Cynarin blocks Ebola virus replication by counteracting VP35 inhibition of interferon-beta production.

Ebola virus (EBOV) is one of the deadliest infective agents whose lethality is linked to the ability to efficiently bypass the host's innate antiviral response. EBOV multifunctional protein VP35 plays a major role in viral replication both as polymerase cofactor and interferon (IFN) antagonist. By hiding the non-self 5'-ppp dsRNA from the cellular receptor RIG-I, VP35 prevents its activation and inhibits IFN-ß production. Blocking VP35-dsRNA interaction and IFN-ß suppression is a validated drug target. We screened a library of natural extracts and found that cynarin inhibits dsRNA-VP35 binding with an IC50 value of 8.5 µM. It reverts the EBOV VP35 inhibition of IFN-ß production, while it does not induce IFN production by itself. Docking experiments suggest that the molecule can bind on the end-capping pocket of VP35 C-terminal Interferon Inhibitory domain (IID), and differential scanning fluorimetry confirmed that cynarin interacts with VP35-IID with a KD of 12 µM. Cynarin was further tested in an EBOV minigenome assay but did not inhibit VP35 polymerase cofactor activity. When evaluated during challenge of IFN-susceptible A549 cells with EBOV isolate derived from the 2014 West African outbreak, cynarin was able to inhibit viral replication with an EC50 value of 9.1 µM, showing no significant cytotoxicity. Our findings show that cynarin blocks EBOV replication by acting directly on VP35 and subverting its IFN antagonism function but not cofactor function, and as such identify the first EBOV inhibitor with this mode of action.

Intracellular human antibody fragments recognizing the VP35 protein of Zaire Ebola filovirus inhibit the protein activity.

BACKGROUND: Ebola hemorrhagic fever is caused by the Ebola filovirus (EBOV), which is one of the most aggressive infectious agents known worldwide. The EBOV pathogenesis starts with uncontrolled viral replication and subversion of both the innate and adaptive host immune response. The multifunctional viral VP35 protein is involved in this process by exerting an antagonistic action against the early antiviral alpha/beta interferon (IFN-α/ß) response, and represents a suitable target for the development of strategies to control EBOV infection. Phage display technology permits to select antibodies as single chain Fragment variable (scFv) from an artificial immune system, due to their ability to specifically recognize the antigen of interest. ScFv is ideal for genetic manipulation and to obtain antibody constructs useful for targeting either antigens expressed on cell surface or intracellular antigens if the scFv is expressed as intracellular antibody (intrabody) or delivered into the cells. RESULTS: Monoclonal antibodies (mAb) in scFv format specific for the EBOV VP35 were isolated from the ETH-2 library of human recombinant antibodies by phage display technology. Five different clones were identified by sequencing, produced in E.coli and expressed in CHO mammalian cells to be characterized in vitro. All the selected scFvs were able to react with recombinant VP35 protein in ELISA, one of the scFvs being also able to react in Western Blot assay (WB). In addition, all scFvs were expressed in cell cytoplasm as intrabodies; a luciferase reporter gene inhibition assay performed in A549 cells showed that two of the scFvs can significantly hamper the inhibition of the IFN-ß-induced RIG-I signaling cascade mediated by EBOV VP35. CONCLUSION: Five antibodies in scFv format recognize an active form of EBOV VP35 in ELISA, while one antibody also recognizes VP35 in WB. Two of these scFvs were also able to interfere with the intracellular activity of VP35 in a cell system in vitro. These findings suggest that such antibodies in scFv format might be employed to develop therapeutic molecules able to hamper EBOV infections.

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. The 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 EBOVencoded 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 is validated target for drug development. Insights into 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.

Identification of Myricetin as an Ebola Virus VP35-Double-Stranded RNA Interaction Inhibitor through a Novel Fluorescence-Based Assay.

