dr. gen. Virol. (I979), 43, 441-445 Printed in Great Britain
44 I
Inhibition of 80S Initiation Complex Formation by Infection with Poliovirus ( A c c e p t e d I2 D e c e m b e r ~978) SUMMARY
Anisomycin has been shown to stabilize ribosome initiation complexes containing messenger RNA and met-tRNA~ °~ to high salt conditions. Extracts from HeLa cells treated with 5 x lO-7 M-anisomycin for 15 rain accumulate 8oS initiation complexes which can be detected by their absorbance in sucrose gradients. Poliovirus-infected cells fail to form the 8oS initiation complex early after infection, when inhibition of host cell protein synthesis occurs. These complexes re-form later in infection after virus RNA is synthesized. No re-formation occurs in the absence of virus replication. Thus, the step in protein synthesis inhibited by poliovirus precedes the entry of components into the 8oS initiation complex. Infection of cells with poliovirus causes a rapid and marked inhibition of cellular protein synthesis (Penman et al. I963). The inhibited step has been narrowed down to the initiation events of protein synthesis (Leibowitz & Penman, ~97L), although the precise reaction which is blocked has not been identified. Recent reports from other laboratories have clarified the temporal sequence of reactions required for initiation of protein synthesis and have identified the initiation factor requirements for these steps (Schreier et al. I977; Trachsel et al. I977; Benne & Hershey, I978). Our analyses of the initiation of protein synthesis by extracts from poliovirus-infected HeLa cells performed in vitro have demonstrated that a crude initiation factor preparation from infected cells contains a messenger RNA (mRNA) discriminatory factor which is totally inactive in stimulating the initiation of protein synthesis directed by cellular mRNA, but which retains activity for stimulating translation of virus mRNA (Helentjaris & Ehrenfeld, I978). On this basis, we suggest that host cell 'shut-off' occurs at a step in the initiation reaction which involves recognition of mRNA or of an initiation complex which contains mRNA. In experiments described in this report, we have attempted to examine the 8oS initiation complex, containing m R N A and met-tRNA~ et, to determine if its formation is inhibited under conditions of virus-induced host cell shut-off. We used the drug anisomycin, which can stabilize the 8oS initiation complex to high salt (o'5 M-NaC1) conditions which would dissociate ribosome complexes not containing mRNA and initiator tRNA (Lodish & Rose, I977), to measure the amount of 8oS initiation complexes in the cell. We show here that the formation of 8oS initiation complexes containing cellular m R N A was inhibited by poliovirus infection and that 8oS initiation complexes containing virus m R N A re-formed after synthesis of virus mRNA synthesis. We conclude that the step in protein synthesis inhibited by poliovirus precedes the entry of components into the 8oS initiation complex. Cytoplasmic extracts of HeLa cells normally contain a significant fraction of 8oS ribosomes, which can be visualized by velocity sedimentation in a sucrose density gradient. However, under high salt conditions (o'5 M-NaC1, o'o3 u-Mg acetate), the absorbance profile of these extracts shows that the majority of the monosomes were dissociated into 4oS and 6oS ribosomal subunits. When cells were treated with 5 × to-~ u-anisomycin for 15 rain prior to harvesting the preparation of cytoplasmic extracts, a portion of their 8oS ribosomes were rendered resistant to dissociation by high salt. Fig. I (a) shows the absoroo22-I317/79/oooo-3482 $02.00 © I979 SGM
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60S
l
(a ) -anisomycin
0.6 0.5 0-4 0,3 0-2 0,1
(b) + anisomycin
80S 60S
0.6 0.5 0.4 0-3 0-2
l
40S
1
