|
|
Determinants of fusogenicity of Syncytin-1, cellular glycoprotein of retroviral origin
Trávníček, Martin ; Trejbalová, Kateřina (advisor) ; Zábranská, Helena (referee)
Syncytin-1 is an endogenous retroviral envelope glycoprotein specifically expressed in human placenta, where the protein was adopted for its physiological function. After interaction with specific receptors, transmembrane proteins ASCT1 and ASCT2, Syncytin-1 initiates cell-cell fusion leading to formation of multinucleated syncytiotrophoblast, which is essential for feto-maternal nutrients exchange. In this diploma thesis a new cell-cell fusion quantification assay was implemented for characterisation of Syncytin-1 fusion determinants. The assay uses Syncytin-1 and ASCT2 expressed separately with fragments of luciferase in heterologous cell-culture system. The assay enables to specifically quantify cell-cell fusions based on activity of reconstituted luciferase reporter. This study discovered new facts about the role of intracytoplasmic tail of Syncytin-1 in the process of the cell- cell fusion. This specific part of protein contains a tandem motif sensitive to changes in amino acid sequence that led to loss of fusogenic potential of Syncytin-1. It was further confirmed, that the protein Suppressyn works as an inhibitor of cell-cell fusions initiated by Syncytin-1. Suppressyn however does not bind to receptors of Syncytin-1 and the mechanism of its inhibition remains unsolved. Finally, it was demonstrated...
|
|
|
Interaction of transmembrane proteins ASCT1 and ASCT2 with retroviral envelope glycoproteins
Trávníček, Martin ; Trejbalová, Kateřina (advisor) ; Mělková, Zora (referee)
Transmembrane proteins ASCT1 and ASCT2 are ubiquitous neutral amino acid transporters. Apart from their transporter function in metabolically active cells, they also serve as receptors for a wide group of retroviruses. All retroviruses recognizing the transmembrane receptor ASCT2/ASCT1 share a similar env gene, encoding the envelope glycoprotein. Syncytin-1 is the envelope glycoprotein, encoded by human endogenous retrovirus type W, produced in placental cytotrophoblasts of primates, including human. Interaction of receptor binding domain of Syncytin-1 and specific extracellular region of ASCT2 is responsible for fusion of neighbouring cells and formation of multinucleated syncytiotrophoblast. The importance of syncytiotrophoblast lies in higher efficiency of feto-maternal exchange of nutrients and simultaneously in modulation of immune response of mother towards fetus. Defect in syncytiotrophoblast differentiation often leads to complications during pregnancy and impairs the proper development of embryo. Characterization of protein domains responsible for the interaction between Syncytin-1 and its receptors is important to uncover genetic causes of these pathologies. Furthermore, understanding the interaction helps us to clarify the mechanism of cell entry and explains the molecular basis of host...
|
|
|
Rous sarcoma virus replication blocks in mammalian cells
Koslová, Anna ; Hejnar, Jiří (advisor) ; Ruml, Tomáš (referee) ; Weber, Jan (referee)
One of the important tasks of virology and immunology is to explore the species- and cell-barriers preventing virus horizontal transmission and reveal the ways how viruses overcome these barriers and "adapt" to different species. This work is based on a well- established retroviral model - avian Rous sarcoma virus (RSV) and studies virus replication blocks in mammalian cells at both pre- and post-integration level. Interaction of the viral envelope glycoprotein (Env) with a specific cellular receptor mediates virus entry into cells. Although mammalian orthologues of specific chicken receptors do not support RSV entry, it was observed that some RSV strains are able to enter mammalian cells. Several RSV-transformed rodent cells lines were described and analysis of provirus H20- RSV in one these cells lines (hamster H-20 tumor cell line) showed multiple mutations including two crucial amino acid substitutions in different regions of Env. Substitutions D32G and L378S confer virus transmission to hamster, human and also chicken cells lacking the appropriate receptor. Altered conformation of H20-RSV Env is similar to a receptor-primed (activated) state of Env. This observation indicates that virus can circumvent the need of original cell receptor because of spontaneous Env activation caused by single...
|
guest ::
