Degradomics : a study of the cellular proteolytic landscape in enterovirus infections

The common human pathogen, enteroviruses (EVs) are a genus of single stranded RNA viruses that include Coxsackie B virus (CVBs). The viral RNA encodes a polyprotein containing two viral proteases 2Apro and 3Cpro, that process the polyprotein into structural and non-structural proteins during virus replication. Previous research has shown that aside from their role in polyprotein processing, the proteases also cleave host proteins in order to modulate different cell functions such as translation, transcription and innate immunity. The innate immune response, and in particular the type 1 interferons (IFN), have an important role in controlling virus spread and protecting neighbouring cells from infection. Similarly, type III IFNs modulate the permissiveness of cells to CVBs. In paper I, we found that CVB3 has evolved a mechanism to evade the type III IFN response in a comparable manner to that previously shown for the type I IFNs. This perturbation is likely mediated via the proteolytic cleavage of the signal transduction proteins IPS-1 and TRIF by 2Apro. When investigating virus-associated diseases, high quality reagents are essential especially when the aim is to detect virus in serum or tissue samples. We recognised a need for reagents capable of detecting CVBs and in paper II we described the development of CVB specific antibodies against 2Apro/3Cpro and show their utility in western blotting, confocal microscopy, immunohistochemistry and flow cytometry. Several observations have suggested a role for CVB infection in the development of type 1 diabetes (T1D). Interestingly, β-cells infected with CVBs in vitro have defective insulin secretion. The aim of paper III was to investigate whether β-cell dysfunction observed during CVB infection could be attributed to the activity of the viral proteases. We found that the viral proteins 2Apro, 3Cpro and 3A individually exert negative effects on glucose stimulated insulin release (GSIS) and voltage stimulated exocytosis in β-cells. Based on the results in paper I and III, it is evident that 2Apro and 3Cpro have multifaceted roles in the viral replication cycle and in the modulation of the host cell. In paper IV we wanted to define the viral proteases specific targets in Coxsackeivirus B3 (CVB3) permissive cell lines of varied origin (HeLa, CaCo-2 and EndoC-βH1). By enriching for the N-terminal peptides using subtiligase labelling combined with LS-MS/MS, we identified and validated 81 host cell protease substrates. Among the substrates we identified, the protein TCF7L2 was a target of 2Apro mediated cleavage. TCF7L2 is an important transcription factor involved in maintaining β-cell functionality and glucose stimulated insulin secretion. This finding provides a potential mechanistic explanation for the observation that β-cells infected with CVBs in vitro are defective in insulin secretion. The studies presented in this thesis increase our understanding the molecular virology of enteroviruses. Moreover, they open a new chapter of research in examining how disease pathology might be caused by the activity of virally encoded proteases

A Proteomic Approach to Elucidating the Function of Picornavirus 2A Protease

Human-infecting viruses have evolved diverse strategies to enter cells and hijack the host machinery to promote their self-replication. Viruses deploy their proteins to subvert a number of host functions, such as the cell cycle, cellular metabolism, protein synthesis, nuclear and RNA transport across the nuclear pore complex, apoptosis, and innate immune responses. Picornaviruses are the most dominant human disease-causing viruses and present an excellent clinical target for research studies into their molecular mechanisms. Picornaviruses have an RNA genome that is translated as a single polyprotein, which is processed into individual components by two proteases, termed 2A and 3C. In addition to polyprotein processing, these proteases also subvert host cell function through cleavage of specific protein targets. The 2A protease is especially critical during the initial stages of picornaviral infections. We developed protein expression platforms to further characterize the 2A protease’s function, interacting partners, and targeting mechanisms, showing that it makes surgical strikes against eIF4G and Nup98, key players in host protein synthesis and nucleocytoplasmic trafficking of proteins and RNA, respectively. We subsequently utilized those protease expression platforms in combination with transport reporter assays to interrogate nuclear import and export through the NPC. By studying the interactome of 2A protease, we discovered that it seems to employ two different cleavage mechanisms for its primary targets, Nup98 and eIF4G. It directly binds and degrades Nup98, and may alternatively bind eIF3L and utilize the eIF3 complex as a targeting platform to cleave eIF4G. Cellular fractionation revealed Nup98 cleavage by 2A protease results in observable dissociation of Rae1 from the NPC as well as cytoplasmic accumulation of proteins normally transported by Karyopherins that interact with Nup98 FG motifs. We developed fluorescent transport reporters with various nuclear import and export signals to elucidate their transport mechanism and dependence on Nup98. Nuclear localization signals promiscuously recognized by a variety of Importins were marginally, if at all, affected by Nup98 depletion, while export and import signals depending on Crm1 or Rae1 mediated RNA export were severely affected by the absence of full-length Nup98. We propose a novel cleavage mechanism for the 2A protease, as well as report on our development of a suite of molecular tools to further characterize 2A protease function and nucleocytoplasmic transport mediated by Nup98. These tools can be adapted to include a diverse variety of viral proteins to further characterize host subversion mechanisms as well as other aspects of nuclear pore complex function

