Screening and Reverse-Engineering of Estrogen Receptor Ligands as Potent Pan-Filovirus Inhibitors.

Filoviridae, including Ebola (EBOV) and Marburg (MARV) viruses, are emerging pathogens that pose a serious threat to public health. No agents have been approved to treat filovirus infections, representing a major unmet medical need. The selective estrogen receptor modulator (SERM) toremifene was previously identified from a screen of FDA-approved drugs as a potent EBOV viral entry inhibitor, via binding to EBOV glycoprotein (GP). A focused screen of ER ligands identified ridaifen-B as a potent dual inhibitor of EBOV and MARV. Optimization and reverse-engineering to remove ER activity led to a novel compound 30 (XL-147) showing potent inhibition against infectious EBOV Zaire (0.09 µM) and MARV (0.64 µM). Mutagenesis studies confirmed that inhibition of EBOV viral entry is mediated by the direct interaction with GP. Importantly, compound 30 displayed a broad-spectrum antifilovirus activity against Bundibugyo, Tai Forest, Reston, and Menglà viruses and is the first submicromolar antiviral agent reported for some of these strains, therefore warranting further development as a pan-filovirus inhibitor.

Development of coumarine derivatives as potent anti-filovirus entry inhibitors targeting viral glycoprotein.

Filoviruses, including Ebolavirus (EBOV), Marburgvirus (MARV) and Cuevavirus, cause hemorrhagic fevers in humans with up to 90% mortality rates. In the 2014-2016 West Africa Ebola epidemic, there are 15,261 laboratory confirmed cases and 11,325 total deaths. The lack of effective vaccines and medicines for the prevention and treatment of filovirus infection in humans stresses the urgency to develop antiviral therapeutics against filovirus-associated diseases. Our previous study identified a histamine receptor antagonist compound CP19 as an entry inhibitor against both EBOV and MARV. The preliminary structure-activity relationship (SAR) studies of CP19 showed that its piperidine, coumarin and linker were related with its antiviral activities. In this study, we performed detailed SAR studies on these groups with synthesized CP19 derivatives. We discovered that 1) the piperidine group could be optimized with heterocycles, 2) the substitution groups of C3 and C4 of coumarin should be relatively large hydrophobic groups and 3) the linker part should be least substituted. Based on the SAR analysis, we synthesized compound 32 as a potent entry inhibitor of EBOV and MARV (IC50 = 0.5 µM for EBOV and 1.5 µM for MARV). The mutation studies of Ebola glycoprotein and molecular docking studies showed that the coumarin and its substituted groups of compound 32 bind to the pocket of Ebola glycoprotein in a similar way to the published entry inhibitor compound 118a. However, the carboxamide group of compound 32 does not have strong interaction with N61 as compound 118a does. The coumarin skeleton structure and the binding model of compound 32 elucidated by this study could be utilized to guide further design and optimization of entry inhibitors targeting the filovirus glycoproteins.

Modular mimicry and engagement of the Hippo pathway by Marburg virus VP40: Implications for filovirus biology and budding.

Ebola (EBOV) and Marburg (MARV) are members of the Filoviridae family, which continue to emerge and cause sporadic outbreaks of hemorrhagic fever with high mortality rates. Filoviruses utilize their VP40 matrix protein to drive virion assembly and budding, in part, by recruitment of specific WW-domain-bearing host proteins via its conserved PPxY Late (L) domain motif. Here, we screened an array of 115 mammalian, bacterially expressed and purified WW-domains using a PPxY-containing peptide from MARV VP40 (mVP40) to identify novel host interactors. Using this unbiased approach, we identified Yes Associated Protein (YAP) and Transcriptional co-Activator with PDZ-binding motif (TAZ) as novel mVP40 PPxY interactors. YAP and TAZ function as downstream transcriptional effectors of the Hippo signaling pathway that regulates cell proliferation, migration and apoptosis. We demonstrate that ectopic expression of YAP or TAZ along with mVP40 leads to significant inhibition of budding of mVP40 VLPs in a WW-domain/PPxY dependent manner. Moreover, YAP colocalized with mVP40 in the cytoplasm, and inhibition of mVP40 VLP budding was more pronounced when YAP was localized predominantly in the cytoplasm rather than in the nucleus. A key regulator of YAP nuclear/cytoplasmic localization and function is angiomotin (Amot); a multi-PPxY containing protein that strongly interacts with YAP WW-domains. Interestingly, we found that expression of PPxY-containing Amot rescued mVP40 VLP egress from either YAP- or TAZ-mediated inhibition in a PPxY-dependent manner. Importantly, using a stable Amot-knockdown cell line, we found that expression of Amot was critical for efficient egress of mVP40 VLPs as well as egress and spread of authentic MARV in infected cell cultures. In sum, we identified novel negative (YAP/TAZ) and positive (Amot) regulators of MARV VP40-mediated egress, that likely function in part, via competition between host and viral PPxY motifs binding to modular host WW-domains. These findings not only impact our mechanistic understanding of virus budding and spread, but also may impact the development of new antiviral strategies.

