Conquering the coronavirus replication centre Coronaviruses use infected cells to make copies of themselves that propagate infection. To replicate their genomes, coronaviruses hijack cellular membranes to build up specialized replication compartments. These work as control centres to make the process efficient and hide it from antiviral cellular defences. Recently, we discovered a unique protein complex that provides a gate in the membranes of these replication compartments. Here, we plan to decipher how this gate and the replication organelles are built and function, which will be key to devise plans to assault these viral fortresses to block virus replication and disease.

Viral infections are a major burden to public health and have a significant socioeconomic impact in the European Union, both through direct disease-associated, but also through indirect costs, most notably loss of labor. Positive-strand RNA (+RNA) viruses are the largest group of human pathogens, comprising viruses causing from severe life-threatening infections such as hepatitis C or SARS over emerging tropical diseases such as Dengue or Chikungunya fever to self-limiting but nonetheless ubiquitous infections such as the common cold or gastro-intestinal infections. For by far the most of these viruses, no vaccines or direct acting antiviral treatments are available, constituting an urgent socioeconomic need in Europe and worldwide. Due to the very limited coding capacity of their genome, RNA viruses strongly depend on host cell functions. The limited number of virus-encoded proteins that can serve as antiviral drug targets and the high mutation rate of RNA viruses, potentially leading to the rapid development of drug resistance, complicate the development of compounds that target viral functions directly and effectively. However, the strong dependency on host cell processes opens up alternative possibilities to inhibit viral replication by interfering with host factors and pathways. Besides a much larger number of potential drug targets, this more indirect approach has the added benefit of a high barrier to resistance, i.e. it is unlikely that the virus can easily escape treatment by acquiring a single point mutation (in contrast to a point mutation at e.g. a drug binding site that could make a virus resistant to a direct-acting antiviral). SysVirDrug aims to identify essential host cell functions, on which all investigated +RNA viruses (and potentially others) critically depend, and to devise strategies to pharmacologically interfere with their function in viral replication. The consortium covers a panel of five diverse and highly relevant plus-strand RNA viruses, including hepatitis C virus, dengue virus, chikungunya virus, SARS-coronavirus and enterovirus (coxsackievirus B3). Using a true systems biology approach, combining available high-throughput experimental data to identify relevant host processes by sophisticated bioinformatics analyses, mechanistic mathematical modeling and detailed quantitative and time-resolved experimentation, SysVirDrug will identify the most sensitive panviral load- and choke points of intracellular replication. Based on an integrative, network-based analysis of published and our own unpublished high-throughput RNAi-screening data, we will identify relevant host processes affecting viral replication in the panel of studied +RNA viruses. Mathematical models of viral replication will be set up based on quantitative, time resolved experimentation, integrating relevant host factors through mechanistic modeling. Models will be iteratively validated and refined and sensitivity analyses will identify the most vulnerable interference points in the developed models as putative targets for antiviral intervention. We hypothesize that targeting common, sensitive host pathways will result in panviral inhibition of replication. We are going to test this hypothesis by silencing relevant genes in implicated pathways or by employing pharmacological inhibitors and assaying for an impact on replication over the full panel of investigated +RNA viruses. Through the involvement of a commercial partner, SysVirDrug will identify and develop small molecule inhibitors of the identified host pathways, which will be translated into marketable pharmaceutical lead compounds. Throughout the entire project, relevant industry users will be integrated through a scientific advisory board, as well as a series of industry workshops and contacts. Through the further integration of an experienced Technology Transfer partner, SysVirDrug will translate cell biological and virological knowledge obtained through iterative cycles of experiment, bioinformatics and mathematical modeling into socioeconomically relevant, industrially exploitable ?antivirotics?, with direct benefits for health and wellbeing in Europe and beyond.  

