Summary The most recent Ebola virus outbreak in West Africa – unprecedented in the number of cases and fatalities, geographic distribution, and number of nations affected – highlights the need for safe, effective, and readily available antiviral agents for treatment and prevention of acute Ebola virus (EBOV) disease (EVD) or sequelae1. No antiviral therapeutics have yet received regulatory approval or demonstrated clinical efficacy. Here we describe the discovery of a novel anti-EBOV small molecule antiviral, GS-5734, a monophosphoramidate prodrug of an adenosine analog. GS-5734 exhibits antiviral activity against multiple variants of EBOV in cell-based assays. The pharmacologically active nucleoside triphosphate (NTP) is efficiently formed in multiple human cell types incubated with GS-5734 in vitro, and the NTP acts as an alternate substrate and RNA-chain terminator in primer-extension assays utilizing a surrogate respiratory syncytial virus RNA polymerase. Intravenous administration of GS-5734 to nonhuman primates resulted in persistent NTP levels in peripheral blood mononuclear cells (half-life = 14 h) and distribution to sanctuary sites for viral replication including testes, eye, and brain. In a rhesus monkey model of EVD, once daily intravenous administration of 10 mg/kg GS-5734 for 12 days resulted in profound suppression of EBOV replication and protected 100% of EBOV-infected animals against lethal disease, ameliorating clinical disease signs and pathophysiological markers, even when treatments were initiated three days after virus exposure when systemic viral RNA was detected in two of six treated animals. These results provide the first substantive, post-exposure protection by a small-molecule antiviral compound against EBOV in nonhuman primates. The broad-spectrum antiviral activity of GS-5734 in vitro against other pathogenic RNA viruses – including filoviruses, arenaviruses, and coronaviruses – suggests the potential for expanded indications. GS-5734 is amenable to large-scale manufacturing, and clinical studies investigating the drug safety and pharmacokinetics are ongoing.

The matrix protein VP40 plays a critical role in Ebola virus assembly and budding, a process that utilizes specialized membrane domains known as lipid rafts. Previous studies with purified protein suggest a role for oligomerization of VP40 in this process. Here, we demonstrate VP40 oligomers in lipid rafts of mammalian cells, virus-like particles, and in the authentic Ebola virus. By mutagenesis, we identify several critical C-terminal sequences that regulate oligomerization at the plasma membrane, association with detergent-resistant membranes, and vesicular release of VP40, directly linking these phenomena. Furthermore, we demonstrate the active recruitment of TSG101 into lipid rafts by VP40. We also report the successful application of the biarsenic fluorophore, FlAsH, combined with a tetracysteine tag for imaging of Ebola VP40 in live cells.rafts ͉ FlAsH ͉ TSG101 ͉ filovirus

SUMMARY RNA viruses exhibit a variety of genome organization strategies, including multicomponent genomes in which each segment is packaged separately. While multicomponent genomes are common among viruses infecting plants and fungi, their prevalence among those infecting animals remains unclear. We characterize a multicomponent RNA virus isolated from mosquitoes, designated Guaico Culex virus (GCXV). GCXV belongs to a diverse clade of segmented viruses (Jingmenvirus) related to the prototypically unsegmented Flaviviridae. The GCXV genome comprises five segments, each of which appears to be separately packaged. The smallest segment is not required for replication, and its presence is variable in natural infections. We also describe a variant of Jingmen tick virus, another Jingmenvirus, sequenced from a Ugandan red colobus monkey, thus expanding the host range of this segmented and likely multicomponent virus group. Collectively, this study provides evidence for the existence of multicomponent animal viruses and their potential relevance for animal and human health.

An efficient research strategy integrating empirically guided, structure-based modeling and chemoinformatics was used to discover potent small molecule inhibitors of the botulinum neurotoxin serotype A light chain. First, a modeled binding mode for inhibitor 2-mercapto-3-phenylpropionyl-RATKML (K i ‫؍‬ 330 nM) was generated, and required the use of a molecular dynamic conformer of the enzyme displaying the reorientation of surface loops bordering the substrate binding cleft. These flexible loops are conformationally variable in x-ray crystal structures, and the model predicted that they were pivotal for providing complementary binding surfaces and solvent shielding for the pseudo-peptide. The docked conformation of 2-mercapto-3-phenylpropionyl-RATKML was then used to refine our pharmacophore for botulinum serotype A light chain inhibition. Data base search queries derived from the pharmacophore were employed to mine small molecule (non-peptidic) inhibitors from the National Cancer Institute's Open Repository. Four of the inhibitors possess K i values ranging from 3.0 to 10.0 M.Of these, NSC 240898 is a promising lead for therapeutic development, as it readily enters neurons, exhibits no neuronal toxicity, and elicits dose-dependent protection of synaptosomalassociated protein (of 25 kDa) in a primary culture of embryonic chicken neurons. Isothermal titration calorimetry showed that the interaction between NSC 240898 and the botulinum A light chain is largely entropy-driven, and occurs with a 1:1 stoichiometry and a dissociation constant of 4.6 M. Botulinum neurotoxins (BoNTs)3 are the most potent of the biological toxins (1), and are listed as category A (highest priority) bioterror agents by the Centers for Disease Control and Prevention. They may be delivered by aerosol route (1, 2), and consequently represent a serious threat to both military personnel and civilians (3, 4). Moreover, BoNTs are now established biotherapeutics for a range of physical ailments and cosmetic treatments (2, 5-8), making their misuse and/or adverse side effects (9) more likely. Neither the currently available BoNT antitoxin nor antibodies can counter these toxins once they are inside neurons; currently, critical care mechanical ventilation is the only life-saving treatment option. However, the effects of internalized BoNTs can last for months (10), and mechanical ventilation would be impractical if even a limited number of individuals were clandestinely/accidentally intoxicated. Thus, there is an urgent need to identify and develop small molecule (non-peptidic) inhibitors (SMNPIs) that can serve as both prophylactics and post-exposure therapeutics.BoNTs are composed of a heavy chain and a light chain (LC) that are connected by a disulfide bridge (11). The heavy chain binds to neurons and transports the LC into the cytosol (12). The LC is a zinc metalloprotease. Each of the seven BoNT serotypes (A-G) cleaves a component of the SNARE (soluble NSFethylmaleimide-sensitive factor attachment protein receptor) proteins (13), which mediate ...

Animal models are needed to better understand the pathogenic mechanisms of Zika virus (ZIKV) and to evaluate candidate medical countermeasures. Adult mice infected with ZIKV develop a transient viremia, but do not demonstrate signs of morbidity or mortality. Mice deficient in type I or a combination of type I and type II interferon (IFN) responses are highly susceptible to ZIKV infection; however, the absence of a competent immune system limits their usefulness for studying medical countermeasures. Here we employ a murine model for ZIKV using wild-type C57BL/6 mice treated with an antibody to disrupt type I IFN signaling to study ZIKV pathogenesis. We observed 40% mortality in antibody treated mice exposed to ZIKV subcutaneously whereas mice exposed by intraperitoneal inoculation were highly susceptible incurring 100% mortality. Mice infected by both exposure routes experienced weight loss, high viremia, and severe neuropathologic changes. The most significant histopathological findings occurred in the central nervous system where lesions represent an acute to subacute encephalitis/encephalomyelitis that is characterized by neuronal death, astrogliosis, microgliosis, scattered necrotic cellular debris, and inflammatory cell infiltrates. This model of ZIKV pathogenesis will be valuable for evaluating medical countermeasures and the pathogenic mechanisms of ZIKV because it allows immune responses to be elicited in immunologically competent mice with IFN I blockade only induced at the time of infection.

Anthrax protective antigen (PA, 83 kDa), a pore-forming protein, upon protease activation to 63 kDa (PA 63 ), translocates lethal factor (LF) and edema factor (EF) from endosomes into the cytosol of the cell. The relatively small size of the heptameric PA 63 pore (ϳ12 Å) raises questions as to how large molecules such as LF and EF can move through the pore. In addition, the reported high binding affinity between PA and EF/LF suggests that EF/LF may not dissociate but remain complexed with activated PA 63 . In this study, we found that purified (PA 63 ) 7 -LF complex exhibited biological and functional activities similar to the free LF. Purified LF complexed with PA 63 heptamer was able to cleave both a synthetic peptide substrate and endogenous mitogenactivated protein kinase kinase substrates and kill susceptible macrophage cells. Electrophysiological studies of the complex showed strong rectification of the ionic current at positive voltages, an effect similar to that observed if LF is added to the channels formed by heptameric PA 63 pore. Complexes of (PA 63 ) 7 -LF found in the plasma of infected animals showed functional activity. Identifying active complex in the blood of infected animals has important implications for therapeutic design, especially those directed against PA and LF. Our studies suggest that the individual toxin components and the complex must be considered as critical targets for anthrax therapeutics.Lethal and edema toxins play key pathogenesis roles as virulence factors produced by Bacillus anthracis, the etiologic agent of anthrax. The toxins show commonality in that both of their enzymatic components, lethal factor (LF, 1 90 kDa) and edema factor (EF, 88 kDa), require protective antigen (PA) for translocation into host cells. LF, a Zn ϩ2 -dependent metalloprotease, cleaves several members of the mitogen-activated protein kinase kinase (MAPKK) family (1-4), and in complex with PA, is responsible for the lethal action of anthrax toxin. EF is a calcium-and calmodulin-dependent adenylate cyclase that elevates intracellular levels of cyclic AMP, causing deregulation of cellular physiology and resulting in edema (5).The proposed in vitro model for the binding, assembly, translocation, and subsequent killing of the target cells by anthrax lethal toxin involves a series of steps (6). PA binds to the ubiquitous cellular receptors tumor endothelial marker 8 (TEM8) (7) and capillary morphogenesis protein 2 (CMG2) (8). Proteolytic cleavage of PA by cell surface proteases such as furin (9, 10) generates a 63-kDa fragment that oligomerizes and forms a ring-shaped heptameric prepore. The LF and EF proteins bind to the prepore, and the whole complex then undergoes receptor-mediated endocytosis. The acidic environment in the endosomes causes PA to undergo a conformational change that promotes membrane insertion and formation of a transmembrane channel. In a recent study, this process of insertion was shown to occur in the early endosomes, whereas the delivery of the enzymatic moieties required the invo...

We previously identified structurally diverse small molecule (non-peptidic) inhibitors (SMNPIs) of the botulinum neurotoxin serotype A (BoNT/A) light chain (LC). Of these, several (including antimalarial drugs) contained a 4-amino-7-chloroquinoline (ACQ) substructure and a separate positive ionizable amine component. The same antimalarials have also been found to interfere with BoNT/A translocation into neurons, via pH elevation of the toxin-mediated endosome. Thus, this structural class of small molecules may serve as dualfunction BoNT/A inhibitors. In this study, we used a refined pharmacophore for BoNT/A LC inhibition to identify four new, potent inhibitors of this structural class (IC 50 's ranged from 3.2 to 17 µM). Molecular docking indicated that the binding modes for the new SMNPIs are consistent with those of other inhibitors that we have identified, further supporting our structure-based pharmacophore. Finally, structural motifs of the new SMNPIs, as well as two structure-based derivatives, were examined for activity, providing valuable information about pharmacophore component contributions to inhibition.

Alveolar macrophages (AM) are very important for pulmonary innate immune responses against invading inhaled pathogens because they directly kill the organisms and initiate a cascade of innate and adaptive immune responses. Although several factors contribute to inhalational anthrax, we hypothesized that unimpeded infection of Bacillus anthracis is directly linked to disabling the innate immune functions contributed by AM. Here, we investigated the effects of lethal toxin (LT), one of the binary complex virulence factors produced by B. anthracis, on freshly isolated nonhuman primate AM. Exposure of AM to doses of LT that killed susceptible macrophages had no effect on the viability of AM, despite complete MEK1 cleavage. Intoxicated AM remained fully capable of B. anthracis spore phagocytosis. However, pretreatment of AM with LT resulted in a significant decrease in the clearance of both the Sterne strain and the fully virulent Ames strain of B. anthracis, which may have been a result of impaired AM secretion of proinflammatory cytokines. Our data imply that cytolysis does not correlate with MEK1 cleavage, and this is the first report of LT-mediated impairment of nonhuman primate AM bactericidal activity against B. anthracis.