involved in the antiviral response against RNA viruses, such as Semliki Forest virus, vesicular stomatitis virus and Sindbis virus (SV), whose genomic RNA is able to bind and activate the kinase, which through the phosphorylation of eIF2alpha inhibits the translation of the genomic mRNA of SV and blocks its replication cycle in cells [14]. It is well known the central role of PKR in the cellular antiviral response as well as the different strategies developed by distinct viruses in order to counteract the negative effects of PKR on virus replication. These evasion strategies include proteins that inhibit PKR, sequester dsRNA, or are pseudosubstrates, and RNA molecules acting as pseudoactivators that compete with activator dsRNA for the binding to PKR [15]. Thus, during HIV-1 infection, PKR is first transiently activated and then inhibited due to viral and virus-activated cellular mechanisms of control [16,17]. Many viruses have evolved mechanisms that modify the activity of cellular translation factors in order to favor viral mRNA translation to the detriment of cellular mRNA translation. The best-characterized example of that is the proteolityc cleavage of eIF4G by proteases of different viruses, leading to the inhibition of capped cellular mRNA translation and to the enhancement of translation of uncapped viral RNAs [18]. Thus, eIF4G is cleaved by 2A protease of rhinovirus or poliovirus, L protease of aphthovirus, and proteases of several retroviruses, including HIV-1 and HIV-2 [19,20,21]. Another protein involved in translation, PABP, is also a target for viral proteases, including 2A and 3C of enterovirus [22,23], and the proteases of HIV-1 and HIV-2 [24]. HIV-1 protein translation occurs late in the viral life cycle in the cytoplasm of cells and is carried out by the host protein synthesis machinery. HIV-1 protease (HIV-1Pro) is a small enzyme that mediates the cleavage of Gag, Gag-Pol, and Nef precursor polyproteins during virion assembly and maturation [16]. In this paper we show that HIV-1 RNA activates GCN2 in vivo and in vitro and that the cleavage of the kinase by HIV-1Pro leads to its inactivation. Synthesis of HIV-1 proteins in cells transfected with a plasmid encoding the viral RNA increased in cells devoid of the GCN2 gene. All these results suggest that GCN2 is involved in the cellular antiviral response against HIV-1 infection. Results HIV-1 RNA promotes GCN2 activation in vitro through its HisRS-related domain Given that the genomic RNA of Sindbis virus activated GCN2 in vitro and that GCN2 have an antiviral effect against this virus [14], we tested whether the genomic RNA of other viruses could activate GCN2 as a sign of a potential antiviral effect against them. We observed that in vitro transcribed HIV-1 genomic RNA, among others, was able to significantly increase the eIF2alpha kinase activity of affinity purified GCN2 in a dose-dependent manner (Figure 1A). This activation was dependent on its HisRS- related domain, as the GCN2-m2 mutant of this domain showed very low autophosphorylation and eIF2alpha kinase activity compared to that of the wild type protein (Figure 1B). As expected, no autophosphorylation or eIF2alpha kinase activity was found when using the GCN2 form bearing a mutation in the lysine residue of the kinase domain responsible for the ATP binding (K618R). GCN2 activation by HIV-1 RNA was comparable to that obtained by using Sindbis virus RNA, which served as a positive control in these assays. HIV-1 Counteracts GCN2 Inhibitory Effect GCN2 cleavage in cells after HIV-1 infection In order to investigate the putative role of GCN2 in the cellular antiviral response against HIV-1, we tested GCN2 activation in MT-2 (human T-lymphocyte cell line) cells infected with HIV-1. To our surprise, the analysis of the cell lysates revealed the disappearance of GCN2 upon HIV-1 infection coinciding with the cleavage and disappearance of full length eIF4GI. Both protein bands were apparently intact at day 1, but disappeared at day 3 after infection, and their cleavage was prevented in presence of the specific inhibitor of the HIV-1 protease (HIV-1Pro) saquinavir (Figure 1 C and D). In the case of GCN2 we were unable to detect any proteolytic fragment, but the cleavage of eIF4GI rendered the expected carboxi-terminal Ct-2 (about 102 kDa) and Ct-3 (about 57 kDa) fragments [20]. Moreover, infection of Peripheral Blood Mononuclear Cells (PBMC) with HIV-1 resulted in a significant proteolytic degradation of GCN2 (45% reduction) and eIF4GI at 4 and 5 dpi, which was prevented in the presence of saquinavir (Figure 1E), strongly suggesting that the low levels of these proteins present in the cells were a consequence of HIV-1Pro cleavage. The level of other proteins such as PKR, actin or eIF2alpha remained unaltered to the same extent throughout the experiment. Note that in Figure 1 D and E, the HIV-1 p24 protein only appeared in the absence of saquinavir as a consequence of HIV-1Pro processing of the Gag (55 kDa) precursor. We also observed increased phosphorylation of eIF2alpha upon HIV-1 infection (Figure 1 C and E), suggesting a possible activation of GCN2. HIV-1 protease directly cleaves GCN2 Next, we tested the ability of HIV-1Pro to cleave GCN2 by using different approaches. First, we checked the cleavage of GCN2 in cells where the protease was expressed in a context in which other HIV-1 proteins were absent. With this aim, we expressed HIV- 1Pro in BHK-21 cells by electroporation of an in vitro transcribed RNA bearing the protease-coding region in the context of SV genomic mRNA [25]. As controls we used similar RNAs in order to express the 2A protease of poliovirus (PV-2APro) or just SV capsid C protein (Figure 2A). At 4 hours post-electroporation, we could observe a marked decrease in the amount of endogenous GCN2 and the appearance of a proteolytic fragment of an apparent molecular weight of 130–140 kDa, accounting for a specific cleavage of GCN2 by HIV-1Pro. In contrast, PV-2APro was unable to cut GCN2, but, as expected, eIF4GI was proteolytically degraded by both PV-2APro and HIV-1Pro. We also assayed the proteolytic activity of HIV-1Pro on GCN2 in COS-7 cells transfected with pTM1-derived plasmids and infected with vaccinia virus encoding T7 polymerase (Figure 2B). Co-expression of HIV-1Pro with mouse or human GCN2 resulted in the production of a cleavage fragment similar to the one obtained in BHK-21 cells expressing the protease. To further study GCN2 proteolytic degradation we performed experiments in which purified HIV-1Pro was added to HeLa cell- free extracts. Complete proteolysis of GCN2 was achieved after only 15 min of incubation, whereas in the same extracts a significant amount of eIF4GI remained intact after 30 min (Figure 2D). The cleavage of both proteins was prevented when the protease was used in the presence of saquinavir. PABP, another known HIV-1Pro substrate, seemed to be processed to a much lower extent and the amount of other proteins such as actin did not experience significant changes. Moreover, affinity-purifed mouse and human GCN2 were cleaved in vitro after incubation with purified HIV-1Pro and HIV- 2Pro (Figure 2C). Both viral proteases rendered GCN2 proteolytic fragments of the same size, suggesting that these proteases directly PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e47272