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Dual mechanism for the translation of subgenomic mRNA from Sindbis virus in infected and uninfected cells.

Sanz MA, Castelló A, Ventoso I, Berlanga JJ, Carrasco L - PLoS ONE (2009)

Bottom Line: Cleavage of eIF4G by poliovirus 2A(pro) does not hamper translation of subgenomic mRNA in SV infected cells, but bisection of this factor blocks subgenomic mRNA translation in uninfected cells or in cell-free systems.Notably, the correct initiation site on the subgenomic mRNA is still partially recognized when the initiation codon AUG is modified to other codons only in infected cells.Finally, immunolocalization of different eIFs reveals that eIF2 alpha and eIF4G are excluded from the foci, where viral RNA replication occurs, while eIF3, eEF2 and ribosomes concentrate in these regions.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain.

ABSTRACT
Infection of BHK cells by Sindbis virus (SV) gives rise to a profound inhibition of cellular protein synthesis, whereas translation of viral subgenomic mRNA that encodes viral structural proteins, continues for hours. To gain further knowledge on the mechanism by which this subgenomic mRNA is translated, the requirements for some initiation factors (eIFs) and for the presence of the initiator AUG were examined both in infected and in uninfected cells. To this end, BHK cells were transfected with different SV replicons or with in vitro made SV subgenomic mRNAs after inactivation of some eIFs. Specifically, eIF4G was cleaved by expression of the poliovirus 2A protease (2A(pro)) and the alpha subunit of eIF2 was inactivated by phosphorylation induced by arsenite treatment. Moreover, cellular location of these and other translation components was analyzed in BHK infected cells by confocal microscopy. Cleavage of eIF4G by poliovirus 2A(pro) does not hamper translation of subgenomic mRNA in SV infected cells, but bisection of this factor blocks subgenomic mRNA translation in uninfected cells or in cell-free systems. SV infection induces phosphorylation of eIF2alpha, a process that is increased by arsenite treatment. Under these conditions, translation of subgenomic mRNA occurs to almost the same extent as controls in the infected cells but is drastically inhibited in uninfected cells. Notably, the correct initiation site on the subgenomic mRNA is still partially recognized when the initiation codon AUG is modified to other codons only in infected cells. Finally, immunolocalization of different eIFs reveals that eIF2 alpha and eIF4G are excluded from the foci, where viral RNA replication occurs, while eIF3, eEF2 and ribosomes concentrate in these regions. These findings support the notion that canonical initiation takes place when the subgenomic mRNA is translated out of the infection context, while initiation can occur without some eIFs and even at non-AUG codons in infected cells.

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Requirement of eIF4G for translation of C+Luc sg-mRNA.Panel A. Protein synthesis and eIF4G cleavage in BHK cells. BHK cells were electroporated with 30 µg of IRES-2A mRNA or transcription buffer as a control. At 2 hpe, cells were again electroporated with 20 µg of C+Luc mRNA or cap-Luc-poly(A) or IRES-Luc-poly(A) as mRNAs control (mRNAs are schematized in the upper part of each figure). Values of luc activity obtained from the different mRNAs are represented. eIF4GI and eIF4GII integrity was analyzed by western blot analysis of extracts recovered at 2 and 4 hpe of IRES-2A. Tubulin was also analyzed to adjust the amount of each sample loaded onto the gel. Panel B. Translation directed by several mRNAs after the cleavage of eIF4G in HeLa S3 extracts. Translation was carried out in Hela S3 extracts pre-treated with purified MBP-2A or MBP proteins. Luc production was determined by measuring luc activity from each translation mixture and the integrity of eIF4GI and eIF4GII was analyzed by western blotting. Panel C. Effect of eIF4G cleavage on protein synthesis in BHK cells transfected with different SV replicons. BHK cells were electroporated with 20 µg of rep C+Luc mRNA (Schematized in the figure). At 2 hpe, cells were again electroporated with 30 µg of IRES-2A mRNA or transcription buffer. At 1, 3 and 5 hpe of IRES-2A mRNA, cell cultures were harvested and luc activity was measured or examined by western blotting with anti-luc antibodies (left panel). eIF4GI and eIF4GII integrity was tested by western-blot analysis. Tubulin was also analyzed to adjust the amount of each sample loaded onto the gel (right panel). α-Tub: α-Tubulin; luc: luciferase; C-t: C-terminal cleavage product of eIF4G; N-t: N-terminal cleavage product of eIF4G.
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pone-0004772-g001: Requirement of eIF4G for translation of C+Luc sg-mRNA.Panel A. Protein synthesis and eIF4G cleavage in BHK cells. BHK cells were electroporated with 30 µg of IRES-2A mRNA or transcription buffer as a control. At 2 hpe, cells were again electroporated with 20 µg of C+Luc mRNA or cap-Luc-poly(A) or IRES-Luc-poly(A) as mRNAs control (mRNAs are schematized in the upper part of each figure). Values of luc activity obtained from the different mRNAs are represented. eIF4GI and eIF4GII integrity was analyzed by western blot analysis of extracts recovered at 2 and 4 hpe of IRES-2A. Tubulin was also analyzed to adjust the amount of each sample loaded onto the gel. Panel B. Translation directed by several mRNAs after the cleavage of eIF4G in HeLa S3 extracts. Translation was carried out in Hela S3 extracts pre-treated with purified MBP-2A or MBP proteins. Luc production was determined by measuring luc activity from each translation mixture and the integrity of eIF4GI and eIF4GII was analyzed by western blotting. Panel C. Effect of eIF4G cleavage on protein synthesis in BHK cells transfected with different SV replicons. BHK cells were electroporated with 20 µg of rep C+Luc mRNA (Schematized in the figure). At 2 hpe, cells were again electroporated with 30 µg of IRES-2A mRNA or transcription buffer. At 1, 3 and 5 hpe of IRES-2A mRNA, cell cultures were harvested and luc activity was measured or examined by western blotting with anti-luc antibodies (left panel). eIF4GI and eIF4GII integrity was tested by western-blot analysis. Tubulin was also analyzed to adjust the amount of each sample loaded onto the gel (right panel). α-Tub: α-Tubulin; luc: luciferase; C-t: C-terminal cleavage product of eIF4G; N-t: N-terminal cleavage product of eIF4G.

