Iván Ventoso

Antiviral effect of the mammalian translation initiation factor 2α kinase GCN2 against RNA viruses

In mammals, four different protein kinases, heme‐regulated inhibitor, double‐stranded RNA‐dependent protein kinase (PKR), general control non‐derepressible‐2 (GCN2) and PKR‐like endoplasmic reticulum kinase, regulate protein synthesis in response to environmental stresses by phosphorylating the α‐subunit of the initiation factor 2 (eIF2α). We now report that mammalian GCN2 is specifically activated in vitro upon binding of two nonadjacent regions of the Sindbis virus (SV) genomic RNA to its histidyl‐tRNA synthetase‐related domain. Moreover, endogenous GCN2 is activated in cells upon SV infection. Strikingly, fibroblasts derived from GCN2−/− mice possess an increased permissiveness to SV or vesicular stomatitis virus infection. We further show that mice lacking GCN2 are extremely susceptible to intranasal SV infection, demonstrating high virus titers in the brain compared to similarly infected control animals. The overexpression of wild‐type GCN2, but not the catalytically inactive GCN2‐K618R variant, in NIH 3T3 cells impaired the replication of a number of RNA viruses. We determined that GCN2 inhibits SV replication by blocking early viral translation of genomic SV RNA. These findings point to a hitherto unrecognized role of GCN2 as an early mediator in the cellular response to RNA viruses.

Poliovirus 2A proteinase cleaves directly the eIF‐4G subunit of eIF‐4F complex

The initiation of translation on eukaryotic mRNA is governed by the concerted action of polypeptides of the eIF‐4F complex. One of these polypeptides, eIF‐4G, is proteolytically inactivated upon infection with several members of the Picornaviridae family. This cleavage occurs by the action of virus‐encoded proteinases: 2Apro (entero‐ and rhinovirus) or Lpro (aphthovirus). An indirect mode of eIF‐4G cleavage through the activation of a second cellular proteinase has been proposed in the case of poliovirus. Although cleavage of eIF‐4G by rhino‐ and coxsackievirus 2Apro has been achieved directly in vitro, a similar activity has not been documented to date for poliovirus 2Apro. We report here that a recombinant form of poliovirus 2Apro fused to maltose binding protein (MBP) directly cleaves human eIF‐4G from a highly purified eIF‐4F complex. Efficient cleavage of eIF‐4G requires magnesium ions. The presence of other initiation factors such as eIF‐3, eIF‐4A or eIF‐4B mimics in part the stimulatory effect of magnesium ions and probably stabilizes the cleavage products of eIF‐4G generated by 2Apro. These results suggest that efficient cleavage of eIF‐4G by MBP‐2Apro requires a proper conformation of this factor. Finally, MBP‐2Apro protein cleaves an eIF‐4G‐derived synthetic peptide at the same site as rhino‐ and coxsackievirus 2Apro (R485‐G486).

Cleavage of eIF4G by HIV-1 protease: effects on translation

We have recently reported that HIV‐1 protease (PR) cleaves the initiation factor of translation eIF4GI [Ventoso et al., Proc. Natl. Acad. Sci. USA 98 (2001) 12966–12971]. Here, we analyze the proteolytic activity of HIV‐1 PR on eIF4GI and eIF4GII and its implications for the translation of mRNAs. HIV‐1 PR efficiently cleaves eIF4GI, but not eIF4GII, in cell‐free systems as well as in transfected mammalian cells. This specific proteolytic activity of the retroviral protease on eIF4GI was more selective than that observed with poliovirus 2Apro. Despite the presence of an intact endogenous eIF4GII, cleavage of eIF4GI by HIV‐1 PR was sufficient to impair drastically the translation of capped and uncapped mRNAs. In contrast, poliovirus IRES‐driven translation was unaffected or even enhanced by HIV‐1 PR after cleavage of eIF4GI. Further support for these in vitro results has been provided by the expression of HIV‐1 PR in COS cells from a Gag‐PR precursor. Our present findings suggest that eIF4GI intactness is necessary to maintain cap‐dependent translation, not only in cell‐free systems but also in mammalian cells.

