title: Studying the dynamics of coronavirus replicative structures.
abstract: Coronaviruses (CoVs) generate specialized membrane compartments, which consist of double membrane vesicles connected to convoluted membranes, the so-called replicative structures, where viral RNA synthesis takes place. These sites harbor the CoV replication-transcription complexes (RTCs): multi-protein complexes consisting of 16 nonstructural proteins (nsps), the CoV nucleocapsid protein (N) and presumably host proteins. To successfully establish functional membrane-bound RTCs all of the viral and host constituents need to be correctly spatiotemporally organized during viral infection. Few studies, however, have investigated the dynamic processes involved in the formation and functioning of the (subunits of) CoV RTCs and the replicative structures in living cells. In this chapter we describe several protocols to perform time-lapse imaging of CoV-infected cells and to study the kinetics of (subunits of) the CoV replicative structures. The approaches described are not limited to CoV-infected cells; they can also be applied to other virus-infected or non-infected cells.
authors.affiliation: Laboratory of Host-Pathogen Dynamics, Cell Biology and Physiology Center (CBPC), National Heart Lung and Blood Institute (NHLBI), National Institutes of Health, Bethesda, MD, USA, marne.hagemeijer@nih.gov.
authors.forename: Marne C
authors.initials: MC
authors.lastname: Hagemeijer
authors.forename: Cornelis A M
authors.initials: CA
authors.lastname: de Haan
mesh_terms: D000818:Animals; D002460:Cell Line; D017934:Coronavirus / Q000502:physiology* / Q000648:ultrastructure; D036681:Fluorescence Recovery After Photobleaching; D051379:Mice; D059010:Single-Cell Analysis; D059008:Time-Lapse Imaging; D014764:Viral Proteins / Q000378:metabolism / Q000648:ultrastructure; D014779:Virus Replication
chemical_list: D014764:Viral Proteins