- Email: [email protected]
Poliovirus-induced inhibition of host-cell protein synthesis
Cell, Vol. 28, 435-436,
March
1982,
Copyright
0 1982
by MIT
Poliovirus-Induced Inhibition Host-Cell Protein Synthesis Ellie Ehrenfeld Department of Biochemistry and Department Cellular, Viral and Molecular Biology University of Utah School of Medicine Salt Lake City, Utah 84132
of
With the earliest molecular biology studies of poliovirus, 20 years ago, came the observation that infection of HeLa cells with this virus results in a rapid and marked inhibition of host-cell protein synthesis. Analysis of the infected cells indicated that the block to cellular protein synthesis is at the initiation step of translation, and that after infection ribosomal initiation complexes fail to form on cellular mRNAs. Despite this clue to the site of action of the inhibition, little progress was made in understanding the mechanism until recently. One obstacle to progress in this field was the assumption made by most investigators that infection of all susceptible cells with all picornaviruses would result in the same display of host-cell shutoff, caused by the same virus-induced mechanism. Surprisingly, however, it now appears that an undefined but rather specific virus-host cell interaction is required. For example, encephalomyocardi!is virus and poliovirus infections of HeLa cells result in different responses by the host with regard to alterations in the cell’s protein-synthesizing machinery (Jen et al., J. Virol. 35, 150-l 58, 1980); and mengovirus produces quite different responses by the host, depending on whether L cells or HeLa cells are infected (Otto and Lucas-Lenard, J. Gen. Virol. 50, 293-307, 1980). The best characterized virus-host cell interaction resulting in specific inhibition of cellular protein synthesis is poliovirus infection of HeLa cells. Examination of the activities of various subcellular fractions of infected cells in vitro implicated some component(s) of the ribosomal salt wash (RSW) as being responsible for the preferential translation of poliovirus RNA over cellular mRNA. Reconstitution of a fractionated protein-synthesizing system prepared with various components from either infected or uninfected cells demonstrated that the RSW from infected cells was incapable of supporting initiation of synthesis of cellular polypeptides, although it did function to support translation of viral mRNA. Two different approaches were taken to identify the “defective” component in the infected-cell RSW. Rose et al. (PNAS 75, 2732-2736, 1978) prepared a protein-synthesizing extract from poliovirus-infected HeLa cells, and showed that as expected, the infected-cell extract was inactive for translation of vesicular stomatitis virus mRNAs, used as a prototype of a normal cellular mRNA. They then tested the ability of purified individual initiation factors from rabbit reticulocytes to restore translational activity to the infected-cell rysate, and found restoring activity in the elF-46 preparation. By this means they concluded
of
that elF-4B was the defective component in poliovirusinfected cells. A somewhat more tedious biochemical approach was taken by Helentjaris et al. (JBC 254, 10973-l 0978, 19791, who attempted to purify individual initiation factors directly from both uninfected and poliovirus-infected HeLa cells, and then to test the activities of individual factors in a fractionated system translating globin mRNA. These investigators found that the RSW from poliovirus-infected cells contained no functional elF-3 activity, although elF-4B activity was present. The resolution of this apparent discrepancy came from work that resulted in the description of a previously unknown factor, the capbinding protein (CBP), which Sonenberg and Shatkin identified as a component of the RSW from rabbit reticulocytes, and which copurified with both elF-4B and elF-3 (Sonenberg et al., PNAS 75, 4843-4847, 1978). The CBP was identified in these preparations as a polypeptide (24 kd) that could be chemically crosslinked to the cap group of oxidized, capped mRNAs. Despite the initial report of restoring activity in elF-4B, all subsequent isolations of the CBP have used crude elF-3 preparations as starting material, since at low ionic strength the CBP appears to bind to or associate with elF-3. Shortly following the initial description of the CBP, the activity that restored vesicular stomatitis virus mRNA translational activity to a poliovirus-infected-cell extract was purified from the rabbit reticulocyte RSW, and was shown to be identical to the CBP, although the activity of the purified CBP was very unstable (Trachsel et al., PNAS 77, 770-774, 1980). The untested but logical conclusion from the above studies is that poliovirus infection somehow resulted in an inactivation of the CBP. The willingness to accept this explanation is