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The hepatitis C virus (HCV) nonstructural protein 3-4A (NS3-4A) protease is a key component of the viral replication complex and the target of protease inhibitors used in current clinical practice. By cleaving and thereby inactivating selected host factors it also plays a role in the persistence and pathogenesis of hepatitis C. Here, we describe ovarian cancer immunoreactive antigen domain containing protein 1 (OCIAD1) as a novel cellular substrate of the HCV NS3-4A protease. OCIAD1 was identified by quantitative proteomics involving stable isotopic labeling using amino acids in cell culture coupled with mass spectrometry. It is a poorly characterized membrane protein believed to be involved in cancer development. OCIAD1 is cleaved by the NS3-4A protease at Cys 38, close to a predicted transmembrane segment. Cleavage was observed in heterologous expression systems, the replicon and cell culture-derived HCV systems, as well as in liver biopsies from patients with chronic hepatitis C. NS3-4A proteases from diverse hepacivirus species efficiently cleaved OCIAD1. The subcellular localization of OCIAD1 on mitochondria was not altered by NS3-4A-mediated cleavage. Interestingly, OCIAD2, a homolog of OCIAD1 with a cysteine residue in a similar position and identical subcellular localization, was not cleaved by NS3-4A. Domain swapping experiments revealed that the sequence surrounding the cleavage site as well as the predicted transmembrane segment contribute to substrate selectivity. Overexpression as well as knock down and rescue experiments did not affect the HCV life cycle in vitro, raising the possibility that OCIAD1 may be involved in the pathogenesis of hepatitis C in vivo.

Apolipoprotein E (ApoE), an exchangeable apolipoprotein, is necessary for production of infectious Hepatitis C virus (HCV) particles. However, ApoE is not the only liver-expressed apolipoprotein and the role of other apolipoproteins for production of infectious HCV progeny is incompletely defined. Therefore, we quantified mRNA expression of human apolipoproteins in primary human hepatocytes. Subsequently, cDNAs encoding apolipoproteins were expressed in 293T/miR-122 cells to explore if they complement HCV virus production in cells that are non-permissive due to limiting endogenous levels of human apolipoproteins. Primary human hepatocytes expressed high mRNA levels of ApoA1, A2, C1, C3, E, and H. ApoA4, A5, B, D, F, J, L1, L2, L3, L4, L6, M, and O were expressed at intermediate levels, and C2, C4, and L5 were not detected. All members of the ApoA and ApoC family of lipoproteins complemented HCV virus production in HCV transfected 293T/miR-122 cells, albeit with significantly lower efficacy compared with ApoE. In contrast, ApoD expression did not support production of infectious HCV. Specific infectivity of released particles complemented with ApoA family members was significantly lower compared with ApoE. Moreover, the ratio of extracellular to intracellular infectious virus was significantly higher for ApoE compared to ApoA2 and ApoC3. Since apolipoproteins complementing HCV virus production share amphipathic alpha helices as common structural features we altered the two alpha helices of ApoC1. Helix breaking mutations in both ApoC1 helices impaired virus assembly highlighting a critical role of alpha helices in apolipoproteins supporting HCV assembly. In summary, various liver expressed apolipoproteins with amphipathic alpha helices complement HCV virus production in human non liver cells. Differences in the efficiency of virus assembly, the specific infectivity of released particles, and the ratio between extracellular and intracellular infectivity point to distinct characteristics of these apolipoproteins that influence HCV assembly and cell entry. This will guide future research to precisely pinpoint how apolipoproteins function during virus assembly and cell entry.

The broad-spectrum antiviral arbidol (Arb) inhibits cell entry of enveloped viruses by blocking viral fusion with host cell membrane. To better understand Arb mechanism of action, we investigated its interactions with phospholipids and membrane peptides. We demonstrate that Arb associates with phospholipids in the micromolar range. NMR reveals that Arb interacts with the polar head-group of phospholipid at the membrane interface. Fluorescence studies of interactions between Arb and either tryptophan derivatives or membrane peptides reconstituted into liposomes show that Arb interacts with tryptophan in the micromolar range. Interestingly, apparent binding affinities between lipids and tryptophan residues are comparable with those of Arb IC50 of the hepatitis C virus (HCV) membrane fusion. Since tryptophan residues of membrane proteins are known to bind preferentially at the membrane interface, these data suggest that Arb could increase the strength of virus glycoprotein's interactions with the membrane, due to a dual binding mode involving aromatic residues and phospholipids. The resulting complexation would inhibit the expected viral glycoprotein conformational changes required during the fusion process. Our findings pave the way towards the design of new drugs exhibiting Arb-like interfacial membrane binding properties to inhibit early steps of virus entry, i.e., attractive targets to combat viral infection.

Staphylococcus aureus necrotizing pneumonia is a severe disease caused by S. aureus strains carrying the Panton Valentine leukocidin (PVL) genes (lukS-PV & lukF-PV) encoded on various bacteriophages (such as phiSLT). Clinical PVL+ strains isolated from necrotizing pneumonia display an increased attachment to matrix molecules (type I and IV collagens and laminin), a phenotype that could play a role in bacterial adhesion to damaged airway epithelium during the early stages of necrotizing pneumonia (J Infect Dis 2004; 190: 1506-15). To investigate the basis of the observed adhesion of S. aureus PVL+ strains, we compared the ability of PVL+ and their isogenic PVL(-) strains to attach to various immobilized matrix molecules. The expression of recombinant fragments of the PVL subunits and the addition of synthetic peptides indicated that the processed LukS-PV signal peptide (LukS-PV SP) was sufficient to significantly enhance the ability of S. aureus to attach to extracellular matrix (ECM) components. Furthermore, we showed that adhesion to ECM components was inhibited by heparin and heparan sulfates (HS) suggesting that in vivo, HS could function as a molecular bridge between the matrix and S. aureus expressing the LukS-PV signal peptide. Site directed mutagenesis, biochemical and structural analyses of the LukS-PV signal peptide indicate that this peptide is present at the S. aureus surface, binds to HS in solid phase assay, and mediates the enhanced S. aureus matrix component adhesion. Our data suggests that after its cleavage by signal peptidase, the signal peptide is released from the membrane and associates to the cell wall through its unique C-terminus sequence, while its highly positively charged N-terminus is exposed on the bacterial surface, allowing its interaction with extracellular matrix-associated HS. This mechanism may provide a molecular bridge that enhances the attachment of the S. aureus PVL+ strains to ECM components exposed at damaged epithelial sites.