Cytokine Effects on the Entry of Filovirus Envelope Pseudotyped Virus-Like Particles into Primary Human Macrophages.

Macrophages are one of the first and also a major site of filovirus replication and, in addition, are a source of multiple cytokines, presumed to play a critical role in the pathogenesis of the viral infection. Some of these cytokines are known to induce macrophage phenotypic changes in vitro, but how macrophage polarization may affect the cell susceptibility to filovirus entry remains largely unstudied. We generated different macrophage subsets using cytokine pre-treatment and subsequently tested their ability to fuse with beta-lactamase containing virus-like particles (VLP), pseudotyped with the surface glycoprotein of Ebola virus (EBOV) or the glycoproteins of other clinically relevant filovirus species. We found that pre-incubation of primary human monocyte-derived macrophages (MDM) with interleukin-10 (IL-10) significantly enhanced filovirus entry into cells obtained from multiple healthy donors, and the IL-10 effect was preserved in the presence of pro-inflammatory cytokines found to be elevated during EBOV disease. In contrast, fusion of IL-10-treated macrophages with influenza hemagglutinin/neuraminidase pseudotyped VLPs was unchanged or slightly reduced. Importantly, our in vitro data showing enhanced virus entry are consistent with the correlation established between elevated serum IL-10 and increased mortality in filovirus infected patients and also reveal a novel mechanism that may account for the IL-10-mediated increase in filovirus pathogenicity.

Assessment of the function and intergenus-compatibility of Ebola and Lloviu virus proteins.

Sequences for Lloviu virus (LLOV), a putative novel filovirus, were first identified in Miniopterus schreibersii bats in Spain following a massive bat die-off in 2002, and also recently found in bats in Hungary. However, until now it is unclear if these sequences correspond to a fully functional, infectious virus, and whether it will show a pathogenic phenotype like African filoviruses, such as ebola- and marburgviruses, or be apathogenic for humans, like the Asian filovirus Reston virus. Since no infectious virus has been recovered, the only opportunity to study infectious LLOV is to use a reverse genetics-based full-length clone system to de novo generate LLOV. As a first step in this process, and to investigate whether the identified sequences indeed correspond to functional viral proteins, we have developed life cycle modelling systems for LLOV, which allow us to study genome replication and transcription as well as entry of this virus. We show that all LLOV proteins fulfill their canonical role in the virus life cycle as expected based on the well-studied related filovirus Ebola virus. Further, we have analysed the intergenus-compatibility of proteins that have to act in concert to facilitate the virus life cycle. We show that some but not all proteins from LLOV and Ebola virus are compatible with each other, emphasizing the close relationship of these viruses, and informing future studies of filovirus biology with respect to the generation of genus-chimeric proteins in order to probe virus protein-protein interactions on a functional level.

Characterization of the Filovirus-Resistant Cell Line SH-SY5Y Reveals Redundant Role of Cell Surface Entry Factors.

Filoviruses infect a wide range of cell types with the exception of lymphocytes. The intracellular proteins cathepsin B and L, two-pore channel 1 and 2, and bona fide receptor Niemann⁻Pick Disease C1 (NPC1) are essential for the endosomal phase of cell entry. However, earlier steps of filoviral infection remain poorly characterized. Numerous plasma membrane proteins have been implicated in attachment but it is still unclear which ones are sufficient for productive entry. To define a minimal set of host factors required for filoviral glycoprotein-driven cell entry, we screened twelve cell lines and identified the nonlymphocytic cell line SH-SY5Y to be specifically resistant to filovirus infection. Heterokaryons of SH-SY5Y cells fused to susceptible cells were susceptible to filoviruses, indicating that SH-SY5Y cells do not express a restriction factor but lack an enabling factor critical for filovirus entry. However, all tested cell lines expressed functional intracellular factors. Global gene expression profiling of known cell surface entry factors and protein expression levels of analyzed attachment factors did not reveal any correlation between susceptibility and expression of a specific host factor. Using binding assays with recombinant filovirus glycoprotein, we identified cell attachment as the step impaired in filovirus entry in SH-SY5Y cells. Individual overexpression of attachment factors T-cell immunoglobulin and mucin domain 1 (TIM-1), Axl, Mer, or dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) rendered SH-SY5Y cells susceptible to filovirus glycoprotein-driven transduction. Our study reveals that a lack of attachment factors limits filovirus entry and provides direct experimental support for a model of filoviral cell attachment where host factor usage at the cell surface is highly promiscuous.