strongly supported by the fact that poliovirus mRNA is not capped. Thus the hypothesis that this virus induced the inactivation of a protein required for translation of capped mRNAs is extremely attractive, since it provides a reasonable means by which the host translational machinery could be modified so as to discriminate between host (capped) and viral (uncapped) mRNAs. Obviously, the missing link lies in the fact that no one has directly isolated the CBP from poliovirus-infected cells and shown it to be defective or modified in any way. Indeed, the first preliminary characterization of the CBP from poliovirus-infected HeLa cells showed it to be present in the RSW and to copurify partially with the fraction of the RSW that contains elF-4B and elF-3 (Hansen and Ehrenfeld, J. Virol. 38, 438-445, 1981). However, when these fractions were sedimented through sucrose gradients so as to separate elF-3 from elF-4B, most of the CBP in the RSW from uninfected HeLa cells cosedimented with elF-3 antigens; all of the CBP in the RSW from poliovirus-infected HeLa cells remained near the top of the gradient (Hansen et al., J. Virol., in press). This difference in sedimentation behavior is the first and only difference thus far detected
Cdl 436
in the CBPs from infected and uninfected cells, and it still remains to be shown that the difference in sedimentation properties is related to any functional differences in activity of the two preparations. Furthermore, failure to find CBP associated with elF-3 in infected cells could be due to alterations either in the CBP per se, or to alterations of some component of elF-3. Thus a rigorous test of the “defective-CBP model” is not yet complete. Two important properties have been characterized and attributed to purified CBP. One is the preferential ability to stimulate translation of capped mRNAs, but not naturally uncapped mRNAs such as encephalomyocarditis virus or satellite tobacco necrosis virus RNA, in protein-synthesizing extracts from uninfected HeLa cells (Sonenberg et al., Nature 285, 331-333, 1980). The other is the ability to restore translation of capped vesicular stomatitis virus mRNA in an extract from poliovirus-infected cells. Tahara et al. (JBC 256, 7691-7894, 1981) have recently shown that a rapidly sedimenting form of the CBP, complexed with other unidentified polypeptides, is responsible for the restoring activity, whereas both slow- and fast-sedimenting forms of the CBP show preferential stimulation of capped over uncapped mRNAs. Whether these different forms of the CBP are related to those described above, one of which was associated with elF-3 antigens, is not clear, because different methods of isolation were used in the two different studies. Additional complications in interpreting the role of the CBP in the shutoff of host-cell protein synthesis by poliovirus are the description by Sonenberg of at least two additional cap-binding proteins whose interaction with the capped end of mRNA is ATP-, Mg’+dependent, and the finding by Trachsel et al. that monoclonal antibodies directed against reticulocyte cap-binding proteins react with several larger polypeptides that share tryptic peptides with the 24 kd CBP. This suggests the possibility of either functional or artifactual proteolytic cleavage in the generation of the CBP. Thus the functional form of CBP in vivo is not yet understood, and the relation of these different forms to the alteration in the poliovirus-infected cell is still to be determined. Although the infected cell is unable to translate capped, cellular mRNAs, translation of poliovirus mRNA occurs efficiently. The mechanism of this translation event is not understood. The AUG initiator codon that begins the coding region for the large poliovirus polyprotein is preceded by approximately 740 nucleotides at the 5’ end of poliovirus RNA, and this untranslated region includes eight AUG codons. Kozak has postulated that the usual sequence of events in eucaryotic ribosome binding to mRNA involves an initial recognition of the capped 5’ terminus and migration of the ribosome in the 3’ direction, with subsequent initiation of translation when the first AUG is encountered, perhaps aided by an additional neighboring sequence. In the case of poliovirus mRNA, the mechanism of initiation of translation may be quite
