HIV-1 Counteracts GCN2 Inhibitory Effect in SDS-PAGE and stained with Coomassie blue. Shown in the lower box is the GCN2 sequence obtained by Edman degradation of the indicated C- terminal fragments. Results are representative of at least three independent experiments. doi:10.1371/journal.pone.0047272.g002 GCN2 fragments detected, in the in vivo and the in vitro experiments, using an anti-GCN2 antibody directed against the central portion of the kinase (aa 419–1024). HIV-1 protease cleavage abrogates GCN2 activity To test whether the proteolytic cleavage by HIV-1Pro led to the inactivation of GCN2, we assayed GCN2 in vitro activity after incubation with HIV-1Pro. Incubation of GCN2 with HIV-1Pro previous to the eIF2alpha kinase assay rendered a cleaved form of GCN2 which was just slightly phosphorylated and unable to phosphorylate eIF2alpha (Figure 3A, lane 3). To make sure that this effect was due to the proteolytic activity of HIV-1Pro, we also performed the assay in the presence of saquinavir, which was added just before the incubation of the protease with GCN2 (Figure 3A, lane 5) or, alternatively, after the incubation of the kinase and the protease, just before initiating the kinase assay (Figure 3A, lane 6). The presence of saquinavir prevented GCN2 cleavage by the viral protease and allowed phosphorylation of the kinase and of eIF2alpha. Thus, we unequivocally concluded that HIV-1Pro activity on GCN2 causes the inactivation of the kinase. To further investigate the observed effect of HIV-1Pro action on GCN2 activity, we expressed, purified and assayed two polypep- tides equivalent to those obtained by HIV-1Pro action on human GCN2, i.e., GCN2 aa 1–560 and aa 561–1649, respectively (Figure 3B). The amino-terminal polypeptide was quite inactive (Figure 3C, lanes 3 and 4), but the carboxi-terminal protein fragment retained some of the ability to phosphorylate eIF2alpha in the presence of HIV-1 RNA (Figure 3C, lanes 5 and 6), although it was significantly lower than the activity shown by the full length GCN2 protein (Figure 3C, lanes 7 and 8). This result corroborates the fact that GCN2 proteolytic cleavage by HIV-1Pro leads to the inactivation of the kinase. GCN2 activity inhibits HIV-1 RNA translation We wanted to test whether the activity of GCN2 had an effect on the synthesis of HIV-1 proteins. To this end, we used two plasmids encoding the HIV-1 genome in which luciferase or GFP genes were inserted into the pNL4-3 nef gene (pNL4-3.Luc.R-E- and pNL4-3.GFP.R-E-). First, we co-transfected HeLa cells with HIV-1 constructs (pNL4-3.Luc.R-E- or pNL4-3.GFP.R-E-) or pcDNA-Luc, along with the plasmids encoding either wild type GCN2 or the inactive mutant GCN2-K618R. Luciferase activity and GFP expression were significantly higher in cells over- expressing GCN2-K618R when compared with the ones bearing wild type GCN2 (Figure 4 A and B), suggesting a possible negative effect of GCN2 activity on HIV-1 protein synthesis. We also wanted to analyze whether the absence of GCN2 in cells could affect HIV-1 protein expression. To do so, we transfected the plasmids pNL4-3.Luc.R-E- or pcDNA-Luc in MEFs devoid of GCN2 (Figure 4E) or in HeLa cells showing low levels of GCN2 due to the stable expression of a specific interferring RNA (RNAi 4) (Figure 4 C and D). In both cases the relative luciferase expression from the HIV-1 clone was significantly increased (more than 2-fold) in cells with low or absent GCN2 activity when compared with wild type MEFs or HeLa cells bearing a unspecific RNAi. Moreover, in HeLa cells where GCN2 was knocked down we found high levels of GFP and HIV-1 p24 proteins compared to cells bearing normal levels of GCN2 (Figure 4C). These results suggest that GCN2 activity inhibits HIV-1 protein synthesis. HIV-1 RNA activates GCN2 in cells We performed transfection with the above mentioned HIV-1 RNA-encoding plasmids in a BHK-21 cell line that over-expresses the regulatory subunit of PP1 phosphatase, GADD34, rendering cells with low levels of phosphorylated eIF2alpha, BHK-A1 (Figure S1). In these cells the expression of HIV-1 constructs induced a significant phosphorylation of GCN2 and the subse- quent increase in the levels of phosphorylated eIF2alpha, which was also observed in MEFs and HeLa cells (Figure 5 A, B and C) transfected with plasmids encoding HIV-1 RNA. It is also noteworthy that the amount of GFP, as a reporter of viral protein synthesis, was higher in the BHK-A1 cell line and in GCN2 knock out (GCN22/2) than in wild-type MEFs (Figure 5 A and B). We also observed that luciferase activity expressed from a HIV-1 RNA was higher in BHK-A1 cells, in cells expressing a non-phosphor- ylatable form of eIF2alpha and in GCN22/2 MEFs than in the respective control cells, BHK-C and wild type MEFs (Figure 5 D, E and F). This increase should be caused by differences in translation, given that the levels of the luciferase-encoding HIV-1 RNA did not rise in those cells (Figure 5 D and E). Thus we can conclude that HIV-1 RNA activates GCN2, which in turn phosphorylates eIF2alpha leading to the inhibition of HIV-1 RNA translation. Discussion In eukaryotic cells the translation of mRNAs into proteins is mainly regulated at the initiation step, where regulation of the eIF2 function by phosphorylation of its alpha subunit by specific protein kinases (eIF2alpha kinases) plays a critical role, given that eIF2alpha phosphorylation leads to a general inhibition of protein synthesis. Synthesis of viral proteins relies on the host cell translation machinery and thus is similarly regulated. In fact, phosphorylation of eIF2alpha by activated eIF2alpha kinases PKR and GCN2 has been shown to be an important mechanism for inhibition of viral protein synthesis and viral cycle progression [14,26]. Here we report that the presence of HIV-1 RNA increases GCN2 eIF2alpha kinase activity in vitro and that this increase is dependent on the m2 motif in the HisRS-related domain of the kinase. This effect of a viral RNA on GCN2 activity was previously observed for the genomic RNA of Sindbis virus [14], suggesting that GCN2 is an RNA binding protein which senses the presence in the cells of viral RNAs through its HisRS-related domain in order to activate an antiviral response. Unlike the previous report on Sindbis virus [14], we have not yet identified the RNA sequences of HIV-1 RNA responsible for the activation of GCN2. As shown in this work, the introduction of HIV-1 RNA by transfection of cells with an HIV-1-encoding cDNA produced the activation of GCN2 and a subsequent increase in the levels of phosphorylated eIF2alpha, similar to what was observed in vitro. The consequence of this fact is a reduction in the levels of proteins whose synthesis depends on the translation of HIV-1 RNA and, finally, it could lead to diminished virus production. Its is not surprising that phosphorylation of eIF2alpha led to the inhibition of the viral protein synthesis, as has been widely proven that over- expression and activation of the eIF2alpha kinase PKR produces the same effect [17,27]. The novelty of our results is that the inhibitory phenomenon is due to the activation of GCN2, given that it has been observed by comparison of HIV-1 protein PLOS ONE | www.plosone.org 5 October 2012 | Volume 7 | Issue 10 | e47272