0.1 Sedimentation Fig. i. Velocity sedimentation analysis in high salt buffer of cytoplasmic extracts from cells treated with anisomycin. 2"5 x Io7 HeLa cells were concentrated to 5 x Io6/ml and incubated with (b) or without (a) 5 x io -7 ra-anisomycin for 15 rain at 37 °C. The cells were collected by centrifugation, suspended in I ml of 0"o5 M-tris-HCl containing 0"5 M-NaCI and 0"03 ~-Mg acetate and 1% Nonidet P-4o. The nuclei were removed by centrifugation and the cytoplasmic extracts in high salt buffer were layered over linear Io to 40% (w/v) sucrose gradients in the same buffer. Centrifugation was for I6 h at I7OOOrev/min in the SW 27 rotor at 4 °C. Gradients were pumped through a Gilford recording spectrophotometer measuring absorbance at 260 nm. bance profiles of ribosomes a n d ribosomal subunits from untreated cells analysed o n sucrose gradients in high salt buffer. I n contrast, in the presence of anisomycin, a large p o r t i o n of the 80S ribosomes were stabilized to high salt (Fig. I b). In different experiments, the absolute a m o u n t of 80S stabilized ribosomes varied according to cell density a n d growth rate, resulting in the most rapidly growing cells showing the highest degree of high salt stabilization of m o n o s o m e s by anisomycin. Lodish & Rose (I977) previously reported that reticulocyte ribosomes which were b o u n d to m R N A a n d initiator t R N A were resistant to dissociation by high salt after a n i s o m y c i n t r e a t m e n t in vitro. W h e n a reticulocyte lysate was i n c u b a t e d with 3 H - m R N A extracted from vesicular stomatitis virus-infected HeLa cells a n d analysed on sucrose gradients in high salt, the Z H - m R N A remained b o u n d to ribosomes a n d sedimented at 80S in the presence o f anisomycin whereas, in the absence o f anisomycin, the 80S ribosomes b o u n d to m R N A
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(a) uninfected 80S 60S
(b) 2 h p.i.
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/
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(c) 2-75 h p.i.
(d) 4 h pA.
]
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0.5 0.4 0.3 0.2 0-1
1
'S virus
(e) 2.75 h p.i. + guanidine
(f) 2-25 h postguanidine reversal
/A/ Sedimentation
Fig. 2. Sedimentation analysis of ribosomes in high salt buffer from poliovirus-infected cells treated with anisomycin. HeLa cells (5 x ior/ml) were infected with Loo p.f.u./cell or poliovirus (type I, Mahoney strain), in the absence (a to d) or presence (e to f ) of I mM-guanidine. At the indicated times after infection, samples were removed and treated for ~5 min with 5× IO-~Manisomycin. The cells were collected by centrifugation and processed for sucrose density gradient sedimentation analysis in high salt buffer as described in the legend to Fig. I. The guanidinetreated culture was incubated for 2"75 h, then the cells were washed free of guanidine and resuspended in fresh medium. At various times, samples were treated with anisomycin and analysed as above. Areas under the peaks were integrated with a Numonics Corp. I224 graphics calculator after estimating peak overlaps. The percentage of total absorbance in ribosomal material which occurred as stabilized 8oS complexes was (a) 36%, (b) 15%, (c) 35%, (d) ]4%, (e) II ~o, (f) 33%. w e r e d i s s o c i a t e d ( d a t a n o t shown). S i m i l a r results w e r e o b t a i n e d u s i n g 3 5 S - f m e t - t R N A to l a b e l i n i t i a t i o n c o m p l e x e s . T h u s the a n i s o m y c i n s t a b i l i z a t i o n o f r i b o s o m e s to h i g h salt a p p e a r s to select f o r t h o s e r i b o s o m e s w h i c h are i n v o l v e d in i n i t i a t i o n c o m p l e x f o r m a t i o n . T h e i n c r e a s e d resistance to h i g h salt o f 80S r i b o s o m e s b o u n d to m R N A a n d m e t - t R N A ~ ~ as i n i t i a t i o n c o m p l e x e s has also b e e n r e p o r t e d by o t h e r s ( F a l v e y & Staehelin, I 9 7 0 ; S c h r e i e r & S t a e h e l i n , I973).
