Insights into Ebola Virus VP35 and VP24 Interferon inhibitory functions and their initial exploitation as drug targets

Upon viral infection, the interferon (IFN) system triggers potent antiviral mechanisms limiting viral growth and spread. Hence, to sustain their infection, viruses evolved efficient counteracting strategies to evade IFN control. Ebola virus (EBOV), member of the family Filoviridae, is one of the most virulent and deadly pathogen ever faced by humans. Etiological agent of the Ebola virus disease (EVD), EBOV can be undoubtedly considered the perfect example of a powerful inhibitor of the host organism immune response activation. Particularly, the efficacious suppression of the IFN cascade contributes to disease progression and severity. Among the EBOV-encoded proteins, the viral proteins 35 (VP35) and 24 (VP24) are responsible for the EBOV extreme virulence, representing the core of such inhibitory function through which EBOV determines its very effective shield to the cellular immune defenses. VP35 inhibits the activation of the cascade leading to IFN production, while VP24 inhibits the activation of the IFN-stimulated genes. A number of studies demonstrated that both VP35 and VP24 are validated target for drug development. Insights of the structural characteristics of VP35 and VP24 domains revealed crucial pockets exploitable for drug development. Considered the lack of therapy for EVD, restoring the immune activation is a promising approach for drug development. In the present review we summarize the importance of VP35 and VP24 proteins in counteracting the host IFN cellular response and discuss their potential as druggable viral targets as a promising approach toward attenuation of EBOV virulence

Exploitation of Ebola Virus VP35 Protein to identify new drugs counteracting its type I IFN antagonism

Ebolaviruses (EBOVs) are among the most virulent and deadly pathogens ever known, causing fulminant haemorrhagic fevers in humans and non-human primates. The 2014 outbreak of Ebola virus disease (EVD) in West Africa has claimed more lives than all previous EVD outbreaks combined. The EBOV high mortality rates have been related to the virus-induced impairment of the host innate immunity reaction due to two virus-coded proteins, VP24 and VP35. EBOV VP35 is a multifunctional protein, it is essential for viral replication as a component of the viral RNA polymerase and it also participates in nucleocapsid assembly. Early during EBOV infection, alpha-beta interferon (IFN-α/β) production would be triggered upon recognition of viral dsRNA products by cytoplasmic retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs). However, this recognition is efficiently prevented by the double-stranded RNA (dsRNA) binding activity of the EBOV VP35 protein, which hides RLRs binding sites on the dsRNA phosphate backbone as well the 5’-triphosphate (5’-ppp) dsRNA ends to RIG-I recognition. In addition to dsRNA binding and sequestration, EBOV VP35 inhibits IFN-α/β production preventing the activation of the IFN regulatory factor 3 (IRF-3) by direct interaction with cellular proteins. Previous studies demonstrated that single amino acid changes in the VP35 dsRNA binding domain reduce EBOV virulence, indicating that VP35 is an attractive target for antiviral drugs development. Within this context, here we report the establishment of a novel method to characterize the EBOV VP35 inhibitory function of the dsRNA-dependent RIG-I-mediated IFN-β signaling pathway in a BLS2 cell culture setting. In such system, a plasmid containing the promoter region of IFN-β gene linked with a luciferase reporter gene was transfected, together with a EBOV VP35 mammalian expression plasmid, into the IFN-sensitive A549 cell line, and the IFN-induction was stimulated through dsRNA transfection. Through alanine scanning mutational studies with biochemical, cellular and computational methods we highlighted the importance of some VP35 residues involved in dsRNA end-capping binding, such as R312, K282 and R322, that may serve as target for the development of small-molecule inhibitors against EBOV. Furthermore, we identified a synthetic compound that increased IFN-induction only under antiviral response stimulation and subverted VP35 inhibition, proving to be very attractive for the development of an antiviral drug. In conclusion, our results provide the establishment of a new assay as a straightforward tool for the screening of antiviral compounds that target i) dsRNA-VP35 or cellular protein-VP35 interaction and ii) dsRNA-dependent RIG-I-mediated IFN signaling pathway, in order to potentiate the IFN response against VP35 inhibition, setting the bases for further drug development

