HIV-1 Counteracts GCN2 Inhibitory Effect Figure 1. HIV-1 RNA activates GCN2 and the kinase is proteolytically cleaved during HIV-1 infection. (A) Purified mouse GCN2 was subjected to in vitro eIF2alpha kinase assay in the presence of increasing concentrations of in vitro transcribed RNAs corresponding to the mouse GCN2 open reading frame (as a negative control), or to the genomic RNAs of Sindbis virus or HIV-1, respectively, as indicated. In this experiment 32P- c-ATP was included in the kinase reaction mix and phosphorylation of eIF2alpha was visualized after analysis by SDS-PAGE and autoradiography. (B) In vitro eIF2alpha kinase assay of purified wild type GCN2 (WT), GCN2-K618R (K618R) or GCN2-m2 (m2) in the presence of Sindbis virus (SV) or HIV-1 RNA, as indicated. Results were obtained by western blot analysis of the samples using different antisera to detect eIF2alpha phosphorylated on serine 51, total eIF2alpha and phosphorylated and total GCN2. (C) MT-2 cells were mock infected (2) or infected with 10 or 100 ng of p24gag per 106 cells of HIV-1NL4–3 and harvested at the indicated days post-infection (D.P.I.). Aliquots of cell lysates containing equal amount of proteins were analyzed by western blot (8% SDS-PAGE) using specific antibodies as indicated. (D) MT-2 cells were mock-infected (2) or infected with 100 ng of HIV- 1 NL4-3 per 106 cells, in the absence or the presence of the indicated concentrations of saquinavir (SQ) and harvested at day 3 post-infection. Aliquots of cell lysates containing equal amounts of proteins were analyzed by immunoblot (10% SDS-PAGE) using specific antibodies as indicated. (E) PHA- stimulated PBMCs were mock-infected (2) or infected with 100 ng of HIV-1 NL4-3 per 106 cells, in the absence or the presence of 0.5 mM SQ and harvested at the indicated D.P.I. Aliquots of cell lysates containing equal amounts of proteins were analyzed by immunoblot using specific antibodies as indicated. Results are representative of at least three independent experiments. doi:10.1371/journal.pone.0047272.g001 cleaved both mouse and human GCN2 at the same, or a very close, site. Identification of the HIV-1 protease cleavage site on GCN2 Given that HIV-1Pro produced a direct proteolytic break in GCN2, we next tried to identify the precise site/s of cleavage. Based on the size of the major GCN2 fragment obtained by the action of the protease, we constructed bacterial expression plasmids in order to express and purify GST fusion proteins bearing a portion of mouse (aa 419–1024) or human (aa 525– 1024) GCN2, which was supposed to contain the cleavage site. We used truncated recombinant proteins due to the low yield of full- length GCN2 protein in bacteria. Protease treatment produced a major carboxy-terminal fragment with an apparent molecular weight of about 67 kDa in both mouse and human proteins (Figure 2E). In the case of the human protein there was a minor additional product of about 73 kDa which could account for a secondary cleavage site. The major 67 kDa polypeptide was subjected to amino-terminal sequencing by using the Edman degradation method, which rendered the amino acid sequences VETVIP for the human protein and IETVIP for the mouse protein, which correspond to a cleavage site between residues 560 and 561 or 559 and 560, respectively. Thus the cleavage sites identified are compatible with the size of the carboxi-terminal PLOS ONE | www.plosone.org 3 October 2012 | Volume 7 | Issue 10 | e47272