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Oddziaływanie pomiędzy peptydem fuzyjnym a domeną transbłonową hemaglutyniny : praca doktorska
Creator: Contributor: Publisher:Instytut Biologii Doświadczalnej im. Marcelego Nenckiego PAN
Place of publishing: Date issued/created: Description:116 pages : illustrations ; 30 cm ; Bibliography ; Summaries in English
Degree name: Degree discipline : Degree grantor:Nencki Institute of Experimental Biology PAS ; degree obtained: 2025
Type of object: Subject and Keywords:Fusion Peptide ; Hemagglutinin ; Influenza viruses ; Transmembrane domain ; Viral fusion
Abstract:
Hemagglutinin (HA) is one of the major surface proteins of the influenza A virus. During maturation, HA0 precursor is cleaved into two subunits, HA1 and HA2 subunits. They perform different functions: HA1 is responsible for the receptor binding at the host cell surface, while HA2 mediates viral fusion in the late endosomes. The N-terminal part of the HA2 subunit acts as a fusion peptide (FP). Under low pH conditions in the late endosomes, FP is exposed and initiates fusion. The C-terminal part of the HA2 is transmembrane domain (TMD), which anchors the protein in the membrane and possibly performs an important function in the fusion process. So far, 18 subtypes of HA have been identified, classified into two phylogenetic groups based on the amino acid sequence of the protein. Currently, the most prevalent subtypes circulating in the human population are H1 (group 1) and H3 (group 2). While FP sequences are conserved between subtypes, TMD sequences diverge significantly. Although the role of FP in the fusion process has been well characterized, the function of TMD still remains poorly understood. It has been shown that the presence of TMD is essential for the complete fusion process of the viral envelope with late endosomal membranes. Some studies suggest that an interaction between FP and TMD is responsible for full fusion. However, the presence of this interaction remains a contentious issue in the literature. The main aim of the dissertation was to examine the potential interaction between FP and TMD. To examine this, series of experiments with peptides and full-length HA were carried out. Peptides with amino acids sequences corresponding to FP and TMD from H1 and H3 subtypes were used in the studies. Synthetic peptides corresponding to TMD were utilized in two versions: shorter (H1, H3) and longer (H1long, H3long). The longer versions were extended with amino acids that can interact with cholesterol. In addition, virus-like particles (VLP) were used, with full-length HA of the H1 subtype (H1-TMD-H1) or HA of the H1 subtype with the TMD fragment replaced by that from the H3 subtype (H1-TMD-H3). Liposomes and supported lipid bilayers (SLB) with different lipid compositions were utilized as artificial lipid membrane models. To examine synergic interaction between peptides, individual peptides and FP:TMD mixtures were used. The work aimed to biophysically characterize the action of the peptides in the membrane. To measure the fusion activity, a series of fluorescence experiments was performed. Moreover, membrane lipid order was investigated by fluorescence lifetime imaging microscopy (FLIM) using a dye sensitive to membrane lipid order. Finally, the fusion activity test of VLP with SLB was carried out using FLIM-FRET (fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer). The research showed a lack of interaction between FP and TMD. However, differences were observed in the membrane activity of TMDs from divergent phylogenetic groups. While H1 induced membrane ordering, H3 caused a decrease in the lipid order. Moreover, the results suggest that TMD affects the fusion activity of HA. Moreover, it was observed that TMD may influence the fusion activity of the full-length protein. The experiments with VLP revealed a tendency suggesting that H1-TMD-H3 exhibits higher fusion activity than H1-TMD-H1.
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Digitizing institution:Nencki Institute of Experimental Biology of the Polish Academy of Sciences
Original in:Library of the Nencki Institute of Experimental Biology PAS
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