A Diacylglycerol Kinase Inhibitor, R-59-022, Blocks Filovirus Internalization in Host Cells.

Filoviruses, such as Ebola virus (EBOV) and Marburg virus, are causative agents of unpredictable outbreaks of severe hemorrhagic fevers in humans and non-human primates. For infection, filoviral particles need to be internalized and delivered to intracellular vesicles containing cathepsin proteases and the viral receptor Niemann-Pick C1. Previous studies have shown that EBOV triggers macropinocytosis of the viral particles in a glycoprotein (GP)-dependent manner, but the molecular events required for filovirus internalization remain mostly unknown. Here we report that the diacylglycerol kinase inhibitor, R-59-022, blocks EBOV GP-mediated entry into Vero cells and bone marrow-derived macrophages. Investigation of the mode of action of the inhibitor revealed that it blocked an early step in entry, more specifically, the internalization of the viral particles via macropinocytosis. Finally, R-59-022 blocked viral entry mediated by a panel of pathogenic filovirus GPs and inhibited growth of replicative Ebola virus. Taken together, our studies suggest that R-59-022 could be used as a tool to investigate macropinocytic uptake of filoviruses and could be a starting point for the development of pan-filoviral therapeutics.

Virus and host interactions critical for filoviral RNA synthesis as therapeutic targets.

Filoviruses, which include Ebola virus (EBOV) and Marburg virus, are negative-sense RNA viruses associated with sporadic outbreaks of severe viral hemorrhagic fever characterized by uncontrolled virus replication. The extreme virulence and emerging nature of these zoonotic pathogens make them a significant threat to human health. Replication of the filovirus genome and production of viral RNAs require the function of a complex of four viral proteins, the nucleoprotein (NP), viral protein 35 (VP35), viral protein 30 (VP30) and large protein (L). The latter performs the enzymatic activities required for production of viral RNAs and capping of viral mRNAs. Although it has been recognized that interactions between the virus-encoded components of the EBOV RNA polymerase complex are required for viral RNA synthesis reactions, specific molecular details have, until recently, been lacking. New efforts have combined structural biology and molecular virology to reveal in great detail the molecular basis for critical protein-protein interactions (PPIs) necessary for viral RNA synthesis. These efforts include recent studies that have identified a range of interacting host factors and in some instances demonstrated unique mechanisms by which they act. For a select number of these interactions, combined use of mutagenesis, over-expressing of peptides corresponding to PPI interfaces and identification of small molecules that disrupt PPIs have demonstrated the functional significance of virus-virus and virus-host PPIs and suggest several as potential targets for therapeutic intervention.

Bipartite promoters and RNA editing of paramyxoviruses and filoviruses.

A primary property of paramyxovirus bipartite promoters is to ensure that their RNA genomes are imprinted with a hexamer phase via their association with nucleoproteins, in part because this phase as well the editing sequence itself controls mRNA editing. The question then arises whether a similar mechanism operates for filoviruses that also contain bipartite promoters that are governed by the "rule of six," even though these genomes need not, and given Ebola virus biology, cannot always be of hexamer genome length. This review suggests that this is possible and describes how it might operate, and that RNA editing may play a role in Ebola virus genome interconversion that helps the virus adapt to different host environments.

Persistence and Sexual Transmission of Filoviruses.

There is an increasing frequency of reports regarding the persistence of the Ebola virus (EBOV) in Ebola virus disease (EVD) survivors. During the 2014⁻2016 West African EVD epidemic, sporadic transmission events resulted in the initiation of new chains of human-to-human transmission. Multiple reports strongly suggest that these re-emergences were linked to persistent EBOV infections and included sexual transmission from EVD survivors. Asymptomatic infection and long-term viral persistence in EVD survivors could result in incidental introductions of the Ebola virus in new geographic regions and raise important national and local public health concerns. Alarmingly, although the persistence of filoviruses and their potential for sexual transmission have been documented since the emergence of such viruses in 1967, there is limited knowledge regarding the events that result in filovirus transmission to, and persistence within, the male reproductive tract. Asymptomatic infection and long-term viral persistence in male EVD survivors could lead to incidental transfer of EBOV to new geographic regions, thereby generating widespread outbreaks that constitute a significant threat to national and global public health. Here, we review filovirus testicular persistence and discuss the current state of knowledge regarding the rates of persistence in male survivors, and mechanisms underlying reproductive tract localization and sexual transmission.

