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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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Related in: MedlinePlus

Translation of SV sg-mRNA containing AUGi replaced by other codons.Panel A. Schematic representation of the first 228 nt from the 5′end of SV sg-mRNA that include the leader sequence and the translation enhancing motif. The mutations introduced in the different constructs are indicated. Panel B. Synthesis of C protein from different SV variants with modified start codons of sg-mRNA. BHK cells were electroporated with the different in vitro transcribed mRNAs and, at the times indicated, cultures were labelled with [35S]Met-Cys for 30 min. Samples were processed by SDS-PAGE, fluorography and autoradiography (upper panel) and also by western blot analysis to detect C products with anti-C antibodies (lower panel). Panel C. Synthesis of C protein from sg-mRNAs with altered AUGi electroporated into BHK cells or translated in HeLa cell extracts. BHK cells were electroporated with the different in vitro prepared sg-mRNAs and, at 3 hpe, C production was analyzed by western blotting (upper panel). Translation was carried out in HeLa S3 extracts programmed with the different mRNAs for 1 h at 30°C in presence of 0.7 µC/µl [35S]Met-Cys. The synthesized proteins were analyzed by autoradiography of SDS-polyacrylamide gels (lower panel). Panel D. Synthesis of C protein from sg-mRNAs that have disrupted the DLP structure. BHK cells were electroporated with different SV genomes or with sg-mRNAs synthesized by in vitro transcription. Cultures were collected at 4 hpe for replicons and at 3 hpe for in vitro transcribed sg-mRNAs. C products were analyzed by western blotting with anti-C antibodies. Samples electroporated with sg-mRNAs (Right panel) were exposed ten times more than samples electroporated with replicons (Left panel).
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pone-0004772-g003: Translation of SV sg-mRNA containing AUGi replaced by other codons.Panel A. Schematic representation of the first 228 nt from the 5′end of SV sg-mRNA that include the leader sequence and the translation enhancing motif. The mutations introduced in the different constructs are indicated. Panel B. Synthesis of C protein from different SV variants with modified start codons of sg-mRNA. BHK cells were electroporated with the different in vitro transcribed mRNAs and, at the times indicated, cultures were labelled with [35S]Met-Cys for 30 min. Samples were processed by SDS-PAGE, fluorography and autoradiography (upper panel) and also by western blot analysis to detect C products with anti-C antibodies (lower panel). Panel C. Synthesis of C protein from sg-mRNAs with altered AUGi electroporated into BHK cells or translated in HeLa cell extracts. BHK cells were electroporated with the different in vitro prepared sg-mRNAs and, at 3 hpe, C production was analyzed by western blotting (upper panel). Translation was carried out in HeLa S3 extracts programmed with the different mRNAs for 1 h at 30°C in presence of 0.7 µC/µl [35S]Met-Cys. The synthesized proteins were analyzed by autoradiography of SDS-polyacrylamide gels (lower panel). Panel D. Synthesis of C protein from sg-mRNAs that have disrupted the DLP structure. BHK cells were electroporated with different SV genomes or with sg-mRNAs synthesized by in vitro transcription. Cultures were collected at 4 hpe for replicons and at 3 hpe for in vitro transcribed sg-mRNAs. C products were analyzed by western blotting with anti-C antibodies. Samples electroporated with sg-mRNAs (Right panel) were exposed ten times more than samples electroporated with replicons (Left panel).

