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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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Effect of arsenite on translation of SV sg-mRNA.Panel A. Effect of arsenite on SV infection. Uninfected or SV-infected BHK cells (10 pfu/cell) were treated with different concentrations of sodium arsenite for 30 min at 4 hpi. Proteins were then labelled with [35S]Met-Cys in presence of the same concentrations of sodium arsenite for 30 min. Samples were collected in the appropriate sample buffer and processed by SDS-PAGE, fluorography and autoradiography. Relative densitometry values obtained from capsid or actin bands are indicated below each lane. Panel B. Translation in SV-replicating BHK cells treated with arsenite. BHK cells were electroplated with 20 µg of rep C+Luc mRNA or transcription buffer as control. At 4 hpe cultures were treated with sodium arsenite for 30 min and then half of the cultures were labelled with [35S]Met-Cys in presence or absence of arsenite for 30 min and the other half treated with arsenite only. Radioactive samples were examined by SDS-PAGE, fluorography and autoradiography (upper panel) and non-radioactive samples were used to analyze phosphorylation of eIF2α by isoelectric focusing (lower panel). Panel C. Translation of different mRNAs transfected in BHK cells treated with arsenite. BHK cells were electroporated with 20 µg of cap-Luc-poly(A) or C+Luc mRNA and at 30 mpe treated with different concentrations of arsenite for 1 hour. Half of the cultures were processed to measure luc activity (upper panel) and the other half to detect phosphorylation of eIF2 α by isoelectric focusing (lower panel). Panel D. Translation of sg-mRNA synthesized in the nucleus of BHK cells treated with arsenite. BHK cells were transfected with pcDNA-Luc or pcDNA C+Luc plasmids and, at 18 hpt, treated or not with arsenite for 30 min. Next, cultures were labelled with [35S]Met-Cys in presence or absence of arsenite for 30 min. One quarter of the samples were directly processed by SDS-PAGE, fluorography and autoradiography (upper panel). The remaining samples were first immunoprecipitated with anti-luc antibodies and then processed by SDS-PAGE, fluorography and autoradiography (lower panel).
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pone-0004772-g002: Effect of arsenite on translation of SV sg-mRNA.Panel A. Effect of arsenite on SV infection. Uninfected or SV-infected BHK cells (10 pfu/cell) were treated with different concentrations of sodium arsenite for 30 min at 4 hpi. Proteins were then labelled with [35S]Met-Cys in presence of the same concentrations of sodium arsenite for 30 min. Samples were collected in the appropriate sample buffer and processed by SDS-PAGE, fluorography and autoradiography. Relative densitometry values obtained from capsid or actin bands are indicated below each lane. Panel B. Translation in SV-replicating BHK cells treated with arsenite. BHK cells were electroplated with 20 µg of rep C+Luc mRNA or transcription buffer as control. At 4 hpe cultures were treated with sodium arsenite for 30 min and then half of the cultures were labelled with [35S]Met-Cys in presence or absence of arsenite for 30 min and the other half treated with arsenite only. Radioactive samples were examined by SDS-PAGE, fluorography and autoradiography (upper panel) and non-radioactive samples were used to analyze phosphorylation of eIF2α by isoelectric focusing (lower panel). Panel C. Translation of different mRNAs transfected in BHK cells treated with arsenite. BHK cells were electroporated with 20 µg of cap-Luc-poly(A) or C+Luc mRNA and at 30 mpe treated with different concentrations of arsenite for 1 hour. Half of the cultures were processed to measure luc activity (upper panel) and the other half to detect phosphorylation of eIF2 α by isoelectric focusing (lower panel). Panel D. Translation of sg-mRNA synthesized in the nucleus of BHK cells treated with arsenite. BHK cells were transfected with pcDNA-Luc or pcDNA C+Luc plasmids and, at 18 hpt, treated or not with arsenite for 30 min. Next, cultures were labelled with [35S]Met-Cys in presence or absence of arsenite for 30 min. One quarter of the samples were directly processed by SDS-PAGE, fluorography and autoradiography (upper panel). The remaining samples were first immunoprecipitated with anti-luc antibodies and then processed by SDS-PAGE, fluorography and autoradiography (lower panel).

Mentions: Arsenite is widely used to induce phosphorylation of eIF2α, leading to the inhibition of translation [13], [25], [26]. Furthermore, culture cells infected by SV exhibit high levels of phosphorylated eIF2α at times when viral structural proteins are being synthesized [7], [9]. We therefore compared the eIF2α requirement for the translation of C+Luc mRNA both in uninfected and SV-replicating cells. First, we assayed the effect of arsenite on SV infection by analyzing protein synthesis in cultures treated with different concentrations of this compound (Fig 2A). Arsenite treatment blocked protein synthesis in control BHK cells in a dose-dependent manner. Thus, actin synthesis decreases by 48 and 83% on treatment with 50 and 200 µM arsenite respectively. However, in infected cells, synthesis of C protein is only reduced by 8 and 29%, respectively, after the same treatment. Moreover, as a consequence of arsenite activity at the endoplasmic reticulum, viral glycoprotein processing was affected, such that the precursor PE26KE1 was not cleaved to produce mature products PE2, 6K and E1.