[Innate immunity evasion mechanisms of filoviruses]. / Mécanismes d'échappement des filovirus à l'immunité innée.

Ebola virus is an important pathogen that emerged in Central Africa where it was responsible of numerous outbreaks of haemorrhagic fevers associated with a extremely high mortality rate (up to 90%). The filovirus pathogenicity is related to an inappropriate antiviral response. Indeed, this family of viruses has developed evasion strategies from early innate immunity mechanisms. As a result, a massive viral replication induces an unsuitable immune response causing an acute inflammatory reaction associated with the haemorrhagic syndrome. In this review, we describe the mechanisms adopted by filoviruses like Ebola virus to escape innate immunity response.

Filovirus proteins for antiviral drug discovery: Structure/function of proteins involved in assembly and budding.

There are no approved medications for the treatment of Marburg or Ebola virus infection. In two previous articles (Martin et al., 2016, Martin et al., 2017), we reviewed surface glycoprotein and replication proteins structure/function relationship to decipher the molecular mechanisms of filovirus life cycle and identify antiviral strategies. In the present article, we recapitulate knowledge about the viral proteins involved in filovirus assembly and budding. First we describe the structural data available for viral proteins associated with virus assembly and virion egress and then, we integrate the structural features of these proteins in the functional context of the viral replication cycle. Finally, we summarize recent advances in the development of innovative antiviral strategies to target filovirus assembly and egress. The development of such prophylactic or post-exposure treatments could help controlling future filovirus outbreaks.

Retro-2 and its dihydroquinazolinone derivatives inhibit filovirus infection.

Members of the family Filoviridae cause severe, often fatal disease in humans, for which there are no approved vaccines and only a few experimental drugs tested in animal models. Retro-2, a small molecule that inhibits retrograde trafficking of bacterial and plant toxins inside host cells, has been demonstrated to be effective against a range of bacterial and virus pathogens, both in vitro and in animal models. Here, we demonstrated that Retro-2 and its derivatives, Retro-2.1 and compound 25, blocked infection by Ebola virus and Marburg virus in vitro. We show that the derivatives were more potent inhibitors of infection as compared to the parent compound. Pseudotyped virus assays indicated that the compounds affected virus entry into cells while virus particle localization to Niemann-Pick C1-positive compartments showed that they acted at a late step in virus entry. Our work demonstrates a potential for Retro-type drugs to be developed into anti-filoviral therapeutics.

Reverse Genetics of Filoviruses.

Reverse genetics systems are used for the generation of recombinant viruses. For filoviruses, this technology has been available for more than 15 years and has been used to investigate questions regarding the molecular biology, pathogenicity, and host adaptation determinants of these viruses. Further, reporter-expressing, recombinant viruses are increasingly used as tools for screening for and characterization of candidate medical countermeasures. Thus, reverse genetics systems represent powerful research tools. Here we provide an overview of available reverse genetics systems for the generation of recombinant filoviruses, potential applications, and the achievements that have been made using these systems.

Filovirus Strategies to Escape Antiviral Responses.

This chapter describes the various strategies filoviruses use to escape host immune responses with a focus on innate immune and cell death pathways. Since filovirus replication can be efficiently blocked by interferon (IFN), filoviruses have evolved mechanisms to counteract both type I IFN induction and IFN response signaling pathways. Intriguingly, marburg- and ebolaviruses use different strategies to inhibit IFN signaling. This chapter also summarizes what is known about the role of IFN-stimulated genes (ISGs) in filovirus infection. These fall into three categories: those that restrict filovirus replication, those whose activation is inhibited by filoviruses, and those that have no measurable effect on viral replication. In addition to innate immunity, mammalian cells have evolved strategies to counter viral infections, including the induction of cell death and stress response pathways, and we summarize our current knowledge of how filoviruses interact with these pathways. Finally, this chapter delves into the interaction of EBOV with myeloid dendritic cells and macrophages and the associated inflammatory response, which differs dramatically between these cell types when they are infected with EBOV. In summary, we highlight the multifaceted nature of the host-viral interactions during filoviral infections.

