were derived from healthy voluntary donors, and obtained from leukophoresed blood by FicollTM gradient (Pharmacia Fine Chemicals, Uppsala, Sweden) and elutriated by centrifugation. After washing with phosphate buffered saline (PBS), cells were seeded in RPMI 1640 medium (Biochrom AG) with 10% FBS, 1% L-glutamine, antibiotics, and 50 IU/ml IL-2. PBMC were stimulated for 48 h with phytohemaglutinine (2 mg/ml) and interleukin 2 (IL-2) (100 IU/ml). All cell lines were cultured at 37uC in a 5% CO2 atmosphere. PBMC were kindly provided by the Spanish HIV BioBank integrated in the Spanish AIDS Research Network (RIS) 1 [41]. PBMC and MT-2 cells were infected or mock-infected with 10 or 100 ng of p24gag per 106 cells of HIV-1NL4–3 for 2 h (100 ng of p24gag per 106 cells has been evaluated to be equivalent to 1–2 viral particles per cell) [42], washed three times and incubated in fresh medium. At 1 to 5 days post-infection (dpi), cells were harvested and washed 3 times with PBS, and the dry pellet was stored at 280uC before lysis. BHK-21 cells were electroporated with 30 mg of recombinant SV genomic RNA in a final volume of 50 ml as described in [25]. Electroporated cells were maintained in growth medium, and, at 4 h post-electroporation, metabolically labelled with [35S]Met/Cys mixture (Promix, Amersham), as described previously [14] and lysed in sample buffer. Coupled infection/DNA transfection of COS-7 cells with recombinant vaccinia virus (vvT7), pTM1- derived plasmids and plasmids encoding human and mouse GCN2, have previously been described in detail [43]. For transient transfections, HEK 293T and HeLa cells were plated on 60-mm dishes and transfected with plasmids (6–12 mg/ dish) using LipofectamineTM and PlusTM Reagents (Invitrogen) according to the manufacturer’s instructions. Similarly, BHK-21 and MEF cells were transfected using the cationic polymer reagent jetPEITM (PolyPlus-transfection) according to the manufacturer’s instructions. Virus production and titration Virus stocks were prepared by amplification of HIV-1NL4-3 in MT-2 cell line (ATCC) or PHA+IL-2 activated PBMC. Physical titers of viral stocks were evaluated by quantification of HIV p24gag by ELISA kit (Innotest HIV-1 antigen mAb; Innogenetics) [44]. Lentivirus and stable transduction of shRNAs The following targeted shRNAs in lentiviral construct plasmid pLKO.1-Puro [45] were purchased from Sigma-Aldrich: GCN2 (XM_496066) Clone 1 TRCN0000078648, Clone 2 TRCN0000078649, Clone 3 TRCN0000078650, Clone 4 TRCN0000078651, Clone 5 TRCN0000078652. Lentiviruses were produced by co-transfection of HEK293T Phoenix cells with pLKO.1-Puro shRNA-encoding plasmids, pMD2G (Addgene) and pSPAX2 (Addgene), using jetPEITM (PolyPlus-transfection). Viruses were harvested at 24 and 48 h post-transfection and infections of HeLa cells were carried out in the presence of 8 mg/ ml of polybrene (Sigma-Aldrich). Following transduction, cells were selected with 1 mg/ml puromycin (Sigma-Aldrich). Protein purification For purification of GST fusion proteins, Escherichia coli BL21 cells were transformed with plasmids pGST-mGCN2 (419–1024) and pGST-hGCN2 (525–1024), lysed by sonication, and the fusion proteins were then purified as previously described [46] using glutathione-sepharose (GE Healthcare). Purification of Myc- and 6xHis-tagged GCN2 full-length, GCN2-K618R, GCN2-m2 and GCN2 fragments was done as previously described [14]. HIV- 1Pro was provided by I. Pichova through the Centralised Facility HIV-1 Counteracts GCN2 Inhibitory Effect for AIDS Reagents. Purified HIV-2 protease (HIV-2Pro) [47] was obtained through the NIH (National Institutes of Health) AIDS Research and Reference Reagent Program, Division of AIDS, NIAID (National Institute of Allergy and Infectious Diseases), NIH, Bethesda, MD, U.S.A., from Dr Bret Shirley and Mr Michael Cappola (Boehringer Ingelheim Pharmaceuticals). Protease cleavage assays Crude HeLa S10 extracts were incubated with 2.5 ng/ml of recombinant HIV-1Pro and reactions were stopped by adding sample buffer. In other experiments, purified preparations of mouse or human Myc- and 6xHis-tagged GCN2 were incubated with 2.5 ng/ml of recombinant HIV-1Pro, or HIV-2Pro in cleavage buffer (50 mM sodium phosphate, pH 6.0, 25 mM NaCl, 5 mM EDTA and 1 mM DTT) for 3 h at 30uC. In order to map the cleavage sites of HIV-1Pro on GCN2, 50 mg of recombinant GST– tagged mouse or human GCN2 protein were incubated with 1 mg of recombinant HIV-1Pro in a total volume of 50 ml for 3 h at 30uC in cleavage buffer. Cleavage products were separated by SDS- PAGE, transferred to a PVDF membrane (ImmobilonH–P, Millipore) and then subjected to automated Edman degradation with an Applied Biosystems Procise Sequencer in the Proteomics Service of Centro de Investigaciones Biolo´ gicas (CSIC). In vitro kinase activity Affinity purified GCN2 wild type and mutants (m2 and K618R) were assayed for their ability to phosphorylate eIF2alpha in the presence or the absence of HIV-1 RNA, as previously described [14]. In some experiments 32P-c-ATP was included in the kinase reaction mix and phosphorylated eIF2alpha was detected by autoradiography of the corresponding SDS-PAGE dried gel. In other kinase assays only non-radiactive ATP was used and phosphorylated eIF2alpha and GCN2 were detected by western blot using phosphospecific antibodies. Western blotting Proteins subjected to SDS-PAGE were transferred to a PVDF membrane (ImmobilonH–P, Millipore) for western blot analysis. Western blots were performed using the following antibodies: mouse anti-myc (Sigma); rabbit anti-eIF2alpha phospho-Ser51 (Cell Signaling); rabbit anti-eIF2alpha (Santa Cruz); rabbit anti- GCN2 phospho-Thr898 (Cell Signaling, Abcam); rabbit anti- GCN2 [8]; eIF4GI anti-serum raised against peptides derived from the C-terminal region of the human eIF4GI [43]; rabbit anti- sera raised against the C- and N-terminal region of human PABP [48]; and mouse ascites of a monoclonal antibody against HIV-1 p24 antigen (Centralised Facility for AIDS Reagents, NIBSC, Potters Bar, Herts., U.K.). Goat anti-(rabbit IgG) and goat anti- (mouse IgG) antibodies coupled to peroxidase (Promega) were also used. Immunoreactive bands were visualized by using ECLTM and ECL Plus Western Blotting Detention Systems (GE Healthcare) and autoradiography (Agfa Curix RP2 Plus). Luciferase activity determination After different times post-transfection, cells were washed twice with PBS and lysed using Passive Lysis Buffer (Promega). After removal of cell debris, equal amounts of total protein were used to measure luciferase activity (Luciferase Assay System, Promega) in the lysates. RNA extraction, reverse transcription and real-time PCR Total RNA was extracted using the SV Total RNA Isolation System (Promega), following the manufacturer’s instructions. For PLOS ONE | www.plosone.org 10 October 2012 | Volume 7 | Issue 10 | e47272