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)

Effect of arsenite on translation of SV sg-mRNA.Panel A. Effect of arsenite on SV infection. Uninfected or SV-infected BHK cells (10 pfu/cell) were treated with different concentrations of sodium arsenite for 30 min at 4 hpi. Proteins were then labelled with [35S]Met-Cys in presence of the same concentrations of sodium arsenite for 30 min. Samples were collected in the appropriate sample buffer and processed by SDS-PAGE, fluorography and autoradiography. Relative densitometry values obtained from capsid or actin bands are indicated below each lane. Panel B. Translation in SV-replicating BHK cells treated with arsenite. BHK cells were electroplated with 20 µg of rep C+Luc mRNA or transcription buffer as control. At 4 hpe cultures were treated with sodium arsenite for 30 min and then half of the cultures were labelled with [35S]Met-Cys in presence or absence of arsenite for 30 min and the other half treated with arsenite only. Radioactive samples were examined by SDS-PAGE, fluorography and autoradiography (upper panel) and non-radioactive samples were used to analyze phosphorylation of eIF2α by isoelectric focusing (lower panel). Panel C. Translation of different mRNAs transfected in BHK cells treated with arsenite. BHK cells were electroporated with 20 µg of cap-Luc-poly(A) or C+Luc mRNA and at 30 mpe treated with different concentrations of arsenite for 1 hour. Half of the cultures were processed to measure luc activity (upper panel) and the other half to detect phosphorylation of eIF2 α by isoelectric focusing (lower panel). Panel D. Translation of sg-mRNA synthesized in the nucleus of BHK cells treated with arsenite. BHK cells were transfected with pcDNA-Luc or pcDNA C+Luc plasmids and, at 18 hpt, treated or not with arsenite for 30 min. Next, cultures were labelled with [35S]Met-Cys in presence or absence of arsenite for 30 min. One quarter of the samples were directly processed by SDS-PAGE, fluorography and autoradiography (upper panel). The remaining samples were first immunoprecipitated with anti-luc antibodies and then processed by SDS-PAGE, fluorography and autoradiography (lower panel).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2651626&req=5

pone-0004772-g002: Effect of arsenite on translation of SV sg-mRNA.Panel A. Effect of arsenite on SV infection. Uninfected or SV-infected BHK cells (10 pfu/cell) were treated with different concentrations of sodium arsenite for 30 min at 4 hpi. Proteins were then labelled with [35S]Met-Cys in presence of the same concentrations of sodium arsenite for 30 min. Samples were collected in the appropriate sample buffer and processed by SDS-PAGE, fluorography and autoradiography. Relative densitometry values obtained from capsid or actin bands are indicated below each lane. Panel B. Translation in SV-replicating BHK cells treated with arsenite. BHK cells were electroplated with 20 µg of rep C+Luc mRNA or transcription buffer as control. At 4 hpe cultures were treated with sodium arsenite for 30 min and then half of the cultures were labelled with [35S]Met-Cys in presence or absence of arsenite for 30 min and the other half treated with arsenite only. Radioactive samples were examined by SDS-PAGE, fluorography and autoradiography (upper panel) and non-radioactive samples were used to analyze phosphorylation of eIF2α by isoelectric focusing (lower panel). Panel C. Translation of different mRNAs transfected in BHK cells treated with arsenite. BHK cells were electroporated with 20 µg of cap-Luc-poly(A) or C+Luc mRNA and at 30 mpe treated with different concentrations of arsenite for 1 hour. Half of the cultures were processed to measure luc activity (upper panel) and the other half to detect phosphorylation of eIF2 α by isoelectric focusing (lower panel). Panel D. Translation of sg-mRNA synthesized in the nucleus of BHK cells treated with arsenite. BHK cells were transfected with pcDNA-Luc or pcDNA C+Luc plasmids and, at 18 hpt, treated or not with arsenite for 30 min. Next, cultures were labelled with [35S]Met-Cys in presence or absence of arsenite for 30 min. One quarter of the samples were directly processed by SDS-PAGE, fluorography and autoradiography (upper panel). The remaining samples were first immunoprecipitated with anti-luc antibodies and then processed by SDS-PAGE, fluorography and autoradiography (lower panel).
Mentions: Arsenite is widely used to induce phosphorylation of eIF2α, leading to the inhibition of translation [13], [25], [26]. Furthermore, culture cells infected by SV exhibit high levels of phosphorylated eIF2α at times when viral structural proteins are being synthesized [7], [9]. We therefore compared the eIF2α requirement for the translation of C+Luc mRNA both in uninfected and SV-replicating cells. First, we assayed the effect of arsenite on SV infection by analyzing protein synthesis in cultures treated with different concentrations of this compound (Fig 2A). Arsenite treatment blocked protein synthesis in control BHK cells in a dose-dependent manner. Thus, actin synthesis decreases by 48 and 83% on treatment with 50 and 200 µM arsenite respectively. However, in infected cells, synthesis of C protein is only reduced by 8 and 29%, respectively, after the same treatment. Moreover, as a consequence of arsenite activity at the endoplasmic reticulum, viral glycoprotein processing was affected, such that the precursor PE26KE1 was not cleaved to produce mature products PE2, 6K and E1.

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