Mechanisms of Filovirus Entry.

Filovirus entry into cells is complex, perhaps as complex as any viral entry mechanism identified to date. However, over the past 10 years, the important events required for filoviruses to enter into the endosomal compartment and fuse with vesicular membranes have been elucidated (Fig. 1). Here, we highlight the important steps that are required for productive entry of filoviruses into mammalian cells.

Therapeutic treatment of Marburg and Ravn virus infection in nonhuman primates with a human monoclonal antibody.

As observed during the 2013-2016 Ebola virus disease epidemic, containment of filovirus outbreaks is challenging and made more difficult by the lack of approved vaccine or therapeutic options. Marburg and Ravn viruses are highly virulent and cause severe and frequently lethal disease in humans. Monoclonal antibodies (mAbs) are a platform technology in wide use for autoimmune and oncology indications. Previously, we described human mAbs that can protect mice from lethal challenge with Marburg virus. We demonstrate that one of these mAbs, MR191-N, can confer a survival benefit of up to 100% to Marburg or Ravn virus-infected rhesus macaques when treatment is initiated up to 5 days post-inoculation. These findings extend the small but growing body of evidence that mAbs can impart therapeutic benefit during advanced stages of disease with highly virulent viruses and could be useful in epidemic settings.

Filovirus proteins for antiviral drug discovery: Structure/function bases of the replication cycle.

Filoviruses are important pathogens that cause severe and often fatal hemorrhagic fever in humans, for which no approved vaccines and antiviral treatments are yet available. In an earlier article (Martin et al., Antiviral Research, 2016), we reviewed the role of the filovirus surface glycoprotein in replication and as a target for drugs and vaccines. In this review, we focus on recent findings on the filovirus replication machinery and how they could be used for the identification of new therapeutic targets and the development of new antiviral compounds. First, we summarize the recent structural and functional advances on the molecules involved in filovirus replication/transcription cycle, particularly the NP, VP30, VP35 proteins, and the "large" protein L, which harbors the RNA-dependent RNA polymerase (RdRp) and mRNA capping activities. These proteins are essential for viral mRNA synthesis and genome replication, and consequently they constitute attractive targets for drug design. We then describe how these insights into filovirus replication mechanisms and the structure/function characterization of the involved proteins have led to the development of new and innovative antiviral strategies that may help reduce the filovirus disease case fatality rate through post-exposure or prophylactic treatments.

Modelling filovirus maintenance in nature by experimental transmission of Marburg virus between Egyptian rousette bats.

The Egyptian rousette bat (ERB) is a natural reservoir host for Marburg virus (MARV); however, the mechanisms by which MARV is transmitted bat-to-bat and to other animals are unclear. Here we co-house MARV-inoculated donor ERBs with naive contact ERBs. MARV shedding is detected in oral, rectal and urine specimens from inoculated bats from 5-19 days post infection. Simultaneously, MARV is detected in oral specimens from contact bats, indicating oral exposure to the virus. In the late study phase, we provide evidence that MARV can be horizontally transmitted from inoculated to contact ERBs by finding MARV RNA in blood and oral specimens from contact bats, followed by MARV IgG antibodies in these same bats. This study demonstrates that MARV can be horizontally transmitted from inoculated to contact ERBs, thereby providing a model for filovirus maintenance in its natural reservoir host and a potential mechanism for virus spillover to other animals.

A Sensitive in Vitro High-Throughput Screen To Identify Pan-filoviral Replication Inhibitors Targeting the VP35-NP Interface.

The 2014 Ebola outbreak in West Africa, the largest outbreak on record, highlighted the need for novel approaches to therapeutics targeting Ebola virus (EBOV). Within the EBOV replication complex, the interaction between polymerase cofactor, viral protein 35 (VP35), and nucleoprotein (NP) is critical for viral RNA synthesis. We recently identified a peptide at the N-terminus of VP35 (termed NPBP) that is sufficient for interaction with NP and suppresses EBOV replication, suggesting that the NPBP binding pocket can serve as a potential drug target. Here we describe the development and validation of a sensitive high-throughput screen (HTS) using a fluorescence polarization assay. Initial hits from this HTS include the FDA-approved compound tolcapone, whose potency against EBOV infection was validated in a nonfluorescent secondary assay. High conservation of the NP-VP35 interface among filoviruses suggests that this assay has the capacity to identify pan-filoviral inhibitors for development as antivirals.