Development of novel probes for enterovirus B group to study uncoating and infection
Julkaistu sarjassaJyväskylä studies in biological and environmental science
OppiaineSolu- ja molekyylibiologia
Enterovirus B group (EV-B) viruses are important human pathogens which cause a variety of diseases from mild respiratory illnesses to more severe acute infections such as myocarditis and meningitis. EV-Bs have also been associated with chronic infections and autoimmune diseases such as type I diabetes. Because of their significance, better and more accurate methods are necessary to track and visualize viruses in vitro and in vivo. This thesis focus on the development of novel probes to label viruses site-specifically and track infection in vitro. First, we established a covalent conjugation between gold nanocluster markers and EV-B viruses. We were able to visualize through electron microscopy site-specific labelling of cysteines on the viral capsid surface. Second, we developed probes targeted to the hydrophobic pocket of the enterovirus capsid. The probes were synthesized based on a hydrophobic pocket binding drug pleconaril. Successful binding of the probe was confirmed with nuclear magnetic resonance measurements. The probe was conjugated to a fluorescent KU Orange 2 dye or Au102 gold nanocluster, and further studied by confocal and electron microscopy. The third section of this thesis studied the infectious entry pathway of three different coxsackievirus B1 (CVB1) strains. All of the studied strains appeared to use the previously suggested EV-B group entry pathway: macropinocytic entry dependent on Na+/H+ and Rac1 and independent of acidification. However, strain specific kinetic differences in the infection of non- polarized cells were revealed. Importantly, one of the CVB1 strains infected cells independently from endosomal sorting complexes required for transport (ESCRT) driven multivesicular bodies (MVBs), and initiated the virus uncoating with slower kinetics. The results indicate that the ESCRT driven biogenesis of MVBs is required for efficient uncoating and infection. Thus, the viruses show remarkable similarities to other well studied EV-B viruses. ...
JulkaisijaUniversity of Jyväskylä
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