic extract obtained by lysis of the cells with Triton in isotonic conditions was treated with EDTA and analyzed by sucrose gradient centrifugation. From the sample harvested immediately after the pulse, about one-third of the radioactivity sedimented at 60S, whereas two-thirds remained at the top of the gradient (Fig. 1A). After increasing chase periods, the amount of radioactivity sedimenting at 60S decreased and another peak with a sedimentation value of about 130S rose (Fig. 1B and C). During the chase, no change in the amount of radioactivity at the top fraction occurred. We have shown previously that the 130S peak consists of capsid protein and a 42S RNA, i.e., cytoplasmic viral nucleocapsid, whereas the 60S peak represents viral capsid protein associated with the large ribosomal subunit (13). The material remaining at the top of the gradient consisted of viral envelope proteins and their precursors (data not shown; see reference 13). Since no capsid protein was found in the top fractions,
the increase in the amount of capsid protein at 130S must be derived from the 60S peak, which showed a corresponding decrease in radioactivity during the chase period (Fig. 1). Quantitation of this shift (Fig. 2) showed that the transfer of capsid protein was initially very fast, about 30% being transferred during the first 2 min of chase. Thereafter, the rate of transfer became considerably slower, so that about 20% of the radioactivity was still sedimenting at 60S after 60 min of chase. A
B
60S
2
60S NC
NC
60S
Vo
u
10
20
30
10 FRACTION
20
30
10
20
30
NUMBER
FIG. 1. Pulse-chase experiment with SFV-infected HeLa cells. (A) Cells, grown as monolayers, were infected at a multiplicity of infection of 50, and virus was propagated in methionine-free Eagle minimum essential medium containing 2 pg of actinomycin D per ml and bovine serum albumin as described (13). At 4.5 h after the infection, the cells were labeled for 1 min with r5S]methionine (475 Ci/mmol; Radiochemical Centre, Amersham, England; 50 ILCi/dish). (B) Labeling as in (A) followed by a chase for 5 min and (C) for 60 min with Eagle minimum essential medium containing I mM unlabeled methionine. The cytoplasmic extracts were prepared using an isotonic buffer (0.15 M NaCI-0.01 M Tris (pH 7.4)-1.5 mM MgC42) containing 0.65% Triton X-100 (6). Ribonucleoproteins were released by adding EDTA (final concentration, 20 mM) and analyzed by centrifugation on 15 to 30% (wt/wt) sucrose gradients made in 0.05 M Tris (pH 7.4)-0.1 M NaCI-0.001 M EDTA for 2 h in a Spinco SW41 rotor at 40,000 rpm and 40C. NC, Nucleocapsid; 60S, large subunit. 907
908
J. VIROL.
NOTES
at0S
A
B os
60C
NC
75
c c *-
2?
>1
c ;
E
u
50
o0
O9
5) co
-o
25
99
91 ~~~~~90
9 o 9
9
000
0 0 u~~9999
de 20 0
5
10
15
30
60
CHASE (min)
FIG. 2. Kinetics of capsid protein transfer from a large ribosomal subunit to a viral nucleocapsid. The mS activity associated with nucleocapsids and large subunits was determined by sucrose gradient centrifugation of EDTA-treated cytoplasmic extracts. The infected cells were labeled with IpS~methionine for I min at 4.5 h after the infection and chased for different
periods. The proportion of label sedimenting
at the position of the nucleocapsid (120 to 140S) or the large subunit (60S) is compared (as percentage) to the total "sedimenting" (50 to 200S) WS activity. The latter corresponds roughly to the total amount ofcapsid protein in the gradient (see text). The circles represent data from one experiment, and the bars
indicate the range from three independent experiments. (0) -S at 120 to 140S (nucleocapsid); (@) 3MS at about 60S (the large subunit).
Similar experiments were performed in which the EDTA treatment was omitted that the polysomes remained intact. After the 1-min [35S]methionine pulse, most of the 'S activity sedimented faster than 160S and could be collected with the polysomes on a cushion of 60% sucrose layered below a 15 to 30% sucrose gradient (Fig. 3A). Essentially, no protein label sedimented with the monosomes, even if the bulk of the ribosomes was recovered at 80S (Fig. 3A). If the cytoplasmic extract was treated with EDTA, most of the sedimenting radioactivity was found at 60S (Fig. 3C) as before. When the cells were chased with unlabeled methionine so
after the pulse, the polysome-associated 35S activity was drastically reduced. The envelope proteins were now found at the top of the gradient, whereas the capsid protein sedimented both with nucleocapsids and with monosomes (Fig.
