Limits...
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.

Show MeSH

Related in: MedlinePlus

Localization analyses of eIF2α and eIF4G in SV-infected cells.BHK cells were infected with SV (100 pfu/cell) and were processed for immunofluorescence at 8 hpi.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2651626&req=5

pone-0004772-g006: Localization analyses of eIF2α and eIF4G in SV-infected cells.BHK cells were infected with SV (100 pfu/cell) and were processed for immunofluorescence at 8 hpi.

Mentions: As shown in this work and in previous articles, synthesis of SV late proteins in infected cells can take place without operative eIF4G and eIF2α [7], [14]. For this reason, it was of interest to analyze the distribution of these initiation factors after SV infection. Both eIF2α and eIF4GI modified their location in SV-infected cells, as compared to the uninfected counterparts. Thus, eIF2α concentrates in a region near the nucleus devoid of ribosomes, presumably the centrosomal region (Fig 6, upper panel). On the other hand, most of eIF4GI is found in cytoplasmic granules (Fig 6, upper panel), which are most probably stress granules since both eIF4GI and TIA markers co-localize in SV-infected cells (Fig 6, lower panel). The presence of eIF2α and eIF4GI in places other than those enriched in ribosomes and other translation factors is consistent with the idea that these two factors do not participate in the initiation of translation of SV sg-mRNA.


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)

Localization analyses of eIF2α and eIF4G in SV-infected cells.BHK cells were infected with SV (100 pfu/cell) and were processed for immunofluorescence at 8 hpi.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004772-g006: Localization analyses of eIF2α and eIF4G in SV-infected cells.BHK cells were infected with SV (100 pfu/cell) and were processed for immunofluorescence at 8 hpi.
Mentions: As shown in this work and in previous articles, synthesis of SV late proteins in infected cells can take place without operative eIF4G and eIF2α [7], [14]. For this reason, it was of interest to analyze the distribution of these initiation factors after SV infection. Both eIF2α and eIF4GI modified their location in SV-infected cells, as compared to the uninfected counterparts. Thus, eIF2α concentrates in a region near the nucleus devoid of ribosomes, presumably the centrosomal region (Fig 6, upper panel). On the other hand, most of eIF4GI is found in cytoplasmic granules (Fig 6, upper panel), which are most probably stress granules since both eIF4GI and TIA markers co-localize in SV-infected cells (Fig 6, lower panel). The presence of eIF2α and eIF4GI in places other than those enriched in ribosomes and other translation factors is consistent with the idea that these two factors do not participate in the initiation of translation of SV sg-mRNA.

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