Cellular cryo-tomography of nidovirus replication organelles

Abstract

This thesis describes the cryo-electron microscopic (cryo-EM) analyses of intracellular viral replication structures. The main methodology used for the analyses was cellular electron cryo-tomography (cryo-ET) to study the replication organelles induced by cornoa- and arteriviruses at macromolecular resolution. Cellular cryo-ET is a young technique incorporating focused ion beam (FIB) milling of 100-300 nm thin cryo-lamellae containing intracellular material readily imageble by high-resolution cryo-EM. However, this workflow is low in throughput and error-prone. Part of the thesis describes an improved sample-preparation strategy that successfully increased the throughput of this workflow. Furthermore, this method was applied on cells infected by coronaviruses and unveiled so far unseen protein complexes that seem to shuttle viral RNA across the double-membranes of the viral replication organelles. It was found that these molecular pores are (partially) formed by viral proteins containing transmembrane-domains. The molecular pores appear to be a central component in the replication cycle of corona- and arteriviruses, two virus families united in the virus order Nidovirales. Finally, the structure and function of the viral replication organelles are reviewed and how past and future technical developments in EM did and likely will contribute to our growing understanding of these.