Dual Mechanism for the Translation of Subgenomic mRNA from Sindbis Virus in Infected and Uninfected Cells

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.

Extensive translatome remodeling during ER stress response in mammalian cells.

In this work we have described the translatome of two mammalian cell lines, NIH3T3 and Jurkat, by scoring the relative polysome association of ∼10,000 mRNA under normal and ER stress conditions. We have found that translation efficiencies of mRNA correlated poorly with transcript abundance, although a general tendency was observed so that the highest translation efficiencies were found in abundant mRNA. Despite the differences found between mouse (NIH3T3) and human (Jurkat) cells, both cell types share a common translatome composed by ∼800–900 mRNA that encode proteins involved in basic cellular functions. Upon stress, an extensive remodeling in translatomes was observed so that translation of ∼50% of mRNA was inhibited in both cell types, this effect being more dramatic for those mRNA that accounted for most of the cell translation. Interestingly, we found two subsets comprising 1000–1500 mRNA whose translation resisted or was induced by stress. Translation arrest resistant class includes many mRNA encoding aminoacyl tRNA synthetases, ATPases and enzymes involved in DNA replication and stress response such as BiP. This class of mRNA is characterized by high translation rates in both control and stress conditions. Translation inducible class includes mRNA whose translation was relieved after stress, showing a high enrichment in early response transcription factors of bZIP and zinc finger C2H2 classes. Unlike yeast, a general coordination between changes in translation and transcription upon stress (potentiation) was not observed in mammalian cells. Among the different features of mRNA analyzed, we found a relevant association of translation efficiency with the presence of upstream ATG in the 5′UTR and with the length of coding sequence of mRNA, and a looser association with other parameters such as the length and the G+C content of 5′UTR. A model for translatome remodeling during the acute phase of stress response in mammalian cells is proposed.

GCN2 Has Inhibitory Effect on Human Immunodeficiency Virus-1 Protein Synthesis and Is Cleaved upon Viral Infection

The reversible phosphorylation of the alpha-subunit of eukaryotic translation initiation factor 2 (eIF2alpha) is a well-characterized mechanism of translational control in response to a wide variety of cellular stresses, including viral infection. Beside PKR, the eIF2alpha kinase GCN2 participates in the cellular response against viral infection by RNA viruses with central nervous system tropism. PKR has also been involved in the antiviral response against HIV-1, although this antiviral effect is very limited due to the distinct mechanisms evolved by the virus to counteract PKR action. Here we report that infection of human cells with HIV-1 conveys the proteolytic cleavage of GCN2 and that purified HIV-1 and HIV-2 proteases produce direct proteolysis of GCN2 in vitro, abrogating the activation of GCN2 by HIV-1 RNA. Transfection of distinct cell lines with a plasmid encoding an HIV-1 cDNA clone competent for a single round of replication resulted in the activation of GCN2 and the subsequent eIF2alpha phosphorylation. Moreover, transfection of GCN2 knockout cells or cells with low levels of phosphorylated eIF2alpha with the same HIV-1 cDNA clone resulted in a marked increase of HIV-1 protein synthesis. Also, the over-expression of GCN2 in cells led to a diminished viral protein synthesis. These findings suggest that viral RNA produced during HIV-1 infection activates GCN2 leading to inhibition of viral RNA translation, and that HIV-1 protease cleaves GCN2 to overcome its antiviral effect.

Adaptive Changes in Alphavirus mRNA Translation Allowed Colonization of Vertebrate Hosts

ABSTRACT Members of the Alphavirus genus are arboviruses that alternate replication in mosquitoes and vertebrate hosts. In vertebrate cells, the alphavirus resists the activation of antiviral RNA-activated protein kinase (PKR) by the presence of a prominent RNA structure (downstream loop [DLP]) located in viral 26S transcripts, which allows an eIF2-independent translation initiation of these mRNAs. This article shows that DLP structure is essential for replication of Sindbis virus (SINV) in vertebrate cell lines and animals but is dispensable for replication in insect cells, where no ortholog of the vertebrate PKR gene has been found. Sequence comparisons and structural RNA analysis revealed the evolutionary conservation of DLP in SINV and predicted the existence of equivalent DLP structures in many members of the Alphavirus genus. A mutant SINV lacking the DLP structure evolved in murine cells to recover a wild-type phenotype by creating an alternative structure in the RNA that restored the translational independence for eIF2. Genetic, phylogenetic, and biochemical data presented here support an evolutionary scenario for the natural history of alphaviruses, in which the acquisition of DLP structure in their mRNAs probably allowed the colonization of vertebrate host and the consequent geographic expansion of some of these viruses worldwide.