In the decades ahead of us industrial enzymes and therapeutic proteins will become undoubtedly key molecular entities used in the food industry and human/animal healthcare. Their role is rapidly increasing in this era of advanced biotechnology, with a large part of the newly approved drugs being already protein based. However, due to their large size, biology driven production, and complicated structural features therapeutic proteins and industrial enzymes form some of the most challenging molecular entities to be functionally and structurally characterized. Such a characterization ideally involves the charting of all functionally important proteoforms (i.e. all protein variants originating from one gene). Due to the plethora of modifications often occurring on a given protein backbone, such as disulphide bridges, widespread and extensive glycosylation, phosphorylation, proteolytic truncation, amidation, oxidation, glycation and conformeric variants, tens to thousands of proteoforms may co-occur for a single natural enzyme or engineered antibody, each with its own bioactivity and clearance spectrum. Moreover, another emerging trend in biotechnology is the chemical modification and/or bioengineering of industrial enzymes and therapeutic proteins to enhance their efficacy, with antibody-drug conjugates and “bio-betters” representing some of the well-known emerging classes of such future medicines. Evidently, the chemical moieties attached may even further increase the structural diversity of these proteins, requiring even more in-depth structural characterization. The main goal in this proposal is to develop much-needed new analytical strategies enabling the separation and characterization of all complex proteoforms of industrial enzymes and biotherapeutics in their intact native state. Distinctively, our novel analytical workflows are aimed at the genuine intact proteins and will allow us to simultaneously establish structure-function relationships of proteoforms and aggregation states of proteins. This information is typically lost in current conventional approaches. We aim through this proposal at developing new mass spectrometric methods, and analytical workflows, optimized to analyze complex and large glycoproteins, led by the group of Heck (UU). Our aim is to detect with higher sensitivity and selectivity intact native industrial and therapeutic glycoproteins and their relevant protein assemblies (e.g. aggregates). Hereby we will develop new approaches to obtain analytical breakthroughs allowing these native proteins to reach with more efficiency the detector. From a mass spectrometric angel, gains in mass resolving power, sensitivity, robustness, selectivity and sequencing efficiency will be some of the main deliverables. Development of intact native protein separation techniques forms a second pillar of this proposal, led by the group of Wuhrer (LUMC). We aim to achieve the separation of proteoforms prior to (native) MS analysis and functional characterization to reduce heterogeneity and allow structure-function relationship determination. Workflows for the separation of the heterogeneous glycoproteins require the full spectrum of separation techniques based on different separation principles. Making use of orthogonality in methods, smart hyphenation via new interfacing, inlet and ion transmission solutions, we aim at resolving, identifying and quantifying the plethora of functionally relevant protein modifications. In separating native proteoforms we foresee a major role for protein affinity chromatography exploring interactions that are relevant for the mode-of-action of biopharmaceuticals. We will work on preparative native separation to generate sufficient materials for relevant functional tests. The more established complementary bottom-up approaches, relying on proteolytic processing for protein backbone and glycosylation analysis, are well-established in the laboratories of the academic and industrial applicants and will be used to complement intact-level analysis. A major aim in this proposal is also to develop algorithms and software allowing us for the first time to integrate the information gathered by different levels of product characterization. Firstly, the group at the UU will extend their data-integration software suite to be able to integrate bottom-up, middle-down, top-down and native MS analysis data on structurally highly complex industrial enzymes and biotherapeutics, as this may provide the only means to get a complete qualitative and quantitative picture of the proteoform profiles of the investigated glycoproteins. An important next aim is to integrate also data obtained with the different applied separation technologies into such analyses as well as the qualitative and quantitative glycan profiles of the same products generated at the LUMC using dedicated glyco-profiling software. The most successful outcomes of the optimization process in mass spectrometry and chromatography will be hyphenated with each other through exchange of technologies and researchers (e.g. PhDs, Master and ASPT students) from UU and LUMC, and made available to the industrial partners within the consortium (DSM, FrieslandCampina and Roche) via secondments and training sessions, enabling the detailed characterization of structural and functional features of industrial and biopharmaceutical proteins for establishing structure-function relationships. These insights will speed up development of new or improved protein products of the different industrial partners. Our research is expected to have significant utilization impact in a wide variety of application fields, three of which are specifically addressed in this proposal: the pharmaceutical industry, represented by Roche, industrial enzymes, represented by DSM and milk proteins, represented by FrieslandCampina.

Viruses and their hosts are constantly trying to outsmart each other. For their replication viruses depend on their host, whereas the host has obvious reasons to want the virus out, or at least minimize the viral abuse of its resources. In some cases, this battle is fierce and the host?s life is at stake, as exemplified by successive virus outbreaks and epidemics in humans and animals. Mammals have evolved elaborate immune systems to defend themselves against viruses, but viruses on the other hand are often equipped with mechanisms to avoid, mislead, or suppress the host?s defenses, in order to persist longer and/or replicate more efficiently. The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003 and the repeated devastating outbreaks of porcine respiratory and reproductive syndrome virus (PRRSV) in Asia illustrate that nidoviruses, the virus group to which these pathogens belong, should be taken into account. After entry into the host cell, the replication of viruses with a positive strand RNA (+RNA) genome, such as nidoviruses, begins with the expression of replicase polyproteins. Individual enzymatic subunits (nonstructural proteins) are embedded in this precursor polypeptide and are scissored out by one or more proteases, which commonly reside in this same polyprotein. Thereby, these proteases direct and control the formation of the viral RNA-synthesizing replication/transcription complex, which consists of the