Egress of non-enveloped enteric RNA viruses

A long-standing paradigm in virology was that non-enveloped viruses induce cell lysis to release progeny virions. However, emerging evidence indicates that some non-enveloped viruses exit cells without inducing cell lysis, while others engage both lytic and non-lytic egress mechanisms. Enteric viruses are transmitted via the faecal-oral route and are important causes of a wide range of human infections, both gastrointestinal and extra-intestinal. Virus cellular egress, when fully understood, may be a relevant target for antiviral therapies, which could minimize the public health impact of these infections. In this review, we outline lytic and non-lytic cell egress mechanisms of non-enveloped enteric RNA viruses belonging to five families: Picornaviridae, Reoviridae, Caliciviridae, Astroviridae and Hepeviridae. We discuss factors that contribute to egress mechanisms and the relevance of these mechanisms to virion stability, infectivity and transmission. Since most data were obtained in traditional two-dimensional cell cultures, we will further attempt to place them into the context of polarized cultures and in vivo pathogenesis. Throughout the review, we highlight numerous knowledge gaps to stimulate future research into the egress mechanisms of these highly prevalent but largely understudied viruses

Insights from structural studies of the cardiovirus 2A protein.

Cardioviruses are single-stranded RNA viruses of the family Picornaviridae. In addition to being the first example of internal ribosome entry site (IRES) utilization, cardioviruses also employ a series of alternative translation strategies, such as Stop-Go translation and programmed ribosome frameshifting. Here, we focus on cardiovirus 2A protein, which is not only a primary virulence factor, but also exerts crucial regulatory functions during translation, including activation of viral ribosome frameshifting and inhibition of host cap-dependent translation. Only recently, biochemical and structural studies have allowed us to close the gaps in our knowledge of how cardiovirus 2A is able to act in diverse translation-related processes as a novel RNA-binding protein. This review will summarize these findings, which ultimately may lead to the discovery of other RNA-mediated gene expression strategies across a broad range of RNA viruses

Molecular docking-based virtual screening and computational investigations of biomolecules (curcumin analogs) as potential lead inhibitors for SARS-CoV-2 papain-like protease

In the effort to combat SARS-CoV-2 infection, researchers are currently exploring the repurposing of conventional antiviral drugs, despite their limited efficacy. The SARS-CoV-2 virus encodes a papain-like protease (PLpro), which not only plays a crucial role in viral replication but also cleaves ubiquitin and interferon-stimulated gene 15 protein (ISG15) from host proteins, making it a prime target for the development of new antiviral medications. In this study, we conducted a multi-step in silico screening to identify novel, noncovalent PLpro inhibitors. Curcumin, an antioxidant derived from turmeric rhizomes (Curcuma longa L.), has undergone extensive preclinical investigations and shown significant efficacy against viruses and other ailments in both laboratory and animal studies. However, the pharmacological limitations of curcumin have prompted the synthesis of numerous novel curcumin analogs, necessitating evaluation for their therapeutic potential. The selectivity of the top-scoring compounds was assessed through molecular docking studies and molecular dynamics simulations to determine their binding affinity to PLpro. As a result, we identified 20 potential, selective PLpro inhibitors, from which the top two compounds (THA111 and THHGV6) were selected based on their binding free energy values towards PLpro as estimated by MM-PBSA calculations. These selected candidates demonstrate promising activity against the protein, with binding free energy values ranging from approximately −105 to −108 kJ/mol, and largely adopt a similar binding mode to known noncovalent SARS-CoV-2 PLpro inhibitors (GRL0617 = −100.98 kJ/mol). We further propose these two most promising compounds for future in vitro evaluation. The findings for the top potential PLpro inhibitors have been deposited in a database (Curcumin Research Center) to aid research on anti-SARS-CoV-2 drugs

On the evolution of genetic diversity in RNA virus species : uncovering barriers to genetic divergence and gene length in picorna- and nidoviruses

This thesis combines the use of standard bioinformatics analyses with the development of new computational techniques to study the evolution and genetic diversity of picornaviruses and nidoviruses. It integrates two lines of research __ genetics-based virus classification and evolutionary dynamics of gene length __ and aims at unveiling commonalities in the biology of these and other RNA viruses as well as assisting applied research in virology.NBIC, European UnionUBL - phd migration 201

Arterivirus replicase processing : regulatory cascade or Gordian knot?

