STUDYING REPLICATION ORGANELLES USING ADVANCED FLUORESCENCE MICROSCOPY
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The enterovirus genus comprises many human pathogens such as poliovirus, coxsackievirus and rhinovirus. For efficient replication, enteroviruses induce membrane rearrangements which require the interaction between viral and host factors such as PI4KB, ACBD3 and 3A. Although interactions between these proteins have been intensively studied, their localization at replication organelle (RO) membranes is unknown. To investigate components of ROs in-depth, we established protocols to study coxsackievirus B3 (CVB3) ROs using expansion microscopy (ExM) and super-resolution microscopy (SRM), i.e. Olympus SoRaSpin10 system. Our data is the first evidence of 2A localization. The use of SRM allowed us to visualize the localization of the viral protease 2A at different time points of infection in greater detail. In addition, we could locate 3A at an early time point after infection (i.e. 3 h p.i.), which was hardly attainable with conventional immunofluorescence microscopy. Our data suggest that imaging at earlier time points might be possible. Moreover, both viral proteins were visualized as dotted structures which have not yet been observed. We demonstrate the potential for in-depth visualization of these viral proteins and their colocalization with host factors using super-resolution microscopy. The Olympus SoRaSpin10 system offers the possibility to combine SRM with live-cell imaging. Thus, tracking viral protein localization, RO formation and its dynamics could be further investigated.