different, and may depend on other features of the RNA structure or sequence. The RSW from poliovirus-infected cells actively stimulates initiation of translation of poliovirus RNA in vitro, in reticulocyte lysates as well as in uninfected or infected HeLa cell extracts. As expected, these RSW preparations do not stimulate translation of capped mRNAs in the same system (see, for example, Brown and Ehrenfeld, Virology 703, 327-339, 1980). The specificity of this stimulation of translation of poliovirus mRNA is not understood, but the simple absence of a cap group is insufficient for recognition by the poliovirus-infected-cell RSW. For example, translation of chemically uncapped @-eliminated) vesicular stomatitis virus mRNA is not supported by the RSW from poliovirus-infected cells. Whether virus-induced changes in initiation-factor activity occur to aid in the specific recognition of this mRNA, and what structural features of the viral mRNA are required for translation to occur with the altered initiation factors in the infected cell, remain to be determined. It is apparent that infection with most viruses requires some form of subordination of the host’s protein-synthesizing machinery to allow for efficient translation of viral proteins. It is also apparent that numerous animal viruses can accomplish this without specific interference with the translational mechanism. However, the occurrence of a virus-induced modification of the host such that its translation specificity is switched from a preference for capped mRNAs to a preference for uncapped mRNAs appears not to be unique for poliovirus infection of HeLa cells. A similar phenomenon appears to occur during reovirus infection of L cells (Skup et al., JMB 757, 35-55, 1981). These results were surprising, since reovirus particles transcribe capped and methylated mRNAs in vitro, and thus would appear to require utilization of a capdependent translation mechanism for synthesis of viral proteins. Although parental, infecting virus particles do produce capped mRNAs, in infected L cells, progeny subviral particles that produce the bulk of the mRNA during infection produce uncapped mRNAs terminating in B’pGpC.. . . L-cell-free extracts prepared late in reovirus infection preferentially translate uncapped mRNAs. Although the similarity of the modification induced in reovirus-infected L cells to that observed in poliovirus-infected HeLa cells is not known, and the role of the CBP has not yet been evaluated, a gradual transition to cap-independent translation does occur. The variation in occurrence of alterations in translational machinery, depending on the virus and the host cell, leads one to speculate that the observed biochemical changes in HeLa cells following poliovirus infection may not be essential for virus replication. Indeed, it will be of much interest to determine whether the host-cell components that are apparently inactivated for cellular mRNA translation are in fact utilized by the virus for some other aspect of its replication.
March
1982,
Copyright
0 1982
by MIT
Poliovirus-Induced Inhibition Host-Cell Protein Synthesis Ellie Ehrenfeld Department of Biochemistry and Department Cellular, Viral and Molecular Biology University of Utah School of Medicine Salt Lake City, Utah 84132
of
With the earliest molecular biology studies of poliovirus, 20 years ago, came the observation that infection of HeLa cells with this virus results in a rapid and marked inhibition of host-cell protein synthesis. Analysis of the infected cells indicated that the block to cellular protein synthesis is at the initiation step of translation, and that after infection ribosomal initiation complexes fail to form on cellular mRNAs. Despite this clue to the site of action of the inhibition, little progress was made in understanding the mechanism until recently. One obstacle to progress in this field was the assumption made by most investigators that infection of all susceptible cells with all picornaviruses would result in the same display of host-cell shutoff, caused by the same virus-induced mechanism. Surprisingly, however, it now appears that an undefined but rather specific virus-host cell interaction is required. For example, encephalomyocardi!is virus and poliovirus infections of HeLa cells result in different responses by the host with regard to alterations in the cell’s protein-synthesizing machinery (Jen et al., J. Virol. 35, 150-l 58, 1980); and mengovirus produces quite different responses by the host, depending on whether L cells or HeLa cells are infected (Otto and Lucas-Lenard, J. Gen. Virol. 50, 293-307, 1980). The best characterized virus-host cell interaction resulting in specific inhibition of cellular protein synthesis is poliovirus infection of HeLa cells. Examination of the activities of various subcellular fractions of infected cells in vitro implicated some component(s) of the ribosomal salt wash (RSW) as being responsible for the preferential translation of poliovirus RNA over cellular mRNA. Reconstitution of a fractionated protein-synthesizing system prepared with various components from either infected or uninfected cells demonstrated that the RSW from infected cells was incapable of supporting initiation of synthesis of cellular polypeptides, although it did function to support translation of viral mRNA. Two different approaches were taken to identify the “defective” component in the infected-cell RSW. Rose et al. (PNAS 75, 2732-2736, 1978) prepared a protein-synthesizing extract from poliovirus-infected HeLa cells, and showed that as expected, the infected-cell extract was inactive for translation of vesicular stomatitis virus mRNAs, used as a prototype of a normal cellular mRNA. They then tested the ability of purified individual initiation factors from rabbit reticulocytes to restore translational activity to the infected-cell rysate, and found restoring activity in the elF-46 preparation. By this means they concluded