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We have utilized the ability to detect 8oS initiation complexes stabilized to high salt by anisomycin to determine whether or not these complexes are formed after poliovirus infection of HeLa cells. Cells were infected with IOO p.f.u./cell o f the Mahoney strain of poliovirus type I and treated at various times after infection with 5 x io -7 i-anisomycin for I5 min. Cytoplasmic extracts were prepared and analysed on sucrose gradients in high salt to determine the proportion of ribosomes which were in initiation complexes. Shortly after infection, the number of 8oS initiation complexes in the infected cells decreased, reaching a minimum at approx. 1.75 h p.i. This correlates precisely with the time course of virus-induced inhibition of host cell protein synthesis (Helentjaris & Ehrenfeld, 1977). Fig. z shows the sucrose gradient profiles of extracts prepared at several different times after infection. By 2"75 h p.i., the number of 8oS initiation complexes again begins to increase, as virus-directed translation also commences (Fig. 2c) and when the virus cycle is nearing completion, at 4 h p.i., initation complexes again disappear (Fig. 2d). When cells were infected in the presence of I mM-guanidine, so that no virus replication occurred, the 8oS initiation complex decreased in keeping with the kinetics of inhibition of host cell protein synthesis (Fig. 2 e) and failed to reappear with further incubation. Removal of guanidine from the medium permits virus replication to proceed and 8oS initiation complexes form again (Fig. 2f). Thus, the formation of cellular 8oS initiation complexes is inhibited by poliovirus infection and the block to initiation of protein synthesis must occur at this or a preceding step. Anisomycin is an inhibitor of peptidyl transferase which has been shown to bind to the large ribosomal subunit of the 8oS ribosome (Barbacid & Vasquez, I974). The concentration of anisomycin required to protect monosomes from high salt dissociation in vivo is critical. Below 5 x IO-s M-anisomycin, almost no ribosome protection is seen. Maximum salt stabilization is observed between 5 × lO-~ M- and I × IO-8 M-anisomycin, but at higher concentrations stabilization is lost. The concentrations which give maximal 8oS ribosome stabilization to high salt are those which just achieve full inhibition of protein synthesis. It appears that when elongation is extensively slowed, a build-up of initiation complexes occurs. The number of 8oS initiation complexes observed in this way increases up to I5 min of anisomycin treatment but then remains stable with further incubation. In addition, no anisomycin-stabilized complexes were observed if the cells were treated with cycloheximide prior to or during the anisomycin treatment. The data described in this report demonstrate that the formation of cellular 8oS initiation complexes fails to occur after poliovirus infection. Recently, evidence from this laboratory has been presented (Helentjaris & Ehrenfeld, 1978) which suggests that the inhibition of host cell protein synthesis induced by poliovirus involves an m R N A recognition step of the initiation reaction. Consistent with this, ternary complex formation between GTP, eIF-2 and met-tRNAp "~ appears to be normal in infected cells (Helentjaris & Ehrenfeld, 1978 ) and in this report we show that a later step in initiation is blocked. The suggestion put forward by Lawrence & Thach (1974) that inhibition of cellular protein synthesis results from a competition between virus m R N A and cellular m R N A for some component(s) of the initiation reaction is not supported by these data, since one would not expect a decrease in initiation complexes but merely a replacement of cellular with virus complexes. On the contrary, we find that initiation complexes disappear in infected cells and only reappear subsequent to virus m R N A synthesis. Previous work has shown that fully intact and functional m R N A can be recovered from cells in which translation is completely inhibited (Ehrenfeld & Lund, 1977; Hackett et al. 1977.) The precise step for inhibition induced by poliovirus is still not determined but it now appears to be narrowed down to an event subsequent to ternary complex formation and prior to or at the 6oS junction reaction. When considered with in vitro studies showing m R N A specificity of the a~tivity of the ribosomal
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salt wash, the available data p o i n t to steps involving m R N A b i n d i n g to the 4oS initiation complex as the site of i n h i b i t i o n of host cell protein synthesis. Attempts to determine whether f o r m a t i o n of the 4oS complex c o n t a i n i n g met-tRNA~ alone or the 4oS complex c o n t a i n i n g met-tRNA~ a n d m R N A is inhibited in infected cells have so far failed. It appears that once a 4oS complex c o n t a i n i n g met-tRNA~ a n d m R N A is formed, the 6oS j u n c t i o n reaction is extremely fast, allowing n o a c c u m u l a t i o n or detection o f the preceding step (our u n p u b l i s h e d observations). This work was supported by grants from the N a t i o n a l Institute of Health (AI 12387) a n d from the N a t i o n a l Science F o u n d a t i o n , a n d by a Teacher-Scholar A w a r d from the Dreyfus F o u n d a t i o n . We are grateful to Betty Brown in this l a b o r a t o r y who prepared the reticulocyte lysate.