Mentions: Since translation of sg-mRNA does not require eIF2 in SV-infected cells, we speculated that a different initiation codon to the canonical model may be used for this mRNA. Perhaps, during infection, when eIF2α is phosphorylated the initiation of translation of sg-mRNA could operate at non-AUG codons. To investigate this possibility, a number of constructs were made containing AUGi and the next AUG present in the region coding for C protein changed to other codons. To maintain the predicted base pairing in this region, several modifications were introduced as depicted in figure 3A. In addition, we tested a variant that contains an altered hairpin structure (SV ΔDLP) [7]. The different viral genomic RNAs bearing altered initiation codons and the variant that contains a modified hairpin structure were electroporated into BHK cells and protein synthesis was estimated at different times by radioactive labelling (Fig 3B, upper panel) and also by western blot analysis using anti-C antibodies (Fig 3B, lower panel). As expected, wt SV RNA was the most efficient and synthesized C as a unique product. SV ΔDLP rendered three products that in total represent 28% of C product from wt SV, as estimated by densitometric analysis of the labelled sample at 10 hpe. The two products with higher molecular weight migrate very closely to each other, but they can be separated by using longer electrophoresis times (data not shown). Most probably, these C-related products are the result of a loss of fidelity in AUGi selection during the initiation of translation. Their sizes are consistent with alternative initiation at the first three AUGs of C sequence. The SV-C-Met,Arg/Lys,Lys variant rendered two products, the one with the lower mobility corresponds in size to wt C and represents 7% of protein C synthesized at 10 hpe. Gel mobility of this product suggests that it is synthesized by initiation at the non-canonical AAG codon (see scheme in Fig 3A). The most abundant C product synthesized by SV-C-Met,Arg/Lys,Lys (19% of wt C) is consistent with a translation initiation from the first AUG of its sequence, that is the third AUG in the wt C (see scheme in Fig 3A). SV-C-Met/Ala and SV-C-Met/Cys synthesized the lower amounts of C products at 10 hpe, 9 and 5% of wt C respectively. However, they synthesized almost exclusively products with similar mobility to wt C protein. These C products are presumably derived from initiation at non-canonical GCG or UGU codons, respectively. Analyses and quantification by western blot analysis yielded similar results to those obtained by radioactive labelling (Fig 3B, lower panel). However, one significant difference was that the products synthesized by translation initiation at the non-canonical codons AAG from SV-C-Met,Arg/Lys,Lys or UGU from SV-C-Met/Cys do not accumulate in cells. As wt C protein does not contain any cysteine, this variant will produce a radioactive C product when labelled with radioactive cysteine if the UGU codon is used. BHK cells electroporated with wt SV and SV-C-Met/Cys RNAs were labelled at 5 hpe with [35S]-Cys (Fig S1). wt SV efficiently incorporated radioactive cysteine into its glycoproteins but not into C protein. Notably, although inefficiently incorporated radioactive cysteine into SV-C-Met/Cys, it could be detected both in glycoproteins and in C protein. The mobility of the labelled C protein suggests that translation initiates at the UGU codon.


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)

