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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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Co-localization analyses of SV capsid protein and ribosomes.BHK cells were infected with SV (100 pfu/cell) and processed for immunofluorescence at 8 hpi.
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pone-0004772-g005: Co-localization analyses of SV capsid protein and ribosomes.BHK cells were infected with SV (100 pfu/cell) and processed for immunofluorescence at 8 hpi.

Mentions: We recently provided evidence that transcription and translation are coupled in SV-infected cells [22]. In such a case, we would predict that viral translation takes place in cytoplasmic regions close to transcription and replication factories. Indeed, electron microscopy of SV-infected cells shows that nucleocapsids are assembled around membranous structures localized at discrete sites in the cytoplasm (Fig S2). Some of these membranous structures resemble replication factories (Fig S3). Our first goal was therefore to determine whether viral nucleocapsids co-localize with active transcription sites. To this end, SV-infected cells were labeled with bromouridine (BrU) in presence of actinomycin D at 6 hpi. Fixed cells were then incubated with specific antibodies against C protein and BrU and analyzed by immunofluorescence (Fig 4, upper panel). Transcription sites detected by anti-BrU antibodies did indeed co-localize with C protein. The next step was to examine co-localization of different translation factors with C protein (Fig 4, lower panel). The subcellular distribution of p-110 subunit of eIF3 in SV-infected cells differs from that observed in uninfected BHK cells. In uninfected cells eIF3 is uniformly distributed throughout the cytoplasm, whereas this factor is concentrated in the region of the nucleus in SV-infected cells. Moreover, eIF3 co-localizes with SV C protein. Translation elongation factor eEF2 has a localization and behaviour similar to eIF3, whereas eIF4E does not modify its distribution. To determine whether ribosomes are redistributed during infection, a monoclonal antibody against the carboxy terminal end of P ribosomal protein was employed. Figure 5 shows that ribosomes appear concentrated near the nucleus in SV-infected cells but are spread throughout the entire cytoplasm in control BHK cells. In SV-infected cells, most of the cytoplasm is devoid of ribosomes, which are concentrated close to the nucleus, near and overlapping the C protein signal (see in more detail lower panels in Fig 5). These findings suggest that components of the protein synthesizing machinery are redistributed after SV infection, localizing to a perinuclear region enriched in C protein, where viral transcription is taking place.


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)

Co-localization analyses of SV capsid protein and ribosomes.BHK cells were infected with SV (100 pfu/cell) and processed for immunofluorescence at 8 hpi.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004772-g005: Co-localization analyses of SV capsid protein and ribosomes.BHK cells were infected with SV (100 pfu/cell) and processed for immunofluorescence at 8 hpi.
Mentions: We recently provided evidence that transcription and translation are coupled in SV-infected cells [22]. In such a case, we would predict that viral translation takes place in cytoplasmic regions close to transcription and replication factories. Indeed, electron microscopy of SV-infected cells shows that nucleocapsids are assembled around membranous structures localized at discrete sites in the cytoplasm (Fig S2). Some of these membranous structures resemble replication factories (Fig S3). Our first goal was therefore to determine whether viral nucleocapsids co-localize with active transcription sites. To this end, SV-infected cells were labeled with bromouridine (BrU) in presence of actinomycin D at 6 hpi. Fixed cells were then incubated with specific antibodies against C protein and BrU and analyzed by immunofluorescence (Fig 4, upper panel). Transcription sites detected by anti-BrU antibodies did indeed co-localize with C protein. The next step was to examine co-localization of different translation factors with C protein (Fig 4, lower panel). The subcellular distribution of p-110 subunit of eIF3 in SV-infected cells differs from that observed in uninfected BHK cells. In uninfected cells eIF3 is uniformly distributed throughout the cytoplasm, whereas this factor is concentrated in the region of the nucleus in SV-infected cells. Moreover, eIF3 co-localizes with SV C protein. Translation elongation factor eEF2 has a localization and behaviour similar to eIF3, whereas eIF4E does not modify its distribution. To determine whether ribosomes are redistributed during infection, a monoclonal antibody against the carboxy terminal end of P ribosomal protein was employed. Figure 5 shows that ribosomes appear concentrated near the nucleus in SV-infected cells but are spread throughout the entire cytoplasm in control BHK cells. In SV-infected cells, most of the cytoplasm is devoid of ribosomes, which are concentrated close to the nucleus, near and overlapping the C protein signal (see in more detail lower panels in Fig 5). These findings suggest that components of the protein synthesizing machinery are redistributed after SV infection, localizing to a perinuclear region enriched in C protein, where viral transcription is taking place.

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