Departments of Biochemistry and Microbiology University of Utah Medical Center Salt Lake City, Utah 84132, U.S.A.
ELLIE EHRENFELD SUE MANIS
REFERENCES nARBAOD, M. & VASQUEZ,D. (I974). [~H]anisomycin binding to eukaryotic ribosomes. Journal of Molecular Biology 84, 603-623. BENNE,R. & HERSHEY,J. W. B. (X978). The mechanism of action of protein synthesis initiation factors from rabbit reticulocytes. Journal of Biological Chemistry 253, 3o78-3o87. EHRENEELD, E. & LUND, H. (1977). Untranslated vesicular stomatitis virus messenger RNA after poliovirus infection. Virology go, 297-308. FALVEY,g. & STAEHELIN,T. (t970). Structure and function of mammalian ribosomes. I. Isolation and characterization of active liver ribosomal subunits. Journal of Molecular Biology 53, I-9. HACKET, P. B., EGBERTS,E., BUCHWALD, I. & TRAUB, P. (1977)- Coding capacity of poly (A) + m R N A isolated
from menogvirus-infected Ehrlich ascites tumor cells. Molecular Biology Reports 3, 387-396. HELENTJARIS,T. & EHRENFELD,E. (1977). Inhibition of host cell protein synthesis by UV-irradiated poliovirus. Journal of Virology 2x, 259-267. HELENTJARIS,T. & EHRENFELD,E. (1978). Control of protein synthesis in extracts from poliovirus-infected cells. I. Messenger RNA discrimination by crude initiation factors. Journal of Virology 26, 5IO-521. LAWRENCE, C. & THACH,R. (I974). Encephalomycarditis virus infection of mouse plasmacytoma cells, I. Inhibition of cellular protein synthesis. Journal of Virology x4, 588-61o. LE1BOWITZ,R. & PENMAN,S. (1971). Regulation of protein synthesis in Hela Cells, III. Inhibition during poliovirus infection. Journal of Virology 8, 661-668. LODISH,M. & ROSE,J. (I977). Relative importance of 7-methyl-guanosine in ribosome binding and translation of vesicular stomatitis virus mRNA in wheat germ and reticulocyte cell-free systems. Journal of Biological Chemistry 252, 1I81-I 188. PENMAN, S., SCHERRER, K., BECKER, Y. & OARNELL, J. E. (1963). Polyribosomes in normal and poliovirus-
infected Hela cells and their relationship to messenger RNA. Proceedings of the National Academy of Sciences of the United States of America, 49, 654-662. SCHREIER,M.&STAEHELIN,T. (1973)- Initiation of mammalian protein synthesis: the importance of ribosome and initiation factor quality for the efficiency of in vitro systems. Journal of Molecular Biology 73, 329-34o. SCHREIER, M. n., ERNI, B. & STAEHELIN,T. 0977). Initiation of m a m m a l i a n protein synthesis. Purification and
characterization of seven initiation factors. Journal of Molecular Biology xx6, 727-753. TRACHSEL, H., ERNI, B., SCHREIER, M. H. & STAEHELIN, T. (1977). Initiation of mammalian protein synthesis