of
that elF-4B was the defective component in poliovirusinfected cells. A somewhat more tedious biochemical approach was taken by Helentjaris et al. (JBC 254, 10973-l 0978, 19791, who attempted to purify individual initiation factors directly from both uninfected and poliovirus-infected HeLa cells, and then to test the activities of individual factors in a fractionated system translating globin mRNA. These investigators found that the RSW from poliovirus-infected cells contained no functional elF-3 activity, although elF-4B activity was present. The resolution of this apparent discrepancy came from work that resulted in the description of a previously unknown factor, the capbinding protein (CBP), which Sonenberg and Shatkin identified as a component of the RSW from rabbit reticulocytes, and which copurified with both elF-4B and elF-3 (Sonenberg et al., PNAS 75, 4843-4847, 1978). The CBP was identified in these preparations as a polypeptide (24 kd) that could be chemically crosslinked to the cap group of oxidized, capped mRNAs. Despite the initial report of restoring activity in elF-4B, all subsequent isolations of the CBP have used crude elF-3 preparations as starting material, since at low ionic strength the CBP appears to bind to or associate with elF-3. Shortly following the initial description of the CBP, the activity that restored vesicular stomatitis virus mRNA translational activity to a poliovirus-infected-cell extract was purified from the rabbit reticulocyte RSW, and was shown to be identical to the CBP, although the activity of the purified CBP was very unstable (Trachsel et al., PNAS 77, 770-774, 1980). The untested but logical conclusion from the above studies is that poliovirus infection somehow resulted in an inactivation of the CBP. The willingness to accept this explanation is strongly supported by the fact that poliovirus mRNA is not capped. Thus the hypothesis that this virus induced the inactivation of a protein required for translation of capped mRNAs is extremely attractive, since it provides a reasonable means by which the host translational machinery could be modified so as to discriminate between host (capped) and viral (uncapped) mRNAs. Obviously, the missing link lies in the fact that no one has directly isolated the CBP from poliovirus-infected cells and shown it to be defective or modified in any way. Indeed, the first preliminary characterization of the CBP from poliovirus-infected HeLa cells showed it to be present in the RSW and to copurify partially with the fraction of the RSW that contains elF-4B and elF-3 (Hansen and Ehrenfeld, J. Virol. 38, 438-445, 1981). However, when these fractions were sedimented through sucrose gradients so as to separate elF-3 from elF-4B, most of the CBP in the RSW from uninfected HeLa cells cosedimented with elF-3 antigens; all of the CBP in the RSW from poliovirus-infected HeLa cells remained near the top of the gradient (Hansen et al., J. Virol., in press). This difference in sedimentation behavior is the first and only difference thus far detected
Cdl 436
in the CBPs from infected and uninfected cells, and it still remains to be shown that the difference in sedimentation properties is related to any functional differences in activity of the two preparations. Furthermore, failure to find CBP associated with elF-3 in infected cells could be due to alterations either in the CBP per se, or to alterations of some component of elF-3. Thus a rigorous test of the “defective-CBP model” is not yet complete. Two important properties have been characterized and attributed to purified CBP. One is the preferential ability to stimulate translation of capped mRNAs, but not naturally uncapped mRNAs such as encephalomyocarditis virus or satellite tobacco necrosis virus RNA, in protein-synthesizing extracts from uninfected HeLa cells (Sonenberg et al., Nature 285, 331-333, 1980). The other is the ability to restore translation of capped vesicular stomatitis virus mRNA in an extract from poliovirus-infected cells. Tahara et al. (JBC 256, 7691-7894, 1981) have recently shown that a rapidly sedimenting form of the CBP, complexed with other unidentified polypeptides, is responsible for the restoring activity, whereas both slow- and fast-sedimenting forms of the CBP show preferential stimulation of capped