Dual Mechanism for the Translation of Subgenomic mRNA from Sindbis Virus in Infected and Uninfected Cells Miguel A´ ngel Sanz, Alfredo Castello´ , Iva´ n Ventoso, Juan Jose´ Berlanga, Luis Carrasco* Centro de Biolog´ıa Molecular ‘‘Severo Ochoa’’ (CSIC-UAM), Universidad Auto´ noma 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 (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 eIF2a, 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 a 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. Citation: Sanz MA´ , Castello´ A, Ventoso I, Berlanga JJ, Carrasco L (2009) Dual Mechanism for the Translation of Subgenomic mRNA from Sindbis Virus in Infected and Uninfected Cells. PLoS ONE 4(3): e4772. doi:10.1371/journal.pone.0004772 Editor: Wang-Shick Ryu, Yonsei University, Republic of Korea Received December 23, 2008; Accepted February 12, 2009; Published March 10, 2009 Copyright: 2009 Sanz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: DGICYT Grant (BFU 2006-02182). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: lcarrasco@cbm.uam.es Introduction 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 decapsida- tion 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 post- infection (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]. After about 2 hpi, cellular translation is drastically inhibited, while viral sg-mRNA translation emerges and continues for hours. SV replicons encoding only the non-structural proteins are still capable of shutting off host translation [4]. Translation directed by sg-mRNA leads to synthesis of a polyprotein that is proteolytically cleaved, rendering the mature proteins C, E3, E2, 6K and E1. As occurs with the 49S RNA genome, the sg-mRNA is capped at its 59 end and polyadenylated at the 39 end. The cap structure is followed by 49-nt leader sequence then the AUG initiation codon. A translation enhancer element located in the first 275 nt of the C sequence confers high translatability on this sg-mRNA [5,6]. In addition, this element is required for translation of sg-mRNA when eIF2a is phosphorylated [7]. Thus, significant phosphorylation of eIF2a is observed after togavirus infection, at times when structural proteins are synthesized [7,8]. This modification of eIF2 is not responsible for the inhibition of host translation in SV-infected cells, since it occurs in cells where eIF2a is not phosphorylated [7,9]. It is possible that the function of eIF2 is replaced by eIF2A in SV-infected cells [7]. As occurs with SV, a number of animal viruses are capable of translating their mRNAs in cells where eIF2 has become phosphorylated [10,11]. In fact, some viral RNAs can direct the binding of Met-tRNAi in an eIF2-independent manner [12,13]. Although sg-mRNA contains a cap structure, cleavage of eIF4G by poliovirus (PV) 2Apro or HIV-1 PR does not impair its translation [14]. These findings suggest that translation of SV sg-mRNA does not require the integrity of eIF4F complex and poly(A)-binding protein (PABP). A variety of animal viruses can translate some of their mRNAs in the absence of an integral eIF4F complex [10,15]. One of the most studied examples is picornavirus mRNA, which directs protein synthesis after eIF4G cleavage by several viral proteases [16,17]. In other examples, a virus-encoded protein interacts with PLoS ONE | www.plosone.org 1 March 2009 | Volume 4 | Issue 3 | e4772