Browsing by Author "Matos, Pedro, 1985-"
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- Shedding light on enveloped viruses entry and its inhibition using optical techniquesPublication . Matos, Pedro, 1985-; Santos, Nuno C., 1972-Enveloped virus attachment and fusion into the host cells constitutes the first and crucial step for the life cycle of a virus and is mediated by the envelope glycoproteins. This Thesis aimed to clarify some important factors that influence this process and the mechanism of action at the molecular level of drugs that inhibit it. First, the mode of action of HIV-1 fusion inhibitor peptides at the membrane level was studied using fluorescence spectroscopy. Interaction of these peptides with human cell membranes was assessed by dipole potential sensitive probes. C34-cholesterol, one of the latest generation fusion inhibitors had the highest cell membrane affinity, followed by T-1249, enfuvirtide and sifuvirtide. Sifuvirtide however showed strong preference for rigid membrane domains, mimicked by dipalmitoylphosphatidylcholine (DPPC), while C34-cholesterol strongly partitioned to cholesterol and sphingomyelin-rich model membranes. The overall results establish a relationship between these drugs antiviral activity and their membranotropism towards raft-like membrane compositions, following the trend C34-cholesterol > T-1249 > enfuvirtide. The capacity of the peptides to concentrate on lipid rafts microdomains, near the cell receptors for the virus, would make them more available to bind the viral gp41 in its exposed conformation. We then focused on another aspect of the HIV-1 entry related to envelopereceptor interactions, namely with glycosaminoglycans. Their general structural requirements of that enable the binding to gp120 were determined by surface plasmon resonance. Binding was found to be dependent on sequence type and size, preferring heparin and heparan sulfate, with an extension of at least 12 monomers. Sulfate groups were essential for the interaction. These results help to understand some basic mechanisms of viral attachment. Although envelope-receptor interactions play a pivotal role in the viral entry, lipids are also relevant. The role of acidic lipids in the viral fusion kinetics of vesicular stomatitis pseudoviruses (VSV) was assessed by single-particle imaging x on supported lipid bilayers. Phosphatidylserine (PS) and bis(monoacylglycero)phosphate (BMP) greatly enhanced the efficiency of hemifusion and enabled full fusion. The lag between lipid and content mixing defining the lifetime of a hemifusion intermediate were significantly shorter for BMP-containing compared to PS-containing bilayers. BMP is a late endosomeresident lipid and its effect on promoting VSV fusion makes it an important modulator on the outcome of viral entry through endocytosis. Evolving from model membranes to cells, the internalization and fusion of a retrovirus through endocytosis was imaged. A pH-sensor consisting of two fused fluorescent proteins mTFP1-eYFP was incorporated in the envelope of an avian sarcoma and leucosis virus (ASLV). The pH values were quantified along the virus track and varied from 5.6 to 6.5 in early endosomes. Single-virus tracking showed that ASLV, upon internalization, are sorted into distinct pools of early endosomes with different dynamics, depending on using the two receptor isoforms, TVA800 or TVA950. Overall, this Thesis sheds light on important host factors that influence enveloped virus entry and fusion, establishing lipid binding as a key player in the action of peptide fusion inhibitors.