Mentions: We previously reported that SV sg-mRNA can be translated when eIF4G is cleaved by viral proteases [14]. Initially two types of mRNAs were generated by in vitro transcription, that is, sg-C+Luc mRNA from pT7 C+Luc plasmid [22] and the replicon rep C+Luc [22]. After transfection, rep C+Luc gives rise to the sg-C+Luc mRNA using the SV transcription machinery. In the case of sg-C+Luc mRNA from pT7 C+Luc plasmid, after electroporation, sg-mRNA will be translated in uninfected cells and whereas in the other it will be translated in an environment that resembles the infected cells because there is viral replication and transcription. Translation of C+Luc mRNA renders a fusion protein that releases C protein and luciferase (luc) through proteolytic activity of C protein. To induce cleavage of translation initiation factor eIF4G, PV 2Apro was expressed on electroporation of synthesized IRES-2A mRNA. This mRNA contains the EMCV IRES followed by the PV 2Apro gene (IRES-2A) [23], [24]. BHK cells were then electroporated with IRES-2A mRNA or transcription buffer as control. At 2 hours post-electroporation (hpe), cells were once again electroporated with in vitro transcribed C+Luc mRNA using cap-Luc-poly(A) or IRES-Luc-poly(A) mRNAs as controls (Fig. 1A). eIF4GI and eIF4GII are both already proteolyzed by 2Apro at 2 hpe (Fig 1A). Under these conditions cap-Luc-poly(A) mRNA translation was strongly inhibited at 4 hpe, suggesting that cap- and poly(A)-dependent translation was hampered in IRES-2A electroporated cells (Fig 1A). A similar situation occurs with C+Luc mRNA translation, which is also deeply inhibited after eIF4G cleavage (Fig 1A). These results support the idea that SV sg-mRNA is translated by a cap- and poly(A)-dependent mechanism in uninfected BHK cells. mRNAs that contain EMCV IRES are able to drive translation even when eIF4G is cleaved by 2Apro [24]. Luc activity measured in cells electroporated with IRES-Luc increased consistently throughout the experimental period, irrespective of 2Apro-expression and eIF4G cleavage (Fig 1A). Similar findings were obtained in HeLa cells (data not shown). In addition, the effect of eIF4G cleavage on the translation of C+Luc mRNA was assayed in HeLa S3 cell extracts. To achieve eIF4G cleavage before mRNA addition, 1 µg of purified MBP-2Apro was added to extracts for 30 min at 30°C, followed by translation of the different mRNAs. Luc activity was then estimated (Fig 1B, upper panel), along with hydrolysis of eIF4G (Fig 1B, lower panel). As with uninfected cells, cleavage of eIF4G strongly inhibits translation both of cap-Luc-poly(A) and C+Luc mRNAs whereas protein synthesis directed by IRES-Luc mRNA was stimulated.


