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... Show moreThis 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. Show less
Two pandemics of respiratory distress diseases associated with zoonotic introductions of the species Severe acute respiratory syndrome-related coronavirus in the human population during 21st... Show moreTwo pandemics of respiratory distress diseases associated with zoonotic introductions of the species Severe acute respiratory syndrome-related coronavirus in the human population during 21st century raised unprecedented interest in coronavirus research and assigned it unseen urgency. The two viruses responsible for the outbreaks, SARS-CoV and SARS-CoV-2, respectively, are in the spotlight, and SARSCoV-2 is the focus of the current fast-paced research. Its foundation was laid down by studies of many coronaand related viruses that collectively form the vast order Nidovirales. Comparative genomics of nidoviruses played a key role in this advancement over more than 30 years. It facilitated the transfer of knowledge from characterized to newly identified viruses, including SARS-CoV and SARS-CoV-2, as well as contributed to the dissection of the nidovirus proteome and identification of patterns of variations between different taxonomic groups, from species to families. This review revisits selected cases of protein conservation and variation that define nidoviruses, illustrates the remarkable plasticity of the proteome during nidovirus adaptation, and asks questions at the interface of the proteome and processes that are vital for nidovirus reproduction and could inform the ongoing research of SARS-CoV-2.(c) 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Show less
The order Nidovirales, including families Coronaviridae and Arteriviridae, is a monophyletic group of highly divergent (+)ssRNA viruses that infect vertebrate and invertebrate hosts; they share... Show moreThe order Nidovirales, including families Coronaviridae and Arteriviridae, is a monophyletic group of highly divergent (+)ssRNA viruses that infect vertebrate and invertebrate hosts; they share conserved genome organization and replication mechanisms. The genome sequence is the only information available about many newly discovered nidoviruses whose number is fast increasing driven by technology advancements. This development makes comparative genomics, an approach that already has been used extensively in nidovirology, increasingly important. In this thesis, diverse methods of comparative genomics were used to address scientific questions about composition and evolution of the nidovirus genome and proteome, and their connection to the biology of nidoviruses. Three studies were conducted in collaboration with experimental researchers, and ranged from the analysis of the highly divergent polyprotein N-terminus in arteriviruses, to identification of the fifth universally conserved domain of nidoviruses, and to characterization of a nidovirus with the largest known RNA genome. The latter study prompted the development of a bioinformatics tool facilitating functional annotation of large multidomain polyproteins. The thesis illustrates how a notion of nidovirus-specific conservation has been steadily refined as a result of recent discoveries. Show less
Rappe, J.C.F.; Wilde, A. de; H. di; Muller, C.; Stalder, H.; V'kovski, P.; ... ; Thiel, V. 2018
All eukaryotic positive-stranded RNA (+RNA) viruses appropriate host cell membranes and transform them into replication organelles, specialized micro-environments that are thought to support viral... Show moreAll eukaryotic positive-stranded RNA (+RNA) viruses appropriate host cell membranes and transform them into replication organelles, specialized micro-environments that are thought to support viral RNA synthesis. Arteriviruses (order Nidovirales) belong to the subset of +RNA viruses that induce double membrane vesicles (DMVs), similar to the structures induced by e.g. coronaviruses, picornaviruses and hepatitis C virus. In the last years, electron tomography has revealed substantial differences between the structures induced by these different virus groups. Arterivirus-induced DMVs appear to be closed compartments that are continuous with endoplasmic reticulum membranes, thus forming an extensive reticulovesicular network (RVN) of intriguing complexity. This RVN is remarkably similar to that described for the distantly related coronaviruses (also order Nidovirales) and sets them apart from other DMV-inducing viruses analysed to date. We review here the current knowledge and open questions on arterivirus replication organelles and discuss them in the light of the latest studies on other DMV-inducing viruses, particularly coronaviruses. Using the equine arteritis virus (EAV) model system and electron tomography, we present new data regarding the biogenesis of arterivirus-induced DMVs and uncover numerous putative intermediates in DMV formation. We generated cell lines that can be induced to express specific EAV replicase proteins and showed that DMVs induced by the transmembrane proteins nsp2 and nsp3 form an RVN and are comparable in topology and architecture to those formed during viral infection. Co-expression of the third EAV transmembrane protein (nsp5), expressed as part of a self-cleaving polypeptide that mimics viral polyprotein processing in infected cells, led to the formation of DMVs whose size was more homogenous and closer to what is observed upon EAV infection, suggesting a regulatory role for nsp5 in modulating membrane curvature and DMV formation. (C) 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license. Show less
Positive-stranded RNA viruses replicate in the cytoplasm of host cells and their replication complexes are associated with modified cell membranes. We investigated the structure of the nidovirus... Show morePositive-stranded RNA viruses replicate in the cytoplasm of host cells and their replication complexes are associated with modified cell membranes. We investigated the structure of the nidovirus-induced membrane modifications and found that nidoviruses transform the endoplasmic reticulum into a reticulovesicular network (RVN) that integrates numerous inter-connected double-membrane vesicles (DMVs), and, in the case of coronaviruses, __vesicle packets__ and convoluted membranes (CM). The membranes of DMVs and CM labeled abundantly for replicase proteins, whereas the DMV interior contained the double-stranded RNA and therefore is the presumed site of viral RNA synthesis. Electron tomography, semi-permeabilization and nuclease digestion experiments suggested that the DMV interior is inaccessible from the cytosol, implying that double-stranded RNA is compartmentalized by membranes. Furthermore, brefeldin A treatment showed that early secretory pathway proteins do not play a direct role in RVN morphogenesis or functionality of the SARS-CoV replication complex. Continued translation was not an absolute requirement for viral RNA synthesis, however, was necessary for RVN development thus suggesting a link between accumulation of replicase proteins and RVN formation. Together with biochemical studies of the viral enzyme complex, our ultrastructural description of the RVN aids to further dissect the early stages of the nidovirus infection and virus-host interactions. Show less
