Benzophenone-containing fatty acids and their related photosensitive fluorescent new probes: design, physico-chemical properties and preliminary functional investigations.

Hydrophobic photoaffinity labeling is a powerful strategy to identify hydrophobic segments within molecules, in particular membrane proteins. Here we report the design and synthesis of a novel family of fluorescent and photosensitive lipid tools, which have a common amino acid scaffold functionalized by three groups: (i) a first fatty acid chain grafted with a photoactivatable benzophenone moiety (Fatty Acid BenzoPhenone, FABP), (ii) a second fatty acid chain to ensure anchoring into a half-bilayer or hydrophobic environment, and (iii) a fluorescent carboxytetramethylrhodamine headgroup (CTMR) to detect the photolabeled compound. We present data of the synthesis and characterization of three lipid tools whose benzophenone ring is situated at various distances from the central scaffold. We could therefore establish structure/properties relationships dependent upon the depth of insertion of benzophenone into the membrane. Our lipid tools were extensively characterized both physico- and bio-chemically, and we assessed their functionality in vitro using bacterioRhodopsin (bR). We thus provide the scientific community with novel and reliable tools for the identification and study of hydrophobic regions in proteins.

Characterization of hepatitis C virus pseudoparticles by cryo-transmission electron microscopy using functionalized magnetic nanobeads.

Cell entry and membrane fusion of the hepatitis C virus (HCV) depend on its envelope glycoproteins E1 and E2. HCV pseudotyped particles (HCVpps) are relevant and popular models to study the early steps of the HCV life cycle. However, no structural characterization of HCVpp has been available so far. Using cryo-transmission electron microscopy (cryo-TEM), providing structural information at nanometric resolution, the molecular details of HCVpps and their fusion with liposomes were studied. Cryo-TEM revealed HCVpps as regular 100 nm spherical structures containing the dense retroviral nucleocapsid surrounded by a lipid bilayer. E1-E2 glycoproteins were not readily visible on the membrane surface. Pseudoparticles bearing the E1-E2 glycoproteins of Semliki forest virus looked similar, whereas avian influenza A virus (fowl plague virus) haemagglutinin/neuraminidase-pseudotyped particles exhibited surface spikes. To further characterize HCVpp structurally, a novel method was designed based on magnetic beads covered with anti-HCV antibodies to enrich the samples with particles containing E1-E2. This strategy efficiently sorted HCVpps, which were then directly observed by cryo-TEM in the presence or absence of liposomes at low or neutral pH. After acidification, HCVpps looked the same as at neutral pH and closely contacted the liposomes. These are the first visualizations of early HCV membrane fusion events at the nanometer scale. Furthermore, fluorimetry analysis revealed a relative resistance of HCVpps regarding their fusion capacity when exposed to low pH. This study therefore brings several new molecular details to HCVpp characterization and this efficient strategy of virion immunosorting with magnetic nanobeads is direct, efficient and adaptable to extensive characterization of any virus at a nanometric resolution.

Bioinformatic analysis of the hepadnavirus e-antigen and its precursor identifies remarkable sequence conservation in all orthohepadnaviruses.

The hepatitis B e-antigen (HBeAg) is a non-particulate secretory protein expressed by all viruses within the family Hepadnaviridae. It is not essential for viral assembly or replication but is important for establishment of persistent infection in vivo. Although the exact mechanism(s) by which the HBeAg manifests chronicity are unclear, the HBeAg elicits both humoral and cell-mediated immunity, down-regulates the innate immune response to infection, as well as functioning as a T cell tolerogen and regulating the immune response to the intracellular nucleocapsid. A bioinformatics approach was used to show that the HBeAg and precursory genetic codes share remarkable sequence conservation in all mammalian-infecting hepadnaviruses, irrespective of host, genotype, or geographic origin. Whilst much of this sequence conservation was within key immunomodulatory epitopes, highest conservation was observed at the unique HBeAg N-terminus, suggesting this sequence in particular may play an important role in HBeAg function.

The hepatitis C virus and its hepatic environment: a toxic but finely tuned partnership.

Twenty years after its discovery, HCV (hepatitis C virus) still infects 170 million people worldwide and cannot be properly treated due to the lack of efficient medication. Its life cycle must be better understood to develop targeted pharmacological arsenals. HCV is an enveloped virus bearing two surface glycoproteins, E1 and E2. It only infects humans through blood transmission, and hepatocytes are its only target cells. Hepatic trabeculae are formed by hepatocyte rows surrounded by sinusoid capillaries, irrigating hepatic cells. Hepatocytes are polarized and have basolateral and apical poles, separated by tight junctions in contact with blood and bile respectively. In blood, HCV remains in contact with lipoproteins. It then navigates through hepatic microenvironment and extracellular matrix, composed of glycosaminoglycans and proteins. HCV then encounters the hepatocyte basolateral membrane, where it interacts with its entry factors: the low-density lipoprotein receptor, CD81 tetraspanin, and the high-density lipoprotein (scavenger) receptor SR-BI (scavenger receptor BI). How these molecules interact with HCV remains unclear; however, a tentative sequence of events has been proposed. Two essential factors of HCV entry are the tight junction proteins claudin-1 and occludin. Cell polarity therefore seems to be a key for HCV entry. This raises several exciting questions on the HCV internalization pathway. Clathrin-dependent endocytosis is probably the route of HCV transport to intracellular compartments, and the ultimate step of its entry is fusion, which probably takes place within endosomes. The mechanisms of HCV membrane fusion are still unclear, notably the nature of the fusion proteins is unknown and the contribution of HCV-associated lipoproteins to this event is currently under investigation.