Dual mechanism for the translation of subgenomic mRNA from Sindbis virus in infected and uninfected cells.

Sanz MA, Castelló A, Ventoso I, Berlanga JJ, Carrasco L - PLoS ONE (2009)

Requirement of eIF4G for translation of C+Luc sg-mRNA.Panel A. Protein synthesis and eIF4G cleavage in BHK cells. BHK cells were electroporated with 30 µg of IRES-2A mRNA or transcription buffer as a control. At 2 hpe, cells were again electroporated with 20 µg of C+Luc mRNA or cap-Luc-poly(A) or IRES-Luc-poly(A) as mRNAs control (mRNAs are schematized in the upper part of each figure). Values of luc activity obtained from the different mRNAs are represented. eIF4GI and eIF4GII integrity was analyzed by western blot analysis of extracts recovered at 2 and 4 hpe of IRES-2A. Tubulin was also analyzed to adjust the amount of each sample loaded onto the gel. Panel B. Translation directed by several mRNAs after the cleavage of eIF4G in HeLa S3 extracts. Translation was carried out in Hela S3 extracts pre-treated with purified MBP-2A or MBP proteins. Luc production was determined by measuring luc activity from each translation mixture and the integrity of eIF4GI and eIF4GII was analyzed by western blotting. Panel C. Effect of eIF4G cleavage on protein synthesis in BHK cells transfected with different SV replicons. BHK cells were electroporated with 20 µg of rep C+Luc mRNA (Schematized in the figure). At 2 hpe, cells were again electroporated with 30 µg of IRES-2A mRNA or transcription buffer. At 1, 3 and 5 hpe of IRES-2A mRNA, cell cultures were harvested and luc activity was measured or examined by western blotting with anti-luc antibodies (left panel). eIF4GI and eIF4GII integrity was tested by western-blot analysis. Tubulin was also analyzed to adjust the amount of each sample loaded onto the gel (right panel). α-Tub: α-Tubulin; luc: luciferase; C-t: C-terminal cleavage product of eIF4G; N-t: N-terminal cleavage product of eIF4G.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2651626&req=5

pone-0004772-g001: Requirement of eIF4G for translation of C+Luc sg-mRNA.Panel A. Protein synthesis and eIF4G cleavage in BHK cells. BHK cells were electroporated with 30 µg of IRES-2A mRNA or transcription buffer as a control. At 2 hpe, cells were again electroporated with 20 µg of C+Luc mRNA or cap-Luc-poly(A) or IRES-Luc-poly(A) as mRNAs control (mRNAs are schematized in the upper part of each figure). Values of luc activity obtained from the different mRNAs are represented. eIF4GI and eIF4GII integrity was analyzed by western blot analysis of extracts recovered at 2 and 4 hpe of IRES-2A. Tubulin was also analyzed to adjust the amount of each sample loaded onto the gel. Panel B. Translation directed by several mRNAs after the cleavage of eIF4G in HeLa S3 extracts. Translation was carried out in Hela S3 extracts pre-treated with purified MBP-2A or MBP proteins. Luc production was determined by measuring luc activity from each translation mixture and the integrity of eIF4GI and eIF4GII was analyzed by western blotting. Panel C. Effect of eIF4G cleavage on protein synthesis in BHK cells transfected with different SV replicons. BHK cells were electroporated with 20 µg of rep C+Luc mRNA (Schematized in the figure). At 2 hpe, cells were again electroporated with 30 µg of IRES-2A mRNA or transcription buffer. At 1, 3 and 5 hpe of IRES-2A mRNA, cell cultures were harvested and luc activity was measured or examined by western blotting with anti-luc antibodies (left panel). eIF4GI and eIF4GII integrity was tested by western-blot analysis. Tubulin was also analyzed to adjust the amount of each sample loaded onto the gel (right panel). α-Tub: α-Tubulin; luc: luciferase; C-t: C-terminal cleavage product of eIF4G; N-t: N-terminal cleavage product of eIF4G.
Mentions: We previously reported that SV sg-mRNA can be translated when eIF4G is cleaved by viral proteases [14]. Initially two types of mRNAs were generated by in vitro transcription, that is, sg-C+Luc mRNA from pT7 C+Luc plasmid [22] and the replicon rep C+Luc [22]. After transfection, rep C+Luc gives rise to the sg-C+Luc mRNA using the SV transcription machinery. In the case of sg-C+Luc mRNA from pT7 C+Luc plasmid, after electroporation, sg-mRNA will be translated in uninfected cells and whereas in the other it will be translated in an environment that resembles the infected cells because there is viral replication and transcription. Translation of C+Luc mRNA renders a fusion protein that releases C protein and luciferase (luc) through proteolytic activity of C protein. To induce cleavage of translation initiation factor eIF4G, PV 2Apro was expressed on electroporation of synthesized IRES-2A mRNA. This mRNA contains the EMCV IRES followed by the PV 2Apro gene (IRES-2A) [23], [24]. BHK cells were then electroporated with IRES-2A mRNA or transcription buffer as control. At 2 hours post-electroporation (hpe), cells were once again electroporated with in vitro transcribed C+Luc mRNA using cap-Luc-poly(A) or IRES-Luc-poly(A) mRNAs as controls (Fig. 1A). eIF4GI and eIF4GII are both already proteolyzed by 2Apro at 2 hpe (Fig 1A). Under these conditions cap-Luc-poly(A) mRNA translation was strongly inhibited at 4 hpe, suggesting that cap- and poly(A)-dependent translation was hampered in IRES-2A electroporated cells (Fig 1A). A similar situation occurs with C+Luc mRNA translation, which is also deeply inhibited after eIF4G cleavage (Fig 1A). These results support the idea that SV sg-mRNA is translated by a cap- and poly(A)-dependent mechanism in uninfected BHK cells. mRNAs that contain EMCV IRES are able to drive translation even when eIF4G is cleaved by 2Apro [24]. Luc activity measured in cells electroporated with IRES-Luc increased consistently throughout the experimental period, irrespective of 2Apro-expression and eIF4G cleavage (Fig 1A). Similar findings were obtained in HeLa cells (data not shown). In addition, the effect of eIF4G cleavage on the translation of C+Luc mRNA was assayed in HeLa S3 cell extracts. To achieve eIF4G cleavage before mRNA addition, 1 µg of purified MBP-2Apro was added to extracts for 30 min at 30°C, followed by translation of the different mRNAs. Luc activity was then estimated (Fig 1B, upper panel), along with hydrolysis of eIF4G (Fig 1B, lower panel). As with uninfected cells, cleavage of eIF4G strongly inhibits translation both of cap-Luc-poly(A) and C+Luc mRNAs whereas protein synthesis directed by IRES-Luc mRNA was stimulated.

