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

Relevance of Ebola virus VP35 homo-dimerization on the type I interferon cascade inhibition

Ebola virus high lethality relies on its ability to efficiently bypass the host innate antiviral response, which senses the viral dsRNA through the RIG-I receptor and induces type I interferon a/b production. In the bypassing action, the Ebola virus protein VP35 plays a pivotal role at multiple levels of the RIG-I cascade, masking the viral 50 -triphosphorylated dsRNA from RIG-I, and interacting with other cascade components. The VP35 type I interferon inhibition is exerted by the C-terminal domain, while the N-terminal domain, containing a coiled-coil region, is primarily required for oligomerization. However, mutations at key VP35 residues L90/93/107A (VP35-3m) in the coiled-coil region were reported to affect oligomerization and reduce type I interferon antagonism, indicating a possible but unclear role of homo-oligomerization on VP35 interaction with the RIG-I pathway components. In this work, we investigated the VP35 dimerization thermodynamics and its contribution to type I interferon antagonism by computational and biological methods. Focusing on the coiled-coil region, we combined coarse-grained and all-atom simulations on wild type VP35 and VP35-3m homo-dimerization. According to our results, wild type VP35 coiled-coil is able to self-assemble into dimers, while VP35-3m coiled-coil shows poor propensity to even dimerize. Free-energy calculations confirmed the key role of L90, L93 and L107 in stabilizing the coiled-coil homo-dimeric structure. In vitro type I interferon antagonism studies, using full-length wild type VP35 and VP35-3m, revealed that VP35 homo-dimerization is an essential preliminary step for dsRNA binding, which appears to be the main factor of the VP35 RIG-I cascade inhibition, while it is not essential to block the other steps

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. ISG152/2 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 pathogensThis work was supported by grants from the Spanish Ministry of Health FIS2011-00127, Comunidad de Madrid UAM-CM-CCG10-4911 and UAM-Banco de Santander to SG. This work was also partly supported by NIAID grant U19AI083025 and by CRIP (Center for Research on Influenza Pathogenesis, HHSN266200700010C), a NIAID Center of Excellence for Influenza Research and Surveillance (CEIRS) to AGS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip

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) Gramnegative 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-β 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

Development of Interfering Strategies Against the Innate Immune Inhibition by the VP35 Ebola virus Protein, a Key Viral Target

Relevant health-threatening pathogens developed during their evolution a number of strategies for counteracting the defense schemes of the host organisms and escape immune response. Recent outbreaks over the last 10 years of Ebola, West Nile, Chikungunya, Zika, Middle Eastern Respiratory and other emerging/re-emerging RNA viruses continue to teach a lesson to humankind and scientific community: we still need to further understand the virus–host interactions that regulate disease severity and infection outcome, in order to develop efficient strategies to counteract their anti-immunogenic activity. As part of the early host antiviral defense, the innate immune system mediates pathogen recognition setting up potent antiviral programs that aid to limit virus replication, virus spread, and activate adaptive immune responses. On the other side of the barricade, even viral pathogens evolved several strategies to counteract pathogen recognition and cell-intrinsic antiviral responses, dramatically orchestrating a profound reorganization of the host cell metabolism to create a favorable environment for viral multiplication. Ebola virus (EBOV), one of the most virulent and deadly pathogen ever known and faced by humankind, etiological agent of hemorrhagic fevers in humans and non-human primates today called Ebola virus disease (EVD), can be undoubtedly considered the perfect example of a potent host organism immune response inhibitor. Thanks to the action, among all the virus-coded proteins, of the viral protein 35 (VP35), EBOV determines a very opportune damage to the innate immune responses, a fundamental step to ensure the maximum efficiency in case of EBOV infection, contributing to disease progression and severity. Therefore, the PhD thesis here presented will focus on the study of different approaches that can be adopted to improve the host innate immune response when inhibited by the presence of the viral protein VP35 and on the development of efficient strategies for subverting this inhibitory blockade, with particular interest to: i. searching for molecules, synthetic as well as of natural origin, capable to stimulate the innate immune response of the host organism as well as to counteract the inhibition of the immune response mediated by EBOV VP35; ii) searching for molecules, synthetic as well as of natural origin, capable to directly interfere with a specific domain of the EBOV VP35 protein and inhibiting its biological anti-immunity function; iii) developing an antibody-based approach to directly inhibit the anti-immunity activity of the VP35 protein

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

Use of a Dual Luciferase Cell-Based Drug Screening Assay to Study VP24 Inhibition of JAK/STAT Pathway and its Reversion by Compounds

Ebola virus has evolved several and diversified strategies to antagonize the interferon (IFN) response in target cells leading to a complete impairment of the innate immune system which contribute to the pathogenesis of infection. A decisive role is exerted by VP24 that disables cells to contrast viral replication and propagation by inhibiting the IFN system at level of JAK/STAT pathway. The early control of viremia is one of the keys for survival, hence, blocking an important determinant of virulence such as VP24 is a valuable subject for investigation and a crucial pharmacological target. No drugs currently have been approved for VP24. Based on these findings, driven by the effort to identify compounds able to inhibit VP24, we developed a new miniaturized dual drug screening assay to quantify the suppression of IFN cascade by VP24 and the effect of potential inhibitors. The assay is based on the transient cotransfection of HEK293T cells with a luciferase reporter under the control of the promoter of IFN stimulated genes (pISRE-luc), a plasmid expressing VP24 and RL-TK, as control of transfection efficiency. The stimulation with IFN- led to the activation of ISRE expression in cells transfected only with pISRE-luc. In contrast, cotransfection with VP24 led to the inhibition of ISRE transcription. Addition of compounds, such the same IFN- , led to the partial restoration of the pathway. We optimized the assay to achieve excellent signal and robust performance. Further, the normalization with a Renilla luciferase control allowed to minimize variability between experiments providing high reproducibility

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. ISG152/2 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

El Correo gallego : diario político de la mañana: Ano LVIII Número 20110 - 1936 xullo 26

<p>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 <i>Uvaria angolensis</i> extracts on the HIV-1 reverse transcriptase-associated DNA polymerase and ribonuclease H activities. The <i>U. angolensis</i> 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.</p