HIV-1 Counteracts GCN2 Inhibitory Effect Figure 3. HIV-1 cleavage inhibits GCN2 eIF2alpha kinase activity. (A) Affinity purified human GCN2 was incubated for 30 min at 30uC in the absence or the presence of HIV-1Pro and in the absence or the presence of 2.5 mM SQ, as indicated, prior to being assayed in an eIF2alpha kinase assay (lanes 1–5). In lane 6, SQ was added after incubation of GCN2 with the protease, just before initiating the eIF2alpha kinase assay. HIV-1 RNA was only present during the kinase assay. Incubation was stopped by addition of SDS-PAGE sample buffer and proteins were analyzed by western blot using specific antisera to detect eIF2alpha phosphorylated on serine 51, total eIF2alpha, and phosphorylated and total GCN2. Similar results were obtained from a duplicate experiment. (B) Schematic representation of the structural domains of human GCN2 and the generated GCN2 fragments according to the HIV-1Pro cleavage site. The 1649 amino acid GCN2 sequence is illustrated by a larger box and the figure is drawn to scale. Highlighted domains include the N-terminal (black); the ‘Pseudokinase’ (dark grey) that is related to sub-domains I–XI of eukaryotic protein kinases; the conserved two lobes of the eIF2alpha kinase domain (black), separated by a large insert (light grey); the HisRS-like domain (dark grey); and a C-terminal domain (light grey). The numbers refer to the amino acid residues. (C) In vitro eIF2alpha kinase assay of purified full-length human GCN2 (1–1649) and the generated GCN2 fragments according to the HIV-1Pro cleavage site (1–560 and 561–1649) in the absence or the presence of HIV-1 RNA as indicated. Proteins were analyzed by western blot using specific antisera to detect eIF2alpha phosphorylated on serine 51, total eIF2alpha, and full-length GCN2 or fragments (anti-myc). Similar results were obtained from a duplicate experiment. doi:10.1371/journal.pone.0047272.g003 expression between wild type and GCN2 knock out cells, and in BHK-21 cells (control and over-expressing an eIF2alpha phos- phatase activator) in which PKR expression is extremely low compared with other cell types [28]. Thus, the absence of GCN2 in normal cells or the maintenance of low levels of phosphorylated eIF2alpha in cells with a very low presence of PKR favors the expression of HIV-1 proteins. The relevance of PKR activation and eIF2alpha phosphoryla- tion in HIV-1 protein expression and viral cycle progression is illustrated by a number of countermeasures employed by the virus to prevent PKR response, including viral products and cellular proteins [17]. Therefore, PKR is inhibited by the presence of large amounts of viral TAR RNA and by binding of the HIV-1 Tat protein [29], but also by direct interaction with two cellular RNA binding proteins, the TAR RNA binding protein (TRBP) [30] and adenosine deaminase acting on RNA 1 (ADAR1) [27,31]. These mechanisms seem to make the innate antiviral response ineffective and allow the virus to replicate in permissive cells. Here we show that in cells (MT-2 and PBMC) infected with HIV-1, GCN2 is proteolytically degraded and that its proteolytic cleavage is prevented by the presence of the specific inhibitor of HIV-1Pro saquinavir. Moreover, the presence of saquinavir increased eIF2alpha phosphorylation, probably due to the activation of uncleaved GCN2. Since GCN2 levels seem to increase with PHA treatment and HIV-1 infection, we can speculate that activation of cells and HIV-1 infection can promote the establishment of an antiviral state that includes GCN2 activation and eIF2alpha phosphorylation, being in part counteracted by the proteolytic degradation of GCN2. Theses findings are consistent with previous reports showing that HIV-1Pro cleaves other proteins involved in the regulation of protein synthesis, such as the translation initiation factors eIF4GI and PABP [20,24]; this cleavage inhibits cellular protein synthesis, but allows translation of viral RNA [32] which in turn also needs active eIF2alpha. In good agreement with these observations, is the fact that during HIV-1 infection the breakage of GCN2 showed kinetics coinciding with those observed for eIF4GI. Our results demonstrate that HIV-1Pro cleavage of GCN2 renders an inactive eIF2alpha kinase, although, according to the break site identified, the C-terminal portion of the protein retains the eIF2alpha kinase and HisRs-related domains, responsible for the activity and the activation of the protein, respectively. In fact, it seems that this fragment has some residual activity compared to the full-length protein. This apparent discrepancy could be explained by the possible existence of an additional minor cleavage site in the kinase or the HisRS-related domain. Due to the low sequence identity between HIV-1Pro known substrates and also to the lack of an obvious consensus cleavage motif [33,34], it is difficult to say if GCN2 is a good substrate for the viral protease, although the cleavage site in GCN2 meets the apparent requirement of hydrophobic amino acids. Our results in HeLa cell-free extracts showed that GCN2 was completely broken after 15 min of protease treatment, whereas after 30 min significant amounts of eIF4GI and, especially, PABP remained intact, suggesting that GCN2 is an excellent substrate for the protease and that its specific breakage could favor HIV-1 cell cycle progression. All these findings taken together would point to the following plausible scenario for the role of GCN2 in the antiviral response against HIV-1: in the late phase of the viral life cycle, when there is a huge production of viral mRNA to be translated into proteins for virion assembly and maturation, viral RNAs could activate GCN2, which subsequently would phosphorylate eIF2alpha, leading to the inhibition of viral protein synthesis. The translation of some viral mRNAs brings about the appearance of HIV-1Pro, which inactivates GCN2 by proteolytic cleavage, preventing phosphor- ylation of eIF2alpha and allowing the translation of viral proteins and the progression of the viral cycle. It is possible that the discovery of specific activators of GCN2 could help prevent virus replication and the spread of infection. Materials and Methods Plasmids and in vitro transcription The pTM1-HIV-1 PR plasmid containing a cDNA fragment encoding the gene of HIV-1 protease in the pTM1 vector [35] has been described previously [20]. The plasmids pT7SV-HIV-1PR, pT7SV-2Apro and pT7SVwt were also described previously [25]. The cDNA containing the entire open reading frame of human GCN2 was subcloned into the plasmid pcDNA3.1/Myc-His (Invitrogen) by PCR amplification combined with restriction endonuclease cuts from different cDNA partial clones. This cDNA was used to generate the plasmids encoding the fragments of human GCN2 corresponding to amino acids 1–560 and 561– 1649. The plasmids pGST-mGCN2 (419–1024) and pGST- hGCN2 (525–1024) were constructed by cloning the PCR- amplified cDNA fragments encoding the amino acids 419 to 1024 of mouse GCN2 and 525 to 1024 of human GCN2, respectively, into the plasmid pGEX-4T (GE Healthcare). The HIV-1 cDNA cloned into the plasmid pBH10 [36] was subcloned into the plasmid BlueScript KS (+/2) (Stratagene) in order to allow its transcription from a T7 promoter (pBS-HIV-1). Plasmids pT7SV-HIV-1PR, pT7SV-2Apro, pT7SVwt and pBS-HIV-1 were linearized and in vitro transcribed using the T7 RNA polymerase kit (Promega) in order to produce capped recombinant genomic Sindbis virus (SV) mRNAs and genomic- length HIV-1 RNA. The following reagent was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: pNL4-3.Luc.R-E- from Dr. Nathaniel Landau [37,38]. An equivalent construct in which the firefly luciferase gene PLOS ONE | www.plosone.org 7 October 2012 | Volume 7 | Issue 10 | e47272