Bottom Line: Cleavage of eIF4G by poliovirus 2A(pro) does not hamper translation of subgenomic mRNA in SV infected cells, but bisection of this factor blocks subgenomic mRNA translation in uninfected cells or in cell-free systems.Notably, the correct initiation site on the subgenomic mRNA is still partially recognized when the initiation codon AUG is modified to other codons only in infected cells.Finally, immunolocalization of different eIFs reveals that eIF2 alpha and eIF4G are excluded from the foci, where viral RNA replication occurs, while eIF3, eEF2 and ribosomes concentrate in these regions.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain.

ABSTRACT
Infection of BHK cells by Sindbis virus (SV) gives rise to a profound inhibition of cellular protein synthesis, whereas translation of viral subgenomic mRNA that encodes viral structural proteins, continues for hours. To gain further knowledge on the mechanism by which this subgenomic mRNA is translated, the requirements for some initiation factors (eIFs) and for the presence of the initiator AUG were examined both in infected and in uninfected cells. To this end, BHK cells were transfected with different SV replicons or with in vitro made SV subgenomic mRNAs after inactivation of some eIFs. Specifically, eIF4G was cleaved by expression of the poliovirus 2A protease (2A(pro)) and the alpha subunit of eIF2 was inactivated by phosphorylation induced by arsenite treatment. Moreover, cellular location of these and other translation components was analyzed in BHK infected cells by confocal microscopy. Cleavage of eIF4G by poliovirus 2A(pro) does not hamper translation of subgenomic mRNA in SV infected cells, but bisection of this factor blocks subgenomic mRNA translation in uninfected cells or in cell-free systems. SV infection induces phosphorylation of eIF2alpha, a process that is increased by arsenite treatment. Under these conditions, translation of subgenomic mRNA occurs to almost the same extent as controls in the infected cells but is drastically inhibited in uninfected cells. Notably, the correct initiation site on the subgenomic mRNA is still partially recognized when the initiation codon AUG is modified to other codons only in infected cells. Finally, immunolocalization of different eIFs reveals that eIF2 alpha and eIF4G are excluded from the foci, where viral RNA replication occurs, while eIF3, eEF2 and ribosomes concentrate in these regions. These findings support the notion that canonical initiation takes place when the subgenomic mRNA is translated out of the infection context, while initiation can occur without some eIFs and even at non-AUG codons in infected cells.

Show MeSH
Related in: MedlinePlus