over uncapped mRNAs. Whether these different forms of the CBP are related to those described above, one of which was associated with elF-3 antigens, is not clear, because different methods of isolation were used in the two different studies. Additional complications in interpreting the role of the CBP in the shutoff of host-cell protein synthesis by poliovirus are the description by Sonenberg of at least two additional cap-binding proteins whose interaction with the capped end of mRNA is ATP-, Mg’+dependent, and the finding by Trachsel et al. that monoclonal antibodies directed against reticulocyte cap-binding proteins react with several larger polypeptides that share tryptic peptides with the 24 kd CBP. This suggests the possibility of either functional or artifactual proteolytic cleavage in the generation of the CBP. Thus the functional form of CBP in vivo is not yet understood, and the relation of these different forms to the alteration in the poliovirus-infected cell is still to be determined. Although the infected cell is unable to translate capped, cellular mRNAs, translation of poliovirus mRNA occurs efficiently. The mechanism of this translation event is not understood. The AUG initiator codon that begins the coding region for the large poliovirus polyprotein is preceded by approximately 740 nucleotides at the 5’ end of poliovirus RNA, and this untranslated region includes eight AUG codons. Kozak has postulated that the usual sequence of events in eucaryotic ribosome binding to mRNA involves an initial recognition of the capped 5’ terminus and migration of the ribosome in the 3’ direction, with subsequent initiation of translation when the first AUG is encountered, perhaps aided by an additional neighboring sequence. In the case of poliovirus mRNA, the mechanism of initiation of translation may be quite
different, and may depend on other features of the RNA structure or sequence. The RSW from poliovirus-infected cells actively stimulates initiation of translation of poliovirus RNA in vitro, in reticulocyte lysates as well as in uninfected or infected HeLa cell extracts. As expected, these RSW preparations do not stimulate translation of capped mRNAs in the same system (see, for example, Brown and Ehrenfeld, Virology 703, 327-339, 1980). The specificity of this stimulation of translation of poliovirus mRNA is not understood, but the simple absence of a cap group is insufficient for recognition by the poliovirus-infected-cell RSW. For example, translation of chemically uncapped @-eliminated) vesicular stomatitis virus mRNA is not supported by the RSW from poliovirus-infected cells. Whether virus-induced changes in initiation-factor activity occur to aid in the specific recognition of this mRNA, and what structural features of the viral mRNA are required for translation to occur with the altered initiation factors in the infected cell, remain to be determined. It is apparent that infection with most viruses requires some form of subordination of the host’s protein-synthesizing machinery to allow for efficient translation of viral proteins. It is also apparent that numerous animal viruses can accomplish this without specific interference with the translational mechanism. However, the occurrence of a virus-induced modification of the host such that its translation specificity is switched from a preference for capped mRNAs to a preference for uncapped mRNAs appears not to be unique for poliovirus infection of HeLa cells. A similar phenomenon appears to occur during reovirus infection of L cells (Skup et al., JMB 757, 35-55, 1981). These results were surprising, since reovirus particles transcribe capped and methylated mRNAs in vitro, and thus would appear to require utilization of a capdependent translation mechanism for synthesis of viral proteins. Although parental, infecting virus particles do produce capped mRNAs, in infected L cells, progeny subviral particles that produce the bulk of the mRNA during infection produce uncapped mRNAs terminating in B’pGpC.. . . L-cell-free extracts prepared late in reovirus infection preferentially translate uncapped mRNAs. Although the similarity of the modification induced in reovirus-infected L cells to that observed in poliovirus-infected HeLa cells is not known, and the role of the CBP has not yet been evaluated, a gradual transition to cap-independent translation does occur. The variation in occurrence of alterations in translational machinery, depending on the virus and the host cell, leads one to speculate that the observed biochemical changes in HeLa cells following poliovirus infection may not be essential for virus replication. Indeed, it will be of much interest to determine whether the host-cell components that are apparently inactivated for cellular mRNA translation are in fact utilized by the virus for some other aspect of its replication.