viral RNA templates in association with a complex of nonstructural proteins. The complex is likely supplemented with host protein factors and is supported by a framework of virus-induced membrane structures. Intriguingly, in addition to doing their crucial job in replicase polyprotein processing, some viral proteases appear to serve additional purposes. Virus infection will trigger the sensors of the cell?s innate immunity system, the first line of defense against invading viruses. A cascade of signal transduction will ensue, which soon will result in a general ?antiviral state?. The signal transduction pathways that regulate innate immunity depend, at multiple levels, on the cell?s ubiquitination system. The covalent linkage of the small ubiquitin polypeptide to key protein factors in the cascade secures downstream signaling and the production of antiviral effectors. Removal of ubiquitin (?deubiquitination?) from these key factors should block the cascades leading towards the antiviral state, and promote efficient virus replication. Recently, based on comparative sequence analysis and structural studies, the papain-like proteases (PLpro?s) of nidoviruses, including SARS-coronavirus and the arteriviruses EAV and PRRSV, were proposed to be deubiquitinating enzymes (DUBs). Interestingly, also in other, distinct, virus families these kind of enzymes were recently found, such as in bunya-, adeno- and herpesviruses. Due to this broad presence, they are presumed to be important viral features, however their functions or possible viral and cellular substrates are unknown to date. It was recently confirmed that nidovirus Plpro?s indeed possess deubiquitinating activity in vitro and in vivo. Moreover, one of the downstream antiviral effectors of the cell, ISG15, a so-called ubiquitin-like molecule, can also be cleaved from its substrates by these nidovirus proteases. Do these nidovirus proteases indeed work as a double-edged sword? Are they not only activating the replicative enzymes by cleaving replicase polyproteins, but are they also deliberately targeting the (iso)peptide bond between cellular ubiquitin or ubiquitin-like molecules and their substrates to counteract the host?s antiviral responses? And what about the threat of ubiquitin-triggered degradation by the host cell?s proteasome; do nidoviruses use papain-like proteases/DUBs to protect their proteins from this fate as well? In this project, we propose to biochemically characterize nidovirus PLpro?s by extending and exploring in vitro and in vivo activity assays. These can be used to e.g. perform site-directed mutagenesis studies and assess substrate specificities. The role of the proteases in suppressing innate immunity will be probed by investigating whether different ubiquitin-regulated innate immunity factors are targeted by nidovirus DUBs. Viral DUB interaction with the interferon-induced ISG15 system will also be assessed. The relevance of the deubiquitinating activity for the virus will be investigated by engineering recombinant viruses that lack this feature. Finally, the ubiquitin-mediated degradation of viral proteins during infection will be monitored, to assess whether nidovirus PLpro?s may influence the turn-over of enzymes that are crucial for virus replication. The proposed research will extend our work on the biochemistry of nidovirus replication in an innovative and topical direction. Our combined track record in +RNA virus research, extensive expertise in nidovirus protease research, and experience in the fields of ubiquitin and immune evasion research guarantee a straightforward start for this proposed Ph.D. project. In addition, a large collection of relevant reagents is available and productive (inter)national collaborations have been set-up, to further ensure the successful development of this new research line.

RNA viruses are characterized by a high mutation frequency, which ? paradoxically ? is key to their success as infectious agents of all living organisms. RNA virus populations are ?quasi-species?, clouds of mutants ready to adapt to changing circumstances like the host?s immune response, antiviral therapy, or a new host species. This potential for rapid evolution is attributed to efficient replication in combination with the lack of a proofreading mechanism for removal of nucleotides that are misincorporated by the viral RNA polymerase (RdRp). Conceptually, RNA viruses are faced with a choice between replication speed (to outrun host responses) and reliable genome replication, reflected in a trade-off between processivity and fidelity of their RdRp. The high mutation frequency has presumably restricted genome size expansion, to prevent the accumulation of too many deleterious mutations (?error catastrophe?). In this light, the extraordinary 27- to 32-kilobase (kb) genome size of coronaviruses is remarkable. The group occupies an isolated position at the upper end of the RNA virus genome size scale and speculations about proofreading during coronavirus replication have been numerous. Recent reports implicate a newly discovered viral exoribonuclease (ExoN) in such a function, although the molecular mechanism remains entirely obscure. Interestingly, a group of distant coronavirus relatives, the arteriviruses, which also belong to the order of nidoviruses, have genomes of only 12-16 kb and lack a homolog of the ExoN domain, supporting the hypothesis that ExoN acquisition was a key event in coronavirus genome expansion. We have recently developed the first in vitro activity assays using purified recombinant RdRps of both SARS-coronavirus and the arterivirus EAV. We will use these biochemical assays, in combination with real-time single-molecule techniques (magnetic tweezers) and deep sequencing analysis of in vivo and in vitro produced viral RNA, to characterize the basic RdRp properties (including processivity and fidelity) of the distantly related SARS-coronavirus and EAV RdRps. The well-characterized poliovirus RdRp will be included for comparison and as positive control. The existence of proofreading during coronavirus RNA synthesis will be evaluated, as well as the putative role of the ExoN exoribonuclease in preventing error catastrophe. The recent description of a unique second RdRp in coronaviruses, presumably synthesizing primers for the viral ?main? RdRp, will be the starting point for an in-depth analysis of coronavirus RdRp initiation and elongation, studies in which also the viral helicase will be taken into account. Finally, we will study another trademark of the nidovirus RdRp: the mechanism of discontinuous minus-strand RNA synthesis that operates to produce the corona- and arterivirus subgenomic mRNAs from which the viral structural proteins are expressed. Our multidisciplinary approach (biochemistry, biophysics, and virology) will explore novel experimental avenues for viral RdRps. Our results will be relevant to the (viral) RdRp field at large, will shed more light on the unusual evolutionary position of coronaviruses, and will directly address key aspects of nidovirus molecular biology. The characterization of viral RdRps is highly relevant for the design of live virus vaccines and antiviral drugs, and for assessing the evolutionary potential of RNA viruses as dynamic infectious agents.