Hypoxia up-regulated protein 1 (HYOU1) is an inducible ER chaperone protein that is upregulated in response to cellular stresses including the unfolded protein response and hypoxia

Hypoxia up-regulated protein 1 (HYOU1) is an inducible ER chaperone protein that is upregulated in response to cellular stresses including the unfolded protein response and hypoxia. dengue virus infection modulated the amounts of proteins involved in TUG-891 the interferon and unfolded protein responses, lipid metabolism and the cell cycle. The SILAC-MS results were validated for a select number of proteins over a time course of infection by Western blotting and immunofluorescence microscopy. Our study demonstrates for the first time the power of SILAC-MS for identifying and quantifying novel changes in cellular protein amounts in response to dengue virus infection. Introduction The four serotypes of dengue virus (DENV types 1C4) cause the most important arthropod-borne viral disease of humans. DENV infection results in a range of clinical outcomes ranging from the milder dengue fever to the potentially life threatening dengue haemorrhagic fever/dengue shock syndrome [1]. A recent study estimates that up to 390 million people are infected with DENV annually [2], making dengue a serious global public-health problem. Despite much effort, there are neither vaccines nor antiviral therapies in clinical use to prevent or treat dengue, and our understanding of dengue pathogenesis is still limited. DENV is a member of the genus of the family and has a RNA genome of 11 kb in size. Translation of the genome results in the production of a TUG-891 single large polyprotein that is subsequently processed by a combination of cellular and the viral NS2B/3 proteinase to yield the three structural proteins capsid (C), pre-membrane (prM) and envelope (E) and the non-structural (NS) proteins, NS1, NS2A, NS2B, NS3, NS4A, 2K, NS4B and NS5 [3]. Replication of the DENV genome occurs in intimate association with perinuclear ER membranes which are modified to form characteristic structures during virus infection [4]. High-throughput RNA interference studies have shown that DENV depends heavily on the cellular machinery for replication [5], [6]. However the mechanisms by which DENV interacts with cellular pathways and the viral and cellular proteins involved, largely remain to be determined. Comparative analysis of the gene expression profiles of a range of cell types infected with DENV for 5 min at 4 C. The cytoplasmic fractions were removed, added to an equal volume of 2X SDS-PAGE sample buffer and heated at 95 C for 10 min. The nuclear pellets were resuspended in 3 ml of buffer S1 (0.25 M sucrose, 10 mM MgCl2), layered over a 3 ml cushion of buffer S2 (0.35 M sucrose, 0.5 mM MgCl2) and centrifuged at 1500 for 5 min at 4C. The supernatant was removed and the nuclear pellet resuspended in 200 l of buffer S2 followed by disruption of the nuclei by sonication (320 sec) using a Bioruptor (Diagenode, Belgium). The protein concentration in each fraction was determined using a BCA Protein Assay kit (Pierce – Thermo Scientific). Twenty g of protein from the cytoplasmic fraction prepared from the DENV-2 infected and mock infected cells were mixed and the process repeated for the nuclear fractions. The proteins in the two samples were then separated by one-dimensional SDS-PAGE and stained using Coomassie blue. Each of the lanes was excised and used for LC-MS/MS analysis. LC-MS/MS analysis Each gel lane was cut into 10 slices and each slice subjected to in-gel tryptic digestion Melanotan II Acetate using a ProGest automated digestion unit (Digilab, UK). The resulting peptides were fractionated using a Dionex Ultimate 3000 nanoHPLC system in line with an LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific). In brief, peptides in 1% (v/v) formic acid were injected onto an Acclaim PepMap C18 nano-trap column (Dionex). After washing with 0.5% (v/v) TUG-891 acetonitrile 0.1% (v/v) formic acid, peptides were resolved on a 250 mm75 m Acclaim PepMap C18 TUG-891 reverse phase analytical column (Dionex) over a 150 min organic gradient, using 7 gradient segments (1C6% solvent B over 1 min, 6C15% B over 58 min, 15C32% B over 58 min, 32C40% B over 3 min, 40C90% B over 1 min, held.