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
The replication of all positive-stranded RNA viruses of eukaryotes is thought to take place at cytoplasmic membranous replication organelles. One of the most prominent types of viral ROs induced by... Show moreThe replication of all positive-stranded RNA viruses of eukaryotes is thought to take place at cytoplasmic membranous replication organelles. One of the most prominent types of viral ROs induced by a number of these viruses, including coronaviruses and arteriviruses, are double-membrane vesicles (DMVs) that contain viral double-stranded RNA. This thesis discusses the formation of these replication organelles by arteri- and coronaviruses and provides new insights in the viral proteins involved. The ultrastructure of the replication organelles was unravelled using both electron microscopy and tomography. Furthermore, this research described in this thesis also shows that the innate immune system in infected cells can prevent the formation of the membrane structures. Show less
In part 1 of the thesis quantitative proteomics was used to determine changes in abundance and phosphorylation status of host proteins during infection with the human pathogen chikungunya virus ... Show moreIn part 1 of the thesis quantitative proteomics was used to determine changes in abundance and phosphorylation status of host proteins during infection with the human pathogen chikungunya virus (CHIKV). Several proteins were identified that may be specifically downregulated during CHIKV infection to create a suitable environment for viral replication. eEF2 was identified as a factor that becomes strongly phosphorylated during infection with several viruses which may be a mechanism to stall translation in response to viral infection. In part 2 of the thesis the discovery of a novel and unusual -2/-1 programmed ribosomal frameshifting (PRF) mechanism is described that is used during translation of the nonstructural open reading frame of the economically important porcine reproductive and respiratory syndrome virus (PRRSV). This mechanism relies on a slippery site and stimulatory RNA signal in the PRRSV genome and is stimulated by the viral protein nsp1beta and host poly (C) binding proteins 1 and 2. Frameshifting results in the synthesis of two previously unidentified proteins, nsp2 TF (-2 PRF) and nsp2N (-1 PRF). Virus mutants that can no longer make the frameshift products are attenuated and may be used for vaccine development. Show less
Kasteren, P.B. van; Knaap, R.C.M.; Elzen, P. van den; Snijder, E.J.; Balasuriya, U.B.R.; Born, E. van den; Kikkert, M. 2015
The order Nidovirales comprises a monophyletic group of viruses with positive-stranded RNA genomes that are classified in the families Arteriviridae, Coronaviridae, Mesoniviridae, and Roniviridae.... Show moreThe order Nidovirales comprises a monophyletic group of viruses with positive-stranded RNA genomes that are classified in the families Arteriviridae, Coronaviridae, Mesoniviridae, and Roniviridae. They share a conserved genome organization and a characteristic set of key replicative proteins. Although, in principle, this suggests a conserved replication mechanism, it is currently unclear how far exactly the resemblance extends on a more detailed level. This is foremost due to our poor understanding of the role of most viral proteins in the replication cycle. In addition, most of the knowledge that was obtained predominantly derives from studies of only few coronaviruses, the nidovirus subgroup with the largest known genome and therefore presumably employing the most complex replication strategy. In contrast, thus far only limited attention was given to the RNA rep_licating and processing enzymes of arteriviruses, and none at all to those of mesoni- and roniviruses, whose genome sizes are (much) smaller than those of coronaviruses. The work described in this thesis addresses some poorly or uncharacterized (domains of ) nonstructural proteins (nsps) that are likely involved in one or multiple steps of RNA replication and/or transcription of the prototypic arterivirus equine arteritis virus (EAV). Show less
Li, Y.H.; Tas, A.; Sun, Z.; Snijder, E.J.; Fang, Y. 2015
The work described in this thesis provides novel insights into the structural and (multi)functional characteristics of arterivirus PLP2. This enzyme plays an essential role in the viral replication... Show moreThe work described in this thesis provides novel insights into the structural and (multi)functional characteristics of arterivirus PLP2. This enzyme plays an essential role in the viral replication cycle by cleaving the viral replicase polyproteins. In addition, there were indications that PLP2 is able to influence certain cellular processes by cleaving ubiquitin. We have now shown that PLP2 indeed functions as a deubiquitinating enzyme (DUB) and that this activity is important for the suppression of the innate immune response in the cell. To be able to separate both functions of PLP2 we have solved the crystal structure of this enzyme in complex with ubiquitin. Based on this structure, we were able to design mutations in PLP2 that selectively disrupt the interaction with ubiquitin, without interfering with cleavage of the viral polyproteins. Using these mutants, we have demonstrated for the first time the importance of a viral DUB in the evasion of innate immunity in the context of an infection. The acquired knowledge can now be applied to the design of improved arterivirus vaccines and studies of other viral DUBs, including those encoded by the zoonotic coronaviruses that cause SARS and MERS. Show less