Ebola virus (EBOV) is a filovirus that causes a severe and rapidly progressing hemorrhagic syndrome; a recent epidemic illustrated the urgent need for novel therapeutic agents because no drugs have been approved for treatment of Ebola virus. A key contribution to the high lethality observed during EBOV outbreaks comes from viral evasion of the host antiviral innate immune response in which viral protein VP35 plays a crucial role, blocking interferon type I production, first by masking the viral double-stranded RNA (dsRNA) and preventing its detection by the pattern recognition receptor RIG-I. Aiming to identify inhibitors of the interaction of VP35 with the viral dsRNA, counteracting the VP35 viral innate immune evasion, we established a new methodology for high-yield recombinant VP35 (rVP35) expression and purification and a novel and robust fluorescence-based rVP35-RNA interaction assay ( Z' factor of 0.69). Taking advantage of such newly established methods, we screened a small library of Sardinian natural extracts, identifying Limonium morisianum as the most potent inhibitor extract. A bioguided fractionation led to the identification of myricetin as the component that can inhibit rVP35-dsRNA interaction with an IC50 value of 2.7 µM. Molecular docking studies showed that myricetin interacts with the highly conserved region of the VP35 RNA binding domain, laying the basis for further structural optimization of potent inhibitors of VP35-dsRNA interaction.

Development and Validation of a Novel Dual Luciferase Reporter Gene Assay to Quantify Ebola Virus VP24 Inhibition of IFN Signaling.

The interferon (IFN) system is the first line of defense against viral infections. Evasion of IFN signaling by Ebola viral protein 24 (VP24) is a critical event in the pathogenesis of the infection and, hence, VP24 is a potential target for drug development. Since no drugs target VP24, the identification of molecules able to inhibit VP24, restoring and possibly enhancing the IFN response, is a goal of concern. Accordingly, we developed a dual signal firefly and Renilla luciferase cell-based drug screening assay able to quantify IFN-mediated induction of Interferon Stimulated Genes (ISGs) and its inhibition by VP24. Human Embryonic Kidney 293T (HEK293T) cells were transiently transfected with a luciferase reporter gene construct driven by the promoter of ISGs, Interferon-Stimulated Response Element (ISRE). Stimulation of cells with IFN-α activated the IFN cascade leading to the expression of ISRE. Cotransfection of cells with a plasmid expressing VP24 cloned from a virus isolated during the last 2014 outbreak led to the inhibition of ISRE transcription, quantified by a luminescent signal. To adapt this system to test a large number of compounds, we performed it in 96-well plates; optimized the assay analyzing different parameters; and validated the system by calculating the Z'- and Z-factor, which showed values of 0.62 and 0.53 for IFN-α stimulation assay and VP24 inhibition assay, respectively, indicative of robust assay performance.

Antiviral Agents Against Ebola Virus Infection: Repositioning Old Drugs and Finding Novel Small Molecules.

Ebola virus (EBOV) causes a deadly hemorrhagic syndrome in humans with mortality rate up to 90%. First reported in Zaire in 1976, EBOV outbreaks showed a fluctuating trend during time and fora long period it was considered a tragic disease confined to the isolated regions of the African continent where the EBOV fear was perpetuated among the poor communities. The extreme severity of the recent 2014-16 EBOV outbreak in terms of fatality rate and rapid spread out of Africa led to the understanding that EBOV is a global health risk and highlights the necessity to find countermeasures against it. In the recent years, several small molecules have been shown to display in vitro and in vivo efficacy against EBOV and some of them have advanced into clinical trials. In addition, also existing drugs have been tested for their anti-EBOV activity and were shown to be promising candidates. However, despite the constant effort addressed to identify anti-EBOV therapeutics, no approved drugs are available against EBOV yet. In this chapter, we describe the main EBOV life cycle steps, providing a detailed picture of the druggable viral and host targets that have been explored so far by different technologies. We then summarize the small molecules, nucleic acid oligomers, and antibody-based therapies reported to have an effect either in in silico, or in biochemical and cell-based assays or in animal models and clinical trials, listing them according to their demonstrated or putative mechanism of action.

Uvaria angolensis as a promising source of inhibitors of HIV-1 RT-associated RNA-dependent DNA polymerase and RNase H functions.