Equine arteritis virus (EAV) is the prototypic virus of the family Arteriviridae. The EAV genome is a positive-sense single-stranded RNA molecule in which two open reading frames (ORFs) encode the large replicase polyproteins pp1a and pp1ab. Processing of pp1a and pp1ab is mediated by three viral proteases of which a predicted chymotrypsin-like protease residing in nsp4 releases most non-structural proteins from the replicase polyproteins. To obtain more insight in the biochemical properties of this protease, and viral chymotrypsin-like proteases in general, the three-dimensional structure of nsp4 was determined by X-ray crystallography. The nsp4 three-dimensional structure revealed that the enzyme adopts a chymotrypsin-like fold and possesses an additional C-terminal domain (CTD) not found in most other chymotrypsin-like proteases. The structure revealed also that a connecting stretch of amino acids might facilitate movement of the CTD relative to the rest of the molecule. A site-directed mutagenesis study showed that the nsp4 CTD played a crucial role in EAV replicase processing, but that it was not required for proteloytic activity of the protease per se. Furthermore, the formation of an ion pair between Asp-119 and either Arg-4 in the N-terminus or Arg-203 in the C-terminus was suggested, which could play a role in alternate nsp4 conformations needed for e.g. different cis and trans cleavage activities. Mutations targeting the residues involved in these interactions affected the proteolytic activity of nsp4, but the data were inconclusive regarding the importance of ion pair formation. Processing of the C-terminal half of pp1a (the nsp3-8 region) by nsp4 can proceed following either of two alternative proteolytic pathways. To address the importance of both pathways, various cleavage site mutations were engineered, which were expected to block cleavage by nsp4. The experiments showed that all mutations that blocked processing of the corresponding site in the nsp3-8 precursor also blocked or severely inhibited EAV RNA synthesis. Moreover, evidence was obtained for the presence of a novel, internal nsp4 cleavage site in nsp7, which appears to be conserved among arteriviruses. The theoretical chapters in this thesis introduce the reader to (nido)viruses and nidovirus replicase maturation in particular.UBL - phd migration 201

A fight against viral infections: host factors and antiviral therapies against positive-strand RNA viruses

Viral outbreaks can have devastating impacts on both human health and society. To develop effective antiviral treatments, it is important to understand the interactions between the host and the virus at the molecular level. In the first part of the thesis, we investigated the relationship between mosquito-borne viruses like chikungunya virus (CHIKV), dengue (DENV), Zika virus (ZIKV) and West Nile virus (WNV), and autophagy, a cellular degradative pathway. We discovered that the role of autophagy and autophagy-related proteins (ATG) can vary depending on the virus. For example, depletion of BNIP3 showed a marked increase in CHIKV replication, suggesting that BNIP3 is a new host factor that inhibits CHIKV early in its replication cycle. Moreover, knockout of ATG7 and ATG16L1 reduced DENV-2 viral particle production, suggesting the pro-viral role of these proteins in post-replicative stages of DENV-2 replication cycle.In the second part of this thesis, we examined the effectiveness of some antiviral agents against SARS-CoV-2. We found that boceprevir can inhibit β-coronaviruses by binding to the SARS-CoV-2 3CLpro protease's catalytic pocket. Resveratrol and pterostilbene also showed promise in inhibiting SARS-CoV-2 production by acting on post-entry stages of the replication cycle. Overall, this thesis provides new insights into the molecular mechanisms of mosquito-borne viruses and highlights potential avenues for developing antiviral therapies against SARS-CoV-2

Enteroviral evolution: interspecies recombination and implications for picornavirus research

The original aim of the project was to determine whether a non-poliovirus HEV could evolve to use the poliovirus receptor (PVR). A variety of methods were used to exploit aspects of the evolutionary capacity of viruses to achieve this goal. Although the aim was not attained, the investigation of recombinants between different HEVs yielded interesting results, which were pursued further. Two approaches were developed: in vitro generation of recombinant viruses and phenotypic analysis of such chimeras and the selection for recombinant viruses in vitro. In vitro generation of reciprocal recombinants between the structural and the non-structural coding region of coxsackievirus A21 (CVA21) and poliovirus type 3 (PV3) was initiated. Transfected and passaged chimeras did not produce infectious virions. Immunofluorescence analysis of VP1 protein expression suggested that the recombinants were not acytopathic. A series of assays were then carried out to investigate the nature of the defect. HeLa S10 translation/transcription reactions of the in vitro generated recombinants expressed the correct protein-processing pattern suggesting efficient processing occurred in vitro. Replication assays demonstrated that the chimeras were replication competent. Trans-encapsidation experiments were then carried out and preliminary results strongly suggested that the defect could lie at the packaging level. Selection of recombinants in vivo, without predetermining the crossover sites, was also conducted. Under the conditions used, recombinant between CVA21 and PV3 impaired genomes and echovirus 7 (EV7) and PV3 impaired genomes proved to be unsuccessful. Characterisation of the impaired parental genomes used for the experiment needs to be carried out. However, recent reports of recombinants between Sabin polioviruses and HEV-C confirm the possibility of such a recombination event occurring and emphasize concerns regarding the success of the polio eradication program