The interplay between nidoviruses and the infected host cell was investigated. Arterivirus RNA-synthesising activity was shown to depend on intact membranes and on a cytosolic host protein which... Show moreThe interplay between nidoviruses and the infected host cell was investigated. Arterivirus RNA-synthesising activity was shown to depend on intact membranes and on a cytosolic host protein which does not cosediment with the RTC. Furthermore, the immunosuppressant drug cyclosporin A (CsA) blocks replication of EAV and the swine arterivirus PRRSV in cell culture. Cyclophilin A appears to be an important host factor for EAV replication. CsA may be a nidovirus-wide inhibitor of replication since this compound also blocked replication of the coronaviruses SARS-CoV, HCoV-229E, and MHV. We further described a kinase siRNA library screen that identified ninety antiviral and forty proviral hits and signalling pathways involved in the SARS-CoV replicative cycle. PKR (antiviral) and COPB2 (proviral) were validated in follow-up experiments. We also investigated MERS-CoV replication characteristics and we described an assay t o screen for compounds that block MERS-CoV infection. CsA and pegylated IFN-_ (PEG-IFN) significantly inhibited infection, and MERS-CoV was shown to be much more sensitive to PEG-IFN treatment than SARS-CoV, an observation that may have implications for the treatment of MERS-CoV infection. The data presented in this thesis might contribute to better understand virus replication and hopefully provide additional starting points for the development of antiviral strategies. Show less
Members of the order Nidovirales (Coronaviridae, Arteriviridae, and Roniviridae) employ genomes with mRNA polarity (plus-strand) and encode one of the most complex RNA virus replicases currently... Show moreMembers of the order Nidovirales (Coronaviridae, Arteriviridae, and Roniviridae) employ genomes with mRNA polarity (plus-strand) and encode one of the most complex RNA virus replicases currently known. This replicase is expressed from the viral genome by translation of two large 5__-proximal ORFs into two polyproteins, which are processed by virus-encoded proteases in 13-16 individual nonstructural proteins (nsps). The nsps direct the formation of an RNA-synthesizing complex that mediates viral genome replication, as well as the synthesis of a 3__-coterminal nested set of subgenomic (sg) mRNAs, from which the viral genes located downstream of the replicase gene are expressed. Arteriviruses and coronaviruses presumably employ a unique mechanism of discontinuous minus-strand extension to generate subgenome-length templates for sg mRNA synthesis. This thesis focused on the functional characterization of two replicase subunits and their roles in coupling different processes in the replicative cycle of equine arteritis virus (EAV), the arterivirus prototype. The biological importance of a conserved domain unique to nidoviruses (NendoU), mapping to arterivirus nsp11 and proposed to function as an endoribonuclease, was addressed. We demonstrated the recombinant nsp11 exhibits pyrimidine-specific endoribonuclease activity, and showed the critical importance of the NendoU domain for EAV RNA synthesis. In addition, we identified a multidomain replicase subunit, EAV nsp1, as a key coordinator of EAV genome replication, sg mRNA synthesis, and virus production. Our results reveal that the relative abundance of EAV mRNAs is tightly controlled by nsp1 and is critical for efficient production of new virus particles. The protein was implicated in modulating the accumulation of full-length and subgenome-length minus-strand templates for EAV mRNA synthesis. A protocol for purification of soluble recombinant nsp1, which can be used in future research on the molecular mechanisms of nsp1 function, is described. Show less
Viruses depend on their host cell for the production of their progeny. The genetic information that is required to regulate this process is contained in the viral genome. In the case of plus... Show moreViruses depend on their host cell for the production of their progeny. The genetic information that is required to regulate this process is contained in the viral genome. In the case of plus-stranded RNA viruses, like nidoviruses, the RNA genome is directly involved in translation (resulting in the synthesis of viral enzymes), replication, transcription and encapsidation into progeny virions. The multifunctional nature of these viral RNA genomes requires the tight control of all these processes for which they are equipped with RNA sequence motifs and higher order RNA structures. At 25-32 kilobases, nidoviruses possess the largest known RNA genomes. One characteristic of nidoviruses is that in infected cells they produce a nested set of subgenomic (sg) mRNAs. The sg mRNAs of two nidovirus families, arteri- and coronaviruses, consist of two RNA stretches that are noncontiguous in the genome. It was demonstrated that primary and higher order RNA structures play a crucial role during the synthesis of these special sg mRNAs. The obtained knowledge of arterivirus RNA synthesis, formed the basis for an virus inhibitor study in which regulatory RNA sequences were targeted in an attempt to block virus replication in cell culture using phosphorodiamidate morpholino oligomers (P-PMOs). Show less