II. The assembly of the initiation complex with purified initiation factors. Journal of Molecular Biology ix6, 755-767.
(Received 25 September I978 )
44 I
Inhibition of 80S Initiation Complex Formation by Infection with Poliovirus ( A c c e p t e d I2 D e c e m b e r ~978) SUMMARY
Anisomycin has been shown to stabilize ribosome initiation complexes containing messenger RNA and met-tRNA~ °~ to high salt conditions. Extracts from HeLa cells treated with 5 x lO-7 M-anisomycin for 15 rain accumulate 8oS initiation complexes which can be detected by their absorbance in sucrose gradients. Poliovirus-infected cells fail to form the 8oS initiation complex early after infection, when inhibition of host cell protein synthesis occurs. These complexes re-form later in infection after virus RNA is synthesized. No re-formation occurs in the absence of virus replication. Thus, the step in protein synthesis inhibited by poliovirus precedes the entry of components into the 8oS initiation complex. Infection of cells with poliovirus causes a rapid and marked inhibition of cellular protein synthesis (Penman et al. I963). The inhibited step has been narrowed down to the initiation events of protein synthesis (Leibowitz & Penman, ~97L), although the precise reaction which is blocked has not been identified. Recent reports from other laboratories have clarified the temporal sequence of reactions required for initiation of protein synthesis and have identified the initiation factor requirements for these steps (Schreier et al. I977; Trachsel et al. I977; Benne & Hershey, I978). Our analyses of the initiation of protein synthesis by extracts from poliovirus-infected HeLa cells performed in vitro have demonstrated that a crude initiation factor preparation from infected cells contains a messenger RNA (mRNA) discriminatory factor which is totally inactive in stimulating the initiation of protein synthesis directed by cellular mRNA, but which retains activity for stimulating translation of virus mRNA (Helentjaris & Ehrenfeld, I978). On this basis, we suggest that host cell 'shut-off' occurs at a step in the initiation reaction which involves recognition of mRNA or of an initiation complex which contains mRNA. In experiments described in this report, we have attempted to examine the 8oS initiation complex, containing m R N A and met-tRNA~ et, to determine if its formation is inhibited under conditions of virus-induced host cell shut-off. We used the drug anisomycin, which can stabilize the 8oS initiation complex to high salt (o'5 M-NaC1) conditions which would dissociate ribosome complexes not containing mRNA and initiator tRNA (Lodish & Rose, I977), to measure the amount of 8oS initiation complexes in the cell. We show here that the formation of 8oS initiation complexes containing cellular m R N A was inhibited by poliovirus infection and that 8oS initiation complexes containing virus m R N A re-formed after synthesis of virus mRNA synthesis. We conclude that the step in protein synthesis inhibited by poliovirus precedes the entry of components into the 8oS initiation complex. Cytoplasmic extracts of HeLa cells normally contain a significant fraction of 8oS ribosomes, which can be visualized by velocity sedimentation in a sucrose density gradient. However, under high salt conditions (o'5 M-NaC1, o'o3 u-Mg acetate), the absorbance profile of these extracts shows that the majority of the monosomes were dissociated into 4oS and 6oS ribosomal subunits. When cells were treated with 5 × to-~ u-anisomycin for 15 rain prior to harvesting the preparation of cytoplasmic extracts, a portion of their 8oS ribosomes were rendered resistant to dissociation by high salt. Fig. I (a) shows the absoroo22-I317/79/oooo-3482 $02.00 © I979 SGM
442
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60S
l
(a ) -anisomycin
0.6 0.5 0-4 0,3 0-2 0,1
(b) + anisomycin
80S 60S
0.6 0.5 0.4 0-3 0-2
l
40S
1
0.1 Sedimentation Fig. i. Velocity sedimentation analysis in high salt buffer of cytoplasmic extracts from cells treated with anisomycin. 2"5 x Io7 HeLa cells were concentrated to 5 x Io6/ml and incubated with (b) or without (a) 5 x io -7 ra-anisomycin for 15 rain at 37 °C. The cells were collected by centrifugation, suspended in I ml of 0"o5 M-tris-HCl containing 0"5 M-NaCI and 0"03 ~-Mg acetate and 1% Nonidet P-4o. The nuclei were removed by centrifugation and the cytoplasmic extracts in high salt buffer were layered over linear Io to 40% (w/v) sucrose gradients in the same buffer. Centrifugation was for I6 h at I7OOOrev/min in the SW 27 rotor at 4 °C. Gradients were pumped through a Gilford recording spectrophotometer measuring absorbance at 260 nm. bance profiles of ribosomes a n d ribosomal subunits from untreated cells analysed o n sucrose gradients in high salt buffer. I n contrast, in the presence of anisomycin, a large p o r t i o n of the 80S ribosomes were stabilized to high salt (Fig. I b). In different experiments, the absolute a m o u n t of 80S stabilized ribosomes varied according to cell density a n d growth rate, resulting in the most rapidly growing cells showing the highest degree of high salt stabilization of m o n o s o m e s by anisomycin. Lodish & Rose (I977) previously reported that reticulocyte ribosomes which were b o u n d to m R N A a n d initiator t R N A were resistant to dissociation by high salt after a n i s o m y c i n t r e a t m e n t in vitro. W h e n a reticulocyte lysate was i n c u b a t e d with 3 H - m R N A extracted from vesicular stomatitis virus-infected HeLa cells a n d analysed on sucrose gradients in high salt, the Z H - m R N A remained b o u n d to ribosomes a n d sedimented at 80S in the presence o f anisomycin whereas, in the absence o f anisomycin, the 80S ribosomes b o u n d to m R N A
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(a) uninfected 80S 60S
(b) 2 h p.i.