Translation of SV sg-mRNA containing AUGi replaced by other codons.Panel A. Schematic representation of the first 228 nt from the 5′end of SV sg-mRNA that include the leader sequence and the translation enhancing motif. The mutations introduced in the different constructs are indicated. Panel B. Synthesis of C protein from different SV variants with modified start codons of sg-mRNA. BHK cells were electroporated with the different in vitro transcribed mRNAs and, at the times indicated, cultures were labelled with [35S]Met-Cys for 30 min. Samples were processed by SDS-PAGE, fluorography and autoradiography (upper panel) and also by western blot analysis to detect C products with anti-C antibodies (lower panel). Panel C. Synthesis of C protein from sg-mRNAs with altered AUGi electroporated into BHK cells or translated in HeLa cell extracts. BHK cells were electroporated with the different in vitro prepared sg-mRNAs and, at 3 hpe, C production was analyzed by western blotting (upper panel). Translation was carried out in HeLa S3 extracts programmed with the different mRNAs for 1 h at 30°C in presence of 0.7 µC/µl [35S]Met-Cys. The synthesized proteins were analyzed by autoradiography of SDS-polyacrylamide gels (lower panel). Panel D. Synthesis of C protein from sg-mRNAs that have disrupted the DLP structure. BHK cells were electroporated with different SV genomes or with sg-mRNAs synthesized by in vitro transcription. Cultures were collected at 4 hpe for replicons and at 3 hpe for in vitro transcribed sg-mRNAs. C products were analyzed by western blotting with anti-C antibodies. Samples electroporated with sg-mRNAs (Right panel) were exposed ten times more than samples electroporated with replicons (Left panel).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004772-g003: Translation of SV sg-mRNA containing AUGi replaced by other codons.Panel A. Schematic representation of the first 228 nt from the 5′end of SV sg-mRNA that include the leader sequence and the translation enhancing motif. The mutations introduced in the different constructs are indicated. Panel B. Synthesis of C protein from different SV variants with modified start codons of sg-mRNA. BHK cells were electroporated with the different in vitro transcribed mRNAs and, at the times indicated, cultures were labelled with [35S]Met-Cys for 30 min. Samples were processed by SDS-PAGE, fluorography and autoradiography (upper panel) and also by western blot analysis to detect C products with anti-C antibodies (lower panel). Panel C. Synthesis of C protein from sg-mRNAs with altered AUGi electroporated into BHK cells or translated in HeLa cell extracts. BHK cells were electroporated with the different in vitro prepared sg-mRNAs and, at 3 hpe, C production was analyzed by western blotting (upper panel). Translation was carried out in HeLa S3 extracts programmed with the different mRNAs for 1 h at 30°C in presence of 0.7 µC/µl [35S]Met-Cys. The synthesized proteins were analyzed by autoradiography of SDS-polyacrylamide gels (lower panel). Panel D. Synthesis of C protein from sg-mRNAs that have disrupted the DLP structure. BHK cells were electroporated with different SV genomes or with sg-mRNAs synthesized by in vitro transcription. Cultures were collected at 4 hpe for replicons and at 3 hpe for in vitro transcribed sg-mRNAs. C products were analyzed by western blotting with anti-C antibodies. Samples electroporated with sg-mRNAs (Right panel) were exposed ten times more than samples electroporated with replicons (Left panel).
Mentions: Since translation of sg-mRNA does not require eIF2 in SV-infected cells, we speculated that a different initiation codon to the canonical model may be used for this mRNA. Perhaps, during infection, when eIF2α is phosphorylated the initiation of translation of sg-mRNA could operate at non-AUG codons. To investigate this possibility, a number of constructs were made containing AUGi and the next AUG present in the region coding for C protein changed to other codons. To maintain the predicted base pairing in this region, several modifications were introduced as depicted in figure 3A. In addition, we tested a variant that contains an altered hairpin structure (SV ΔDLP) [7]. The different viral genomic RNAs bearing altered initiation codons and the variant that contains a modified hairpin structure were electroporated into BHK cells and protein synthesis was estimated at different times by radioactive labelling (Fig 3B, upper panel) and also by western blot analysis using anti-C antibodies (Fig 3B, lower panel). As expected, wt SV RNA was the most efficient and synthesized C as a unique product. SV ΔDLP rendered three products that in total represent 28% of C product from wt SV, as estimated by densitometric analysis of the labelled sample at 10 hpe. The two products with higher molecular weight migrate very closely to each other, but they can be separated by using longer electrophoresis times (data not shown). Most probably, these C-related products are the result of a loss of fidelity in AUGi selection during the initiation of translation. Their sizes are consistent with alternative initiation at the first three AUGs of C sequence. The SV-C-Met,Arg/Lys,Lys variant rendered two products, the one with the lower mobility corresponds in size to wt C and represents 7% of protein C synthesized at 10 hpe. Gel mobility of this product suggests that it is synthesized by initiation at the non-canonical AAG codon (see scheme in Fig 3A). The most abundant C product synthesized by SV-C-Met,Arg/Lys,Lys (19% of wt C) is consistent with a translation initiation from the first AUG of its sequence, that is the third AUG in the wt C (see scheme in Fig 3A). SV-C-Met/Ala and SV-C-Met/Cys synthesized the lower amounts of C products at 10 hpe, 9 and 5% of wt C respectively. However, they synthesized almost exclusively products with similar mobility to wt C protein. These C products are presumably derived from initiation at non-canonical GCG or UGU codons, respectively. Analyses and quantification by western blot analysis yielded similar results to those obtained by radioactive labelling (Fig 3B, lower panel). However, one significant difference was that the products synthesized by translation initiation at the non-canonical codons AAG from SV-C-Met,Arg/Lys,Lys or UGU from SV-C-Met/Cys do not accumulate in cells. As wt C protein does not contain any cysteine, this variant will produce a radioactive C product when labelled with radioactive cysteine if the UGU codon is used. BHK cells electroporated with wt SV and SV-C-Met/Cys RNAs were labelled at 5 hpe with [35S]-Cys (Fig S1). wt SV efficiently incorporated radioactive cysteine into its glycoproteins but not into C protein. Notably, although inefficiently incorporated radioactive cysteine into SV-C-Met/Cys, it could be detected both in glycoproteins and in C protein. The mobility of the labelled C protein suggests that translation initiates at the UGU codon.

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