3B). When polysomes, isolated immediately after the pulse, were treated with EDTA and the resultant components were analyzed, the capsid protein sedimented with the large subunit as expected, and the nascent chains were found at the top of the gradient. These data suggest that the newly formed capsid protein first associates with those ribosomes actively engaged in protein synthesis. It
30 0
10 20 30 FRACTION NUMBER
10
20
30
FIG. 3. Isolation of polysomes from SFV-infected HeLa cells. (A) Cells were prelabeled with [3H]uridine (25 to 29 Ci/mmol; Radiochemical Centre, Amersham, England; 5 ,&Ci/dish) in Eagle minimum essential medium containing 1% fetal calf serum for 16 h before infection and pulse-labeled for I min with [3Smethionine at 4.5 h postinfection. (B) The infected cells were pulse-labeled for 1 min with rSJmethionine and chased for 15 min. Cytoplasmic extracts were prepared and centrifuged at 40,000 rpm and 40C in a Spinco SW41 rotor for 2 h and 20 min on a 15 to 30% (wt/wt) sucrose gradient made in isotonic buffer with a 0.5-ml cushion of 60% sucrose on the bottom. (C) Part of the cytoplasmic extract in (A) was treated with EDTA (20 mM) and analyzed similarly in a sucrose gradient made in 0.05 M Tris (pH 7.4)-O.1 M NaCI-0.001 M EDTA. 0, 35S; *, 3H. may then be released with the ribosomes to the monosome pool or directly transferred to assembling nucleocapsids. Thus, one function for the ribosome-capsid protein complex seems to be
protein transport in the nucleocapsid assembly. We thank L Ki .en for fruitful discussions and M. Lehtonen and R. Rajala for skillful technical assistance. Actinomyci D was a kind gift from Merck Sharp & Dohme. This work was supported by grants from the Finnish Academy and from the Leo & Regina Weinstein Foundation. LITERATURE CITED 1. Compans, R. W. 1973. Influenza vimr proteins. II. Association with components of the cytoplasm. Virology 51:56-70. 2. Glanville, N., and I. Ulnen 1976. Biological activity ofin vitro synthesized protein: binding of Semiiki Forest virus capsid protein to the large ribosomal subunit. Biochem. Biophys. Res. Commun. 71:393-399. 3. Kaie, L, 8. Ker ]en,B.ehi S8d rhund, 1975. Replication of SemK. T1oml, and L liki Forest virus. Med. BioL 53:342-362. 4. Ksarlam~en, L, K. Simons, and C.-H. von Bonsdorff. 1969. Studies in subviral components of Semliki Forest virus Ann. Med. Exp. Biol. Fenn. 47:235-248. 5. Krug, R. AL, and P. R. Etkind. 1973. Cytoplasmic and nuclear virus-specific proteins in influenza virus-infected MDCK-cells. Virology 5:334-348. 6. Kumar, A., and U. Lindberg. 1972. Characterization of messenger ribonucleoprotein and messenger RNA from KB cells. Proc. Natl. Acad. Sci. U.S.A. 60:681-85. 7. Manak, X. I., S. L Abreu, and J. Lucas-Lenard. 1975. Association of mengo-virus proteins with host cell native 408 ribosomal subunit. INSERM 47:387-396. 8. Matthewsa, B., E. Butterworth, L Chaffin, and R. R.
VOL. 24, 1977 Rueckert. 1973. Encephalomyocarditis (EMC) virus and rhinovirus 1A (HRV-1A) peptides associated with the infected cell ribosomes. Fed. Proc. Fed. Am. Soc. Exp. Biol. 32:461. 9. Medvedkina, 0. A., L V. Scarlat, N. 0. Kalina, and V. I. Agol. 1974. Virus specific proteins associated with ribosomes of Krebs cells infected with Encephalomyocarditis virus& FEBS Lett. 39:4-8. 10. Pfefferkorn, E. R., and D. Shapiro. 1974. Reproduction of togaviruses, p. 171-230. In H. Fraenkel-Conrat and R. R Wagner (ed.), Comprehensive virology, vol. 2. Plenum Publishing Corp., New York.
NOTES 11. Sdderlund,
909
H. 1973. Kinetics of formation of Semliki Forest virus nucleocapsid. Intervirology 1:354-361. 12. Strauss, J. IL, and E. G. Strauss. 1977. Togaviruses, p. 111-166. In D. Nayak (ed.), The molecular biology of animal viruses. Marcel Dekker, Inc., New York. 13. Ulnen, L, H. S6derlund, and L Kiiriinen. 1976. Semliki Forest virus capsid protein associates with the 608 ribosomal subunit in infected cells J. Virol. 20:203-210. 14. Wright, P. J., and P. D. Cooper. 1974. Poliovirus proteins associated with ribosomal structures in infected cells. Virology 59:1-20.
