Dynamics of the coronavirus replicative structures

Abstract

Coronaviruses (CoV) are positive-strand RNA (+RNA) viruses that are important infectious agents in both animals and man. Upon infection, CoVs generate large multicomponent protein complexes, consisting of 16 nonstructural proteins (nsp’s) and yet to be identified cellular proteins, dedicated to the replication and transcription of the viral genome. These complexes are associated with modified cellular membranes, which consist of a network of double-membrane vesicles (DMVs) and convoluted membranes (CMs), collectively referred to as replicative structures. Still not much is known with respect to (i) the mechanisms underlying the formation of the replicative structures, (ii) the interactions and dynamics of replicative structure-associated proteins and (iii) the exact subcellular location of the RNA synthesizing activity. The research described in this thesis focuses on these three topics, thereby using innovative techniques such as live-cell imaging. We started our studies by establishing the topology of the three nsp’s that were predicted to be integral membrane proteins; nsp3, nsp4 and nsp6. Our results indicate that both nsp3 and nsp6 contain conserved non-membrane-spanning hydrophobic domains that we hypothesize to play an important role in the formation of the replicative structures. The dynamics of the replicative structures were investigated by focusing on three replication-associated proteins: the soluble nsp2, the transmembrane protein nsp4 and the structural nucleocapsid (N) protein. Live-cell imaging of infected cells demonstrated that small nsp2-positive structures move through the cytoplasm in a microtubule-dependent manner. In contrast, large fluorescent structures are rather immobile. Nsp2, once recruited to the replicative structures, is not exchanged with nsp2 present in the cytoplasm or at other replicative structures. In contrast, the N protein is dynamically associated with these structures, which is likely correlated with this protein being involved in both replication and assembly. Furthermore, although the membranes of the endoplasmic reticulum (ER) are continuous with those harboring the replicative structures, the mobility of nsp4 at the latter structures is relatively restricted. In agreement herewith, nsp4 was shown to be engaged in homotypic and heterotypic interactions, the latter with nsp3 and nsp6. The location of viral RNA synthesis was studied with a new approach in which cells are fed with an uridine analogue, after which nascent viral RNAs are detected using click chemistry. Early in infection nascent viral RNA and nsp’s colocalized with or occurred adjacent to dsRNA positive sites. Late in infection the correlation between such dsRNA dots, then found dispersed throughout the cytoplasm, nsp’s and nascent RNAs was less obvious. This result is suggestive of maturation of the replicative structures and indicates that dsRNA dots not necessarily correspond with sites active in viral RNA synthesis. The research described in this thesis provides novel insights in the assembly, dynamics and functioning of the replicative structures of CoVs in particularly, but also for +RNA viruses in general.