Influenza

Biography

Biography: Remigiusz Worch

Abstract

Membrane fusion induced by hemagglutinin fragment, the so-called fusion peptide (HAfp), and host RNA cleavage by viral polymerase are the key steps of influenza replication. A 20-amino acid HAfp1-20 peptide has a boomerang-like shape, however it has been shown recently that extending HAfp1-20 by three more conservative residues (to HAfp1-23) leads to a helical hairpin formation. We determined partition coefficients Kx for a series of peptides in their native forms using tryptophan fluorescence. HAfp1-23 showed more favorable interaction than HAfp1-20 with DOPC at pH 7.4, but at endosomal pH 5.0 the difference was negligible. Both peptides lead to liposome content leakage in a similar fashion, as measured in single giant unilamellar vesicles (GUV) using fluorescence microscopy. Nevertheless HAfp1-23 had larger liposome fusion capacity, as concluded from FRET experiments and showed a distinct lipid bilayer distortion by fluorescence lifetime imaging. Despite intensive studies on endonucleolitic polymerase domain (PA-Nter), the existing results on divalent ion preference are contradictory. We quantified the PA-Nter cleavage reaction rates by fluorescence cross-correlation spectroscopy (FCCS). This microscopic technique provides rapid, highly sensitive and real-time monitoring of single molecule interactions. In the regime of enzyme excess, using ss-DNA at nanomolar concentrations, we determined the maximum reaction rates at 0.51 and 0.77 nM/min for Mg2+ and Mn2+, respectively. Our results show the superiority of FCCS technique for real-time kinetic analysis over the electrophoretic assays. Presented studies constitute a step towards better understanding of fusion and RNA cleavage mechanisms. Supported by Scientific Polpharma Foundation, 2012/07/D/NZ1/04255 and Foundation for Polish Science grants.