Reverse transcriptase (RT)-associated DNA polymerase (RDDP) and ribonucleaser H (RNase H) functions are both essential for HIV-1 genome replication, and the identification of new inhibitors to block both of them is a goal actively pursued by the scientific community. In this field, natural extracts have shown a great potential as source of new antivirals. In the present work, we investigated the effect of Uvaria angolensis extracts on the HIV-1 reverse transcriptase-associated DNA polymerase and ribonuclease H activities. The U. angolensis stem bark methanol extract inhibit both HIV-1 RNase H function and RDDP activity with IC50 values of 1.0 ± 0.2 and 0.62 ± 0.15 µg/mL, respectively and, after been fractionated with different solvents, its solid residue showed an IC50 of 0.10 ± 0.03 and of 0.23 ± 0.04 µg/mL against RNase H and RDDP, respectively, hence laying the bases for further studies for identification of single active components.

Broad-range potential of Asphodelus microcarpus leaves extract for drug development.

BACKGROUND: Many plants have been used in traditional medicine for their antibacterial, antifungal, antiprotozoal, antiviral, antidiarrhoeal, analgesic, antimalarial, antioxidant, anti-inflammatory and anticancer activities. In order to find novel antimicrobial and antiviral agents, the aim of the present study was the evaluation of the antibacterial and antibiofilm susceptibility of Asphodelus microcarpus leaves extract. Moreover, the antiviral activity and the phytochemical composition of the active extract were also determined. METHODS: Antimicrobial and antibiofilm activities of leaves ethanol extract of A. microcarpus were evaluated on 13 different microbial strains. We selected three different sets of microorganisms: (i) Gram-positive bacteria, (ii) Gram-negative bacteria and (iii) yeasts. The potential antiviral activity of A. microcarpus leaves ethanol extract was evaluated with a luciferase reporter gene assay in which the dsRNA-dependent RIG-I-mediated IFN-ß activation was inducted or inhibited by the Ebola virus VP35 protein. HPLC-DAD-MS was used to identify phenolic profile of the active extract. RESULTS: A. microcarpus leaves extract showed a potent inhibitory activity on Gram-positive bacteria while only a reduced inhibition was observed on Gram-negative bacteria. No activity was detected against Yeasts. The extract also showed an interesting antibiofilm motif on various bacterial strains (E. coli, S. aureus, S. haemolyticus and B. clausii). Moreover, this extract significantly affected the Ebola virus VP35 inhibition of the viral RNA (vRNA) induced IFN response. CONCLUSIONS: The overall results provide supportive data on the use of A. microcarpus as antimicrobial agent and a potential source of anti-viral natural products. Data collected set the bases for further studies for the identification of single active components and the development of new pharmaceuticals.

Correction: ISG15 Regulates Peritoneal Macrophages Functionality against Viral Infection.

[This corrects the article DOI: 10.1371/journal.ppat.1003632.].

ISG15 regulates peritoneal macrophages functionality against viral infection.

Upon viral infection, the production of type I interferon (IFN) and the subsequent upregulation of IFN stimulated genes (ISGs) generate an antiviral state with an important role in the activation of innate and adaptive host immune responses. The ubiquitin-like protein (UBL) ISG15 is a critical IFN-induced antiviral molecule that protects against several viral infections, but the mechanism by which ISG15 exerts its antiviral function is not completely understood. Here, we report that ISG15 plays an important role in the regulation of macrophage responses. ISG15-/- macrophages display reduced activation, phagocytic capacity and programmed cell death activation in response to vaccinia virus (VACV) infection. Moreover, peritoneal macrophages from mice lacking ISG15 are neither able to phagocyte infected cells nor to block viral infection in co-culture experiments with VACV-infected murine embryonic fibroblast (MEFs). This phenotype is independent of cytokine production and secretion, but clearly correlates with impaired activation of the protein kinase AKT in ISG15 knock-out (KO) macrophages. Altogether, these results indicate an essential role of ISG15 in the cellular immune antiviral response and point out that a better understanding of the antiviral responses triggered by ISG15 may lead to the development of therapies against important human pathogens.