Diversity in viral anti-PKR mechanisms: A remarkable case of evolutionary convergence

Most viruses express during infection products that prevent or neutralize the effect of the host dsRNA activated protein kinase (PKR). Translation of Sindbis virus (SINV) mRNA escapes to PKR activation and eIF2 phosphorylation in infected cells by a mechanism that requires a stem loop structure in viral 26S mRNA termed DLP to initiate translation in the absence of functional eIF2. Unlike the rest of viruses tested, we found that Alphavirus infection allowed a strong PKR activation and eIF2α phosphorylation in vitro and in infected animals so that the presence of DLP structure in mRNA was critical for translation and replication of SINV. Interestingly, infection of MEFs with some viruses that express PKR inhibitors prevented eIF2α phosphorylation after superinfection with SINV, suggesting that viral anti-PKR mechanisms could be exchangeable. Thus, translation of SINV mutant lacking the DLP structure (ΔDLP) in 26S mRNA was partially rescued in cells expressing vaccinia virus (VV) E3 protein, a known inhibitor of PKR. This case of heterotypic complementation among evolutionary distant viruses confirmed experimentally a remarkable case of convergent evolution in viral anti-PKR mechanisms. Our data reinforce the critical role of PKR in regulating virus-host interaction and reveal the versatility of viruses to find different solutions to solve the same conflict.

An RNA trapping mechanism in Alphavirus mRNA promotes ribosome stalling and translation initiation

During translation initiation, eukaryotic initiation factor 2 (eIF2) delivers the Met-tRNA to the 40S ribosomal subunit to locate the initiation codon (AUGi) of mRNA during the scanning process. Stress-induced eIF2 phosphorylation leads to a general blockade of translation initiation and represents a key antiviral pathway in mammals. However, some viral mRNAs can initiate translation in the presence of phosphorylated eIF2 via stable RNA stem-loop structures (DLP; Downstream LooP) located in their coding sequence (CDS), which promote 43S preinitiation complex stalling on the initiation codon. We show here that during the scanning process, DLPs of Alphavirus mRNA become trapped in ES6S region (680–914 nt) of 18S rRNA that are projected from the solvent side of 40S subunit. This trapping can lock the progress of the 40S subunit on the mRNA in a way that places the upstream initiator AUGi on the P site of 40S subunit, obviating the participation of eIF2. Notably, the DLP structure is released from 18S rRNA upon 60S ribosomal subunit joining, suggesting conformational changes in ES6Ss during the initiation process. These novel findings illustrate how viral mRNA is threaded into the 40S subunit during the scanning process, exploiting the topology of the 40S subunit solvent side to enhance its translation in vertebrate hosts.

New insights into the topology of the scanning ribosome during translation initiation: Lessons from viruses

ABSTRACT Location of the translation initiation codon generally requires scanning of the 43S ribosomal preinitiation complex (43S PIC) from the 5′ of the mRNA. Associated RNA helicases can facilitate movement of the 43S PIC by removing secondary structure present in the 5′ UTR of mRNA, which is required for codon inspection. The canonical RNA-dependent helicase eIF4A is directly involved in this process, as part of the eIF4F complex (eIF4G + eIF4A + eIF4E) that associates first with mRNA and then recruits the 43S PIC to initiate scanning. The topology and operational mechanism of the scanning PIC are probably the least understood aspects of the initiation step. Recent findings from translation of alphavirus mRNA, together with new biochemical and structural data of the 43S PIC, suggest a role for the ES6S region of 40S as the gateway for mRNA entry during scanning. The presence of eIF4G-eIF4A complex in this region, interacting with the incoming mRNA, supports a model where eIF4A could work ahead of the scanning complex during translation initiation. Here we present additional data supporting this model.