Evolutionary Targeted Discovery of Influenza A Virus Replication Inhibitors

Influenza A is one of the most prevalent and significant viral infections worldwide, resulting in annual epidemics and occasional pandemics. Upon infection, antiviral drugs targeting the neuraminidase protein and M2 protein are the only treatment options available. However, the emergence of antiviral drug resistance is concerning, therefore the aim of this work was to identify inhibitor molecules that may bind to highly conserved regions of selected internal influenza A proteins. Sequences of the non-structural protein 1 (NS1), nuclear export protein (NEP) and polymerase basic protein 2 (PB2) from all hosts and subtypes were aligned and the degree of amino acid conservation was calculated based on Valdar’s scoring method. Missing parts of the experimental structures were predicted using the I-TASSER server and ligand binding hot spots were identified with computational solvent mapping. Selected binding sites in conserved regions were subjected to virtual screening against two compound libraries using AutoDock Vina and AutoDock 4. Two out of twelve top hit compounds predicted to target the NS1 protein showed capability of reducing influenza A H1N1 replication in plaque reduction assays at concentrations below 100 µM, although the target protein and mechanism of action could not be confirmed. For the NEP, conservation analysis was based on 3000 sequences and binding hot spots were located in common areas amongst three structures. Docking results revealed predicted binding affinities of up to -8.95 kcal/mol, and conserved amino acid residues interacting with top compounds include Arg42, Asp43, Lys39, Ile80, Gln101, Leu105, and Val109. For the PB2 protein, conservation analysis was based on ~12,000 sequences and fifteen potential binding hot spots were identified. Docking results revealed predicted binding affinities of up to -10.3 kcal/mol, with top molecules interacting with the highly conserved residues Gln138, Gly222, Ile539, Asn540, Gly541, Tyr531 and Thr530. The findings from this research could provide starting points for in vitro experiments, as well as the development of antiviral drugs that function to inhibit influenza A replication without leading to resistance

Vývoj testovací metody pro identifikaci inhibitorů chřipkové polymerasy

Virus chřipky způsobuje infekční oněmocnění, postihující 3 až 5 milionů jedinců ročně. V současné době se k lečbě chřipkových infekcí používají dva druhy léčiv: inhibitory neuraminidasy a inhibitory iontového kanálu M2. Vzrůstající počet kmenů resistentních vůči těmto inhibitorům a riziko vzniku nových pandemických kmenů však vytvářejí dosud neuspokojenou potřebu nového typu inhibitorů. RNA-dependentní RNA polymerasa viru chřipky je novým cílem pro vývoj inhibitorů směrovaných proti viru chřipky. Cílem této diplomové práce bylo vyvinout vysokokapacitní testovací metody pro nalezení potenciálních inhibitorů dvou aktivit chřipkové polymerázy - vazby 7-methylguanosinové čepičky hostitelských RNA a jejich enodukleasové štěpění. Pro doménu vázající 7-methylguanosinovou čepičku bylo testování založeno na metodě DIANA (DNA-linked Inhibitor ANtibody Assay), která byla nedávno vyvinuta v laboratoři školitele; testování inhibitorů endonukleasové domény pak bylo založeno na technologii AlphaScreen. Pro potřeby vývoje velkokapacitních testovacích metod byla připravena rekombinantní doména vázající 7-methylguanosinovou čepičku z podjednotky PB2 a N-koncová doména PA podjednotky chřipkové polymerasy, nesoucí endonukleasovou aktivitu. Oba rekombinantní proteiny byly exprimovány s příslušnými afinitními značkami a...Influenza virus A circulates in birds and mammals and causes severe infectious disease that affects from 3 to 5 million people each year. There are two classes of anti-influenza drugs currently available: neuraminidase and M2 channel inhibitors. However, increasing resistance against these two types of inhibitors along with the potential emergence of new viral strains and unpredictability of pandemic outbreaks emphasize an unmet need for new types of inhibitors. RNA-dependent influenza polymerase serves as a novel promising target for the development of anti-influenza medications. The aim of this master thesis is to develop in vitro high-throughput assays for screening of compounds targeting influenza RNA polymerase, particularly, its cap binding and endonuclease domains. For cap-binding domain the screening is based on DIANA (DNA-linked Inhibitor ANtibody Assay) method that was recently developed in our laboratory; for endonuclease domain, the method is based on AlphaScreen technology. For the purposes of the methods development, recombinant cap binding domain of PB2 subunit and N-terminal endonuclease domain of PA subunit of influenza polymerase were expressed with appropriate fusion tags and purified using affinity and gel permeation chromatography. The probes for the screening assays were...Department of Physical and Macromolecular ChemistryKatedra fyzikální a makromol. chemiePřírodovědecká fakultaFaculty of Scienc

Interferon, viruses and drug discovery

The interferon (IFN) response is a crucial component of cellular innate immunity, vital for controlling virus infections. Dysregulation of the IFN response however can lead to serious medical conditions including autoimmune disorders. Modulators of IFN induction and signalling could be used to treat these diseases and as tools to further understand the IFN response and viral infections. We have developed cell-based assays to identify modulators of IFN induction and signalling, based on A549 cell lines where a GFP gene is under the control of the IFN-β promoter (A549/pr(IFN-β).GFP) and the ISRE containing MxA promoter (A549/pr(ISRE).GFP) respectively. The assays were optimized, miniaturized and validated as suitable for HTS by achieving Z’ Factor scores >0.6. A diversity screen of 15,667 compounds using the IFN induction reporter assay identified 2 hit compounds (StA-IFN-1 and StA-IFN-4) that were validated as specifically inhibiting IFNβ induction. Characterisation of these molecules demonstrated that StA-IFN-4 potently acts at, or upstream, of IRF3 phosphorylation. We successfully expanded this HTS platform to target viral interferon antagonists acting upon IFN-signalling. An additional assay was developed where the A549/pr(ISRE).GFP.RBV-P reporter cell line constitutively expresses the Rabies virus phosphoprotein. A compound inhibiting viral protein function will restore GFP expression. The assay was successfully optimized for HTS and used in an in-house screen. We further expanded this assay by placing the expression of RBV-P under the control of an inducible promoter. This demonstrates a convenient approach for assay development and potentiates the targeting of a variety of viral IFN antagonists for the identification of compounds with the potential to develop a novel class of antiviral drugs

Discovery and characterisation of host-factors involved in Zika virus

Zika virus (ZIKV, Flaviviridae), like other emerging arboviruses, poses a considerable threat to human health. It is estimated that approximately half of the world’s population is at risk from contracting a mosquito-borne arboviral infection, and this was exemplified during the 2015/16 ZIKV outbreak in the Americas. ZIKV infection is thought to be largely asymptomatic, although ZIKV disease has previously been characterised by mild symptoms such as a maculopapular rash, conjunctivitis, and fever. However, recent outbreaks have been associated with an increased incidence of Guillain-Barré syndrome, and a pattern of neurological and developmental symptoms in neonates which is now termed congenital Zika syndrome. Despite intense efforts, no therapeutic or vaccine has been developed. As such, it is vital that further fundamental research is conducted to discover novel host-virus interactions in both vector and host systems, which may allow development of targeted interventions. Here, multiple approaches were used to generate basic tools for ZIKV research, and siRNA screens and data from mass-spectrometry based proteomics were utilised to uncover important host interactors of ZIKV. A study investigating the Aedes aegypti immune response was conducted, and the classical RNAi effector Argonaute 2 (Ago2) was not found to be antiviral, whereas PIWI 4 was. Data from a previous proteomics experiment suggested that glucose-regulated protein 78 kDa (GRP78) may interact with ZIKV E. In this study, co-immunoprecipitation and immunofluorescence was used to verify that GRP78 interacts with ZIKV E in both mammalian and Aedes aegypti cell culture. GRP78 is a key modulator of the unfolded protein response (UPR), and while small-molecule inhibitors (EGCG and HNK) of the GRP78-mediated UPR did not inhibit ZIKV infection, EGCG was able to inhibit ZIKV entry independent of GRP78, likely through direct binding of the virion. Further study of GRP78 revealed that while it is not important for entry, replication, or egress of ZIKV, it did aid viral translation. Depletion of GRP78 with siRNA resulted in a loss of coordination of viral replication factories and relieved a virus-specific inhibition of host translation. Furthermore, STRING analysis of GRP78 host-interactors followed by a targeted siRNA screen revealed that DnaJC1 is also a pro-viral factor. DnaJC1 has previously been shown to coordinate GRP78 localisation to ribosome exit tunnels, and so may contribute to ZIKV infection through GRP78, though this was not assessed in this study. Additionally, by using a circular polymerase extension reaction (CPER) system, a reverse genetics ZIKV was generated. This CPER ZIKV represents a genetically stable source of virus which can be easily modified and can support future research. Collectively, the data herein informs on important ZIKV interactions in both arthropod vector and mammalian systems, and highlights tools and techniques that can be used to conduct future fundamental ZIKV research

New Advances on Zika Virus Research

Zika virus (ZIKV) is a mosquito-borne member of the Flaviviridae family that historically has been associated with mild febrile illness. However, the recent outbreaks in Brazil in 2015 and its rapid spread throughout South and Central America and the Caribbean, together with its association with severe neurological disorders—including fetal microcephaly and Guillain-Barré syndrome in adults—have changed the historic perspective of ZIKV. Currently, ZIKV is considered an important public health concern that has the potential to affect millions of people worldwide. The significance of ZIKV in human health and the lack of approved vaccines and/or antiviral drugs to combat ZIKV infection have triggered a global effort to develop effective countermeasures to prevent and/or treat ZIKV infection. In this Special Issue of Viruses, we have assembled a collection of 32 research and review articles that cover the more recent advances on ZIKV molecular biology, replication and transmission, virus–host interactions, pathogenesis, epidemiology, vaccine development, antivirals, and viral diagnosis

Meeting report: 34th international conference on antiviral research

As a result of the multiple gathering and travels restrictions during the SARS-CoV-2 pandemic, the annual meeting of the International Society for Antiviral Research (ISAR), the International Conference on Antiviral Research (ICAR), could not be held in person in 2021. Nonetheless, ISAR successfully organized a remote conference, retaining the most critical aspects of all ICARs, a collegiate gathering of researchers in academia, industry, government and non-governmental institutions working to develop, identify, and evaluate effective antiviral therapy for the benefit of all human beings. This article highlights the 2021 remote meeting, which presented the advances and objectives of antiviral and vaccine discovery, research, and development. The meeting resulted in a dynamic and effective exchange of ideas and information, positively impacting the prompt progress towards new and effective prophylaxis and therapeutics

Targeting cellular nuclear export to inhibit influenza A virus replication

Influenza A virus (IAV) is a global health threat, causing seasonal epidemics and potential pandemics leading to morbidity, death and economic losses. Currently, there are two main classes of licensed antivirals against IAV available in the US and Europe; adamantanes and neuraminidase inhibitors, both of which are hindered by the generation of resistant virus variants. The viral polymerase has a high error rate leading to mutations that allow the virus to overcome selection pressures directed at its own genome from conventional antivirals. The prospect of inhibiting host proteins that the virus exploits to facilitate its replication is of increasing interest as an antiviral strategy as the emergence of resistance has been predicted to be slower when targeting a host cellular factor. IAV utilizes the host nuclear export protein CRM1 to transport viral ribonucleoproteins (vRNPs) from the nucleus to the cytoplasm of an infected cell, a critical late stage of the influenza lifecycle. Leptomycin B (LMB), a Streptomyces metabolite, has been previously shown to target this pathway, resulting in reduced viral propagation; however, LMB’s potent cytotoxicity has limited its use as a therapeutic agent. This thesis examined two novel selective inhibitors of nuclear export (SINE), KPT-335 and KPT-185, with less cytotoxicity. In vitro, KPT-335 inhibited replication of human and animal IAV strains in a dose-dependent manner with minimal cytotoxicity. To assess the resistance potential of KPT-335, IAV viruses were serially passaged in the presence of a sub-optimal concentration of the compound and assayed for the development of resistance. Resistance to KPT-335 became evident at 8-10 rounds of passage. Sequencing analysis of independently derived resistant virus clones identified 4 single amino acid changes on a surface exposed patch of the viral nucleoprotein (NP). Introduction of these amino acid changes, into otherwise wild type viruses by reverse genetics, confirmed that changes Q311R and N309T conferred a drug-resistant phenotype. However, these substitutions came at a fitness cost to virus replication. The molecular basis for resistance was unclear but Q311R and N309T NP-mutant viruses produced increased levels of M1 during infection as well as producing virus particles with increased M1:NP ratios. Furthermore, the KPT-335-resistance mutations were surprisingly similar to NP sequence polymorphisms previously associated with susceptibility to the innate defence protein MxA. Consistent with this, viruses harbouring the Q311R mutation displayed increased susceptibility to MxA inhibition compared to wild-type virus. Altogether this study confirms that SINEs have the potential to be successful therapeutic agents against IAV replication and that although resistance could be generated, it may be difficult for the virus to overcome both drug selection pressures and the human innate immune response restrictions by escape mutations

Gastrointestinal viruses and beyond: antiviral development and molecular epidemiology

With over 27 viral families known to infect humans, viral pathogens impose a significant global public health and economic burden. Despite this, only a small fraction of human viruses possess antiviral treatments or vaccines. Whilst antiviral development efforts are crucial to the host-pathogen arms race, so too is the molecular surveillance of these viruses to identify prevalent and virulent strains for vaccine development. This thesis begins, in chapter four, with the development of broad-spectrum non-nucleoside inhibitor compounds using a complex-based pharmacophore and virtual screening approach. This virtual screen identified one compound, NCS-013, which demonstrated broad-spectrum inhibition of the transcriptional activity of human norovirus and feline calicivirus from the Caliciviridae, Zika virus and hepatitis C virus from the Flaviviridae and hepatitis A virus from the Picornaviridae. The second half of the thesis focuses on molecular epidemiology of norovirus and adenovirus, two of the leading causes of viral gastroenteritis worldwide. In chapter five, we observed an interesting dynamic of GII.4 Sydney 2012 [P16] co-dominance in clinical samples throughout the study period. We also enhanced our sewage surveillance capabilities through the addition of partial ORF1 sequencing enabling the identification of recombinant strains. The role of non-group F adenoviruses in gastroenteritis although often reported, remains an area of controversy. In chapter six, we analysed sewage to complement clinical samples and better understand the diversity of adenovirus within the population, including from healthy individuals. In summary, this thesis approached the problem of viral pathogens from both the angle of antiviral development and through understanding of population-level molecular epidemiology, which can contribute to future vaccine development efforts