GCN2 Has Inhibitory Effect on Human Immunodeficiency Virus-1 Protein Synthesis and Is Cleaved upon Viral Infection Javier del Pino1, Jose´ Luis Jime´ nez2, Iva´ n Ventoso1, Alfredo Castello´ 1¤, Ma A´ ngeles Mun˜ oz-Ferna´ ndez3, Ce´ sar de Haro1, Juan Jose´ Berlanga1* 1 Centro de Biolog´ıa Molecular Severo Ochoa (CSIC-UAM), Universidad Auto´ noma de Madrid, Madrid, Spain, 2 Plataforma de Laboratorio, Hospital General Universitario Gregorio Maran˜ o´ n, Madrid, Spain, 3 Laboratory of Molecular Immunobiology, Hospital General Universitario Gregorio Maran˜ o´ n, Madrid, Spain Abstract 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. Citation: del Pino J, Jime´nez JL, Ventoso I, Castello´ A, Mun˜ oz-Ferna´ndez MA´ , et al. (2012) GCN2 Has Inhibitory Effect on Human Immunodeficiency Virus-1 Protein Synthesis and Is Cleaved upon Viral Infection. PLoS ONE 7(10): e47272. doi:10.1371/journal.pone.0047272 Editor: Jean-Luc E. P. H. Darlix, Institut National de la Sante´ et de la Recherche Me´dicale, France Received June 27, 2012; Accepted September 10, 2012; Published October 23, 2012 Copyright: 2012 del Pino 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: This work was supported in part by grants BFU2007-62987 (to CDH) BFU2006-12822 and BFU2009-09469 (to JJB) from the Direccio´ n General de Investigacio´ n Cient´ıfica y Te´cnica (DGICYT), a grant from the Fundacio´ n Ramo´ n Areces (to CDH), a grant from Universidad Auto´ noma de Madrid and the Government of Madrid (to JJB), grants PS09-02029 and RD06-0006-0035 (to MAM-F) and PI081495 (to JLJ) from Fondo de Investigaciones Sanitarias, and an institutional grant from the Fundacio´ n Ramo´ n Areces to the Centro de Biolog´ıa Molecular Severo Ochoa. JDP was supported by a Ph.D. fellowship from the Ministerio de Ciencia y Tecnolog´ıa (Government of Spain). JLJ is supported by Programa de Investigacio´ n de la Consejer´ıa de Sanidad de la Comunidad de Madrid. 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: jberlanga@cbm.uam.es. € Current address: European Molecular Biology Laboratory, Heidelberg, Germany Introduction The control of protein synthesis is central to the global process of regulation of gene expression, first leading to translational reprogramming and, as a consequence, affecting the transcrip- tional profile of cells. Protein synthesis is basically regulated at the initiation step, where phosphorylation of the alpha subunit of initiation factor eIF2 (eIF2alpha) at residue Ser-51 by specific protein kinases represents one of the best-characterized mecha- nisms regulating mRNA translation in eukaryotic cells in response to various stress conditions, such as lack of nutrients, endoplasmic reticulum stress, iron deficiency, heat shock and viral infection [1,2]. In mammalian cells, four different eIF2alpha kinases regulated by specific signals have been identified: HRI (iron deficiency) [3,4]; PKR (double-stranded RNA produced in cells infected by viruses) [5]; PERK (stress situations in the endoplasmic reticulum) [1]; and GCN2 (amino acid or serum deprivation and ultraviolet light irradiation) [6,7,8]. Some members of this family of eIF2alpha kinases are also present in other eukaryotic organisms: PERK and GCN2 in Drosophila melanogaster [5,9,10]; GCN2 in the yeast Saccharomyces cerevisiae [11] and two eIF2alpha kinases related to mammalian HRI (Hri1 and Hri2), in addition to Gcn2, in the fission yeast Schizosaccharomyces pombe [12]. eIF2 forms a ternary complex with GTP and the initiator methionyl-tRNA, which delivers the latter to the small ribosomal subunit with the subsequent hydrolisis of GTP. The conversion of inactive eIF2-GDP into active eIF2-GTP is catalyzed by the guanine nucleotide exchange factor eIF2B. Phosphorylation of eIF2alpha at Ser-51 increases its affinity for eIF2B, which then becomes sequestered in a stable and inactive complex, leading to the inhibition of protein synthesis due to the lack of ternary complexes. GCN2 was first described in the budding yeast Saccharomyces cerevisiae where the kinase is activated in response to amino acid starvation through the binding of uncharged tRNA to a region homologous to the histidyl-tRNA synthetases (HisRS) [13]. In mammals, this region is also responsible for the in vitro activation of GCN2 by tRNA and viral RNA [14]. Thus GCN2 has been PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e47272