The Combined Use of Alphavirus Replicons and Pseudoinfectious Particles for the Discovery of Antivirals Derived from Natural Products

Alphaviruses are a prominent class of reemergent pathogens due to their globally expanding ranges, potential for lethality, and possible use as bioweapons. The absence of effective treatments for alphaviruses highlights the need for innovative strategies to identify antiviral agents. Primary screens that use noninfectious self-replicating RNAs, termed replicons, have been used to identify potential antiviral compounds for alphaviruses. Only inhibitors of viral genome replication, however, will be identified using replicons, which excludes many other druggable steps in the viral life cycle. To address this limitation, we developed a western equine encephalitis virus pseudoinfectious particle system that reproduces several crucial viral life cycle steps in addition to genome replication. We used this system to screen a library containing ~26,000 extracts derived from marine microbes, and we identified multiple bacterial strains that produce compounds with potential antiviral activity. We subsequently used pseudoinfectious particle and replicon assays in parallel to counterscreen candidate extracts, and followed antiviral activity during biochemical fractionation and purification to differentiate between inhibitors of viral entry and genome replication. This novel process led to the isolation of a known alphavirus entry inhibitor, bafilomycin, thereby validating the approach for the screening and identification of potential antiviral compounds.Universidad de Costa RicaUCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Químic

Discovery of potent broad spectrum antivirals derived from marine actinobacteria.

Natural products provide a vast array of chemical structures to explore in the discovery of new medicines. Although secondary metabolites produced by microbes have been developed to treat a variety of diseases, including bacterial and fungal infections, to date there has been limited investigation of natural products with antiviral activity. In this report, we used a phenotypic cell-based replicon assay coupled with an iterative biochemical fractionation process to identify, purify, and characterize antiviral compounds produced by marine microbes. We isolated a compound from Streptomyces kaviengensis, a novel actinomycetes isolated from marine sediments obtained off the coast of New Ireland, Papua New Guinea, which we identified as antimycin A1a. This compound displays potent activity against western equine encephalitis virus in cultured cells with half-maximal inhibitory concentrations of less than 4 nM and a selectivity index of greater than 550. Our efforts also revealed that several antimycin A analogues display antiviral activity, and mechanism of action studies confirmed that these Streptomyces-derived secondary metabolites function by inhibiting the cellular mitochondrial electron transport chain, thereby suppressing de novo pyrimidine synthesis. Furthermore, we found that antimycin A functions as a broad spectrum agent with activity against a wide range of RNA viruses in cultured cells, including members of the Togaviridae, Flaviviridae, Bunyaviridae, Picornaviridae, and Paramyxoviridae families. Finally, we demonstrate that antimycin A reduces central nervous system viral titers, improves clinical disease severity, and enhances survival in mice given a lethal challenge with western equine encephalitis virus. Our results provide conclusive validation for using natural product resources derived from marine microbes as source material for antiviral drug discovery, and they indicate that host mitochondrial electron transport is a viable target for the continued development of broadly active antiviral compounds

Widely applicable PCR markers for sex identification in birds

To aid in avian sex determination if birds are not sexually dimorphic and/or they are sexually immature, several molecular assays involving the polymerase chain reaction (PCR) have been developed. To test in a variety of domestic and wild avian species applicability of five sexing assays: previously described four assays based on nucleotide sequence differences between the Z and W copy of the chicken chromodomainhelicase-DNA-binding protein gene (CHD1Z and CHD1W), and a new sexing marker using the ubiquitin associated protein 2 (UBAP2) gene sequence. At least one molecular sexing marker was successful in 84 out of 88 examined species across 13 avian orders. These assays may be useful in breeding management of domestic and wild birds as well as in studies of avian ecology, population genetics, embryology and transgenesis

Disruption of mitochondrial electron transport suppresses WEEV replication.

<div><p>(A) Schematic of mETC enzyme complexes. The known targets for the inhibitors shown in italics are indicated by the cross bars. Cyt c, cytochrome C; CoQ, coenzyme Q.</p> <p>(B) Antiviral activity and toxicity of mETC inhibitors. Cells were treated with increasing concentrations of the indicated inhibitors, and replicon inhibition, total cellular ATP production, and cytotoxicity were measured in separate assays. Results are presented as IC₅₀ or CC₅₀ values for the indicated parameter, and represent the mean ± SEM from at least three independent experiments. The numerical values on the graph indicate fold-differences in IC₅₀ values between replicon inhibition and ATP production suppression for the indicated select compounds. For rotenone, the comparison was made with CC₅₀ values, since we were unable to calculate reliable IC₅₀ values for ATP production suppression.</p> <p>(C) Complementation assays with select mETC inhibitors and WEEV replicons. Cells were treated with 100 μM of the indicated supplement or antioxidant and antimycin A (AA), CCCP, or mycophenolic acid (MPA) at 2X or 5X replicon IC₅₀ concentrations, and replicon activity was measured 16-20 h later. Results represent the mean ± SEM from four independent experiments. <i>p</i>-value < 0.05* or 0.005** compared to supplement- or antioxidant-only treated controls. 2-MPG, <i>N</i>-(2-mercaptopropionyl)glycine.</p> <p>(D) Complementation assay with antimycin A and infectious virus. BE(2)-C cells were infected with FMV at an MOI = 1, treated simultaneously with 100 μM of the indicated supplement or antioxidant and control DMSO or antimycin A at 5X replicon IC₅₀ concentration, and viral titers in tissue culture supernatants were measured at 24 hpi. Results represent the mean ± SEM from four independent experiments. **<i>p</i>-value < 0.005 compared to inhibitor-treated controls without supplementation (open bars).</p></div

Antimycin A derivatives produced by <i>Streptomyces</i> have potent antiviral activity against WEEV serogroup alphaviruses.

<div><p>(A) Molecular structure of antimycin A. Core structure is shown at the top, and the individual R1 and R2 constituents of derivatives A1a, A2a, A3a, A4a, and A10a are shown below the core structure. Specific atom designations correspond to the NMR results in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082318#pone.0082318.s005" target="_blank">Table S1</a>.</p> <p>(B) Antiviral activity of commercial antimycin A (AA) and mycophenolic acid (MPA) analyzed with WEEV replicons. Dose titration results for both replicon activity (closed symbols) and viability (open symbols) are presented as the percent untreated control cells and represent the mean ± SEM from at least five independent experiments. Calculated IC₅₀ values for anti-replicon activity are shown on the graph for both compounds, and an average MW of 550 g/mol was used to estimate molar concentrations for commercial antimycin A.</p> <p>(C and D) Antiviral activity of commercial AA and MPA analyzed with infectious WEEV (C) or FMV (D) in BE(2)-C neuronal cells. Cells were infected with WEEV (MOI = 0.1) or FMV (MOI = 1), treated simultaneously with compounds at the indicated concentrations, and virus production was measured by plaque assay at 24 hpi. Results are presented as infectious virion concentration in tissue culture supernatants and represent the mean ± SEM from at least three independent experiments. Calculated IC₅₀ values are shown on the graph for both compounds, and for commercial antimycin A these values were determined as described above in (B). The dashed reference lines represent results from infected cells treated with DMSO control.</p> <p>(E) HPLC separation of individual antimycin A derivatives from commercial stock compound. Only the select portion of an HPLC tracing that contained the four most prominent peaks is shown, and the various grey scale tracings represent different absorbance wavelengths. The identification of individual antimycin A derivatives represented by the four most prominent peaks is shown, where structures were determined by NMR analysis of purified fractions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082318#pone.0082318.s005" target="_blank">Table S1</a>).</p> <p>(F) Antiviral activity of individual antimycin A derivatives analyzed with WEEV replicons. Dose titration results are presented as the percent untreated control cells and represent the mean ± SEM from at least four independent experiments. Calculated IC₅₀ values for individual derivatives are shown on the graph, and were calculated using MWs of 548.63, 534.61, 520.58, and 506.55 g/mol for antimycins A1a, A2a, A3a, and A4a, respectively. The methoxy group in 2-methoxyantimycin A3 (MeO-AA3) is located at the 6’ position in the core antimycin structure shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082318#pone-0082318-g005" target="_blank">Figure 5A</a>. ND, not determined.</p></div

Antimycin A improves clinical disease and survival and reduces CNS titers in mice infected with WEEV.

<div><p>(A and B) Clinical disease severity and survival in WEEV-infected mice. C57BL/6 mice were infected with 10³ pfu WEEV, treated twice daily with DMSO or the indicated dose of antimycin A via intraperitoneal injection, and both clinical disease (A) and mortality (B) were monitored for 14 days post-infection. Representative results from one of two independent experiments are shown (N = 7-8 mice per group). *<i>p</i>-value < 0.05 compared to DMSO-treated mice.</p> <p>(C) Virus titers in the CNS of WEEV-infected mice. Mice were infected and treated as described above, and virus titers in brain were determined at 6 days post-infection. N = 4 mice per group. **p-value < 0.01 compared to DMSO control.</p></div

Schematic of marine microbe-based natural product extract production, screening, and validation.

<p>Individual steps are indicated in the left column, with explanatory comments provided on the right. The number of extracts and corresponding number of individual strains, where appropriate, are indicated in bold type between steps.</p

Purified antiviral compound from <i>S. kaviengensis</i> suppresses WEEV RNA replication and virus production in single-step growth assays.

<div><p>(A) Infectious virion production. BE(2)-C cells were infected with WEEV at an MOI = 10, treated with DMSO, 25 μM mycophenolic acid (MPA), or 100 ng/ml (~200 nM) purified compound F7E2e, and virus titers is tissue culture supernatants were determined by plaque assay at 6, 12, 24, and 48 hpi. Plaque assay sensitivity was 10² pfu/ml. Results represent the mean ± SEM from three independent experiments. *<i>p</i>-value < 0.05 compared to DMSO-treated controls for both MPA- and F7E2e-treated samples.</p> <p>(B) Quantitative RT-PCR analysis of WEEV RNA accumulation. Cells were infected and treated as above in (A), total RNA was harvested at the indicated time points, and primers corresponding to either the nsP1 or E1 WEEV genome were used to amplify and quantify either genomic (nsP1) or genomic plus subgenomic (E1) RNA accumulation. Results are presented as WEEV RNA levels relative to infected DMSO-treated control cells, and represent the mean ± SEM from six independent experiments. <i>p</i>-value < 0.001* or 0.0001** compared to DMSO-treated controls.</p> <p>(C) Northern blot analysis of WEEV RNA accumulation. Mock-infected cells (lane 1) or cells infected and treated as above in (A) with DMSO (lane 2), MPA (lane 3) or F7E2e (lane 4) were harvested at 12 hpi, and total RNA was analyzed by Northern blotting with a strand-specific ³²P-labelled riboprobe that detected both positive-sense genomic and subgenomic viral RNA (vRNA). The location and relative size of genomic