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 (2Apro) 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 2Apro 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 eIF2α, 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 α 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.
Highlights
The genome of sindbis virus (SV), a member of the Alphavirus genus, contains a single-stranded RNA molecule of positive polarity [1]
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
To induce cleavage of translation initiation factor eIF4G, PV 2A protease (2Apro) was expressed on electroporation of synthesized IRES followed by the PV 2Apro gene (IRES-2A) mRNA
Summary
The genome of sindbis virus (SV), a member of the Alphavirus genus, contains a single-stranded RNA molecule of positive polarity [1]. After virus entry into susceptible cells and decapsidation the viral genome of 11.5 kb, acting as mRNA, directs the synthesis of early non-structural proteins (nsp1-4) involved in viral RNA replication and transcription. About 2–3 hours postinfection (hpi), synthesis of late SV proteins commences under the direction of 26S subgenomic (sg)-mRNA. This mRNA corresponds to the 39 third of the genome and is transcribed from an internal promoter present on the minus strand RNA [2,3]. A translation enhancer element located in the first 275 nt of the C sequence confers high translatability on this sg-mRNA [5,6] This element is required for translation of sg-mRNA when eIF2a is phosphorylated [7]. Significant phosphorylation of eIF2a is observed after togavirus infection, at times when structural proteins are synthesized [7,8]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