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'S virus
(e) 2.75 h p.i. + guanidine
(f) 2-25 h postguanidine reversal
/A/ Sedimentation
Fig. 2. Sedimentation analysis of ribosomes in high salt buffer from poliovirus-infected cells treated with anisomycin. HeLa cells (5 x ior/ml) were infected with Loo p.f.u./cell or poliovirus (type I, Mahoney strain), in the absence (a to d) or presence (e to f ) of I mM-guanidine. At the indicated times after infection, samples were removed and treated for ~5 min with 5× IO-~Manisomycin. The cells were collected by centrifugation and processed for sucrose density gradient sedimentation analysis in high salt buffer as described in the legend to Fig. I. The guanidinetreated culture was incubated for 2"75 h, then the cells were washed free of guanidine and resuspended in fresh medium. At various times, samples were treated with anisomycin and analysed as above. Areas under the peaks were integrated with a Numonics Corp. I224 graphics calculator after estimating peak overlaps. The percentage of total absorbance in ribosomal material which occurred as stabilized 8oS complexes was (a) 36%, (b) 15%, (c) 35%, (d) ]4%, (e) II ~o, (f) 33%. w e r e d i s s o c i a t e d ( d a t a n o t shown). S i m i l a r results w e r e o b t a i n e d u s i n g 3 5 S - f m e t - t R N A to l a b e l i n i t i a t i o n c o m p l e x e s . T h u s the a n i s o m y c i n s t a b i l i z a t i o n o f r i b o s o m e s to h i g h salt a p p e a r s to select f o r t h o s e r i b o s o m e s w h i c h are i n v o l v e d in i n i t i a t i o n c o m p l e x f o r m a t i o n . T h e i n c r e a s e d resistance to h i g h salt o f 80S r i b o s o m e s b o u n d to m R N A a n d m e t - t R N A ~ ~ as i n i t i a t i o n c o m p l e x e s has also b e e n r e p o r t e d by o t h e r s ( F a l v e y & Staehelin, I 9 7 0 ; S c h r e i e r & S t a e h e l i n , I973).
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We have utilized the ability to detect 8oS initiation complexes stabilized to high salt by anisomycin to determine whether or not these complexes are formed after poliovirus infection of HeLa cells. Cells were infected with IOO p.f.u./cell o f the Mahoney strain of poliovirus type I and treated at various times after infection with 5 x io -7 i-anisomycin for I5 min. Cytoplasmic extracts were prepared and analysed on sucrose gradients in high salt to determine the proportion of ribosomes which were in initiation complexes. Shortly after infection, the number of 8oS initiation complexes in the infected cells decreased, reaching a minimum at approx. 1.75 h p.i. This correlates precisely with the time course of virus-induced inhibition of host cell protein synthesis (Helentjaris & Ehrenfeld, 1977). Fig. z shows the sucrose gradient profiles of extracts prepared at several different times after infection. By 2"75 h p.i., the number of 8oS initiation complexes again begins to increase, as virus-directed translation also commences (Fig. 2c) and when the virus cycle is nearing completion, at 4 h p.i., initation complexes again disappear (Fig. 2d). When cells were infected in the presence of I mM-guanidine, so that no virus replication occurred, the 8oS initiation complex decreased in keeping with the kinetics of inhibition of host cell protein synthesis (Fig. 2 e) and failed to reappear with further incubation. Removal of guanidine from the medium permits virus replication to proceed and 8oS initiation complexes form again (Fig. 2f). Thus, the formation of cellular 8oS initiation complexes is inhibited by poliovirus infection and the block to initiation of protein synthesis must occur at this or a preceding step. Anisomycin is an inhibitor of peptidyl transferase which has been shown to bind to the large ribosomal subunit of the 8oS ribosome (Barbacid & Vasquez, I974). The concentration of anisomycin required to protect monosomes from high salt dissociation in vivo is critical. Below 5 x IO-s M-anisomycin, almost no ribosome protection is seen. Maximum salt stabilization is observed between 5 × lO-~ M- and I × IO-8 M-anisomycin, but at higher concentrations stabilization is lost. The concentrations which give maximal 8oS ribosome stabilization to high salt are those which just achieve full inhibition of protein synthesis. It appears that when elongation is extensively slowed, a build-up of initiation complexes occurs. The number of 8oS initiation complexes observed in this way increases up to I5 min of anisomycin treatment but then remains stable with further incubation. In addition, no anisomycin-stabilized complexes were observed if the cells were treated with cycloheximide prior to or during the anisomycin treatment. The data described in this report demonstrate that the formation of cellular 8oS initiation complexes fails to occur after poliovirus infection. Recently, evidence from this laboratory has been presented (Helentjaris & Ehrenfeld, 1978) which suggests that the inhibition of host cell protein synthesis induced by poliovirus involves an m R N A recognition step of the initiation reaction. Consistent with this, ternary complex formation between GTP, eIF-2 and met-tRNAp "~ appears to be normal in infected cells (Helentjaris & Ehrenfeld, 1978 ) and in this report we show that a later step in initiation is blocked. The suggestion put forward by Lawrence & Thach (1974) that inhibition of cellular protein synthesis results from a competition between virus m R N A and cellular m R N A for some component(s) of the initiation reaction is not supported by these data, since one would not expect a decrease in initiation complexes but merely a replacement of cellular with virus complexes. On the contrary, we find that initiation complexes disappear in infected cells and only reappear subsequent to virus m R N A synthesis. Previous work has shown that fully intact and functional m R N A can be recovered from cells in which translation is completely inhibited (Ehrenfeld & Lund, 1977; Hackett et al. 1977.) The precise step for inhibition induced by poliovirus is still not determined but it now appears to be narrowed down to an event subsequent to ternary complex formation and prior to or at the 6oS junction reaction. When considered with in vitro studies showing m R N A specificity of the a~tivity of the ribosomal
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salt wash, the available data p o i n t to steps involving m R N A b i n d i n g to the 4oS initiation complex as the site of i n h i b i t i o n of host cell protein synthesis. Attempts to determine whether f o r m a t i o n of the 4oS complex c o n t a i n i n g met-tRNA~ alone or the 4oS complex c o n t a i n i n g met-tRNA~ a n d m R N A is inhibited in infected cells have so far failed. It appears that once a 4oS complex c o n t a i n i n g met-tRNA~ a n d m R N A is formed, the 6oS j u n c t i o n reaction is extremely fast, allowing n o a c c u m u l a t i o n or detection o f the preceding step (our u n p u b l i s h e d observations). This work was supported by grants from the N a t i o n a l Institute of Health (AI 12387) a n d from the N a t i o n a l Science F o u n d a t i o n , a n d by a Teacher-Scholar A w a r d from the Dreyfus F o u n d a t i o n . We are grateful to Betty Brown in this l a b o r a t o r y who prepared the reticulocyte lysate.
Departments of Biochemistry and Microbiology University of Utah Medical Center Salt Lake City, Utah 84132, U.S.A.
ELLIE EHRENFELD SUE MANIS
REFERENCES nARBAOD, M. & VASQUEZ,D. (I974). [~H]anisomycin binding to eukaryotic ribosomes. Journal of Molecular Biology 84, 603-623. BENNE,R. & HERSHEY,J. W. B. (X978). The mechanism of action of protein synthesis initiation factors from rabbit reticulocytes. Journal of Biological Chemistry 253, 3o78-3o87. EHRENEELD, E. & LUND, H. (1977). Untranslated vesicular stomatitis virus messenger RNA after poliovirus infection. Virology go, 297-308. FALVEY,g. & STAEHELIN,T. (t970). Structure and function of mammalian ribosomes. I. Isolation and characterization of active liver ribosomal subunits. Journal of Molecular Biology 53, I-9. HACKET, P. B., EGBERTS,E., BUCHWALD, I. & TRAUB, P. (1977)- Coding capacity of poly (A) + m R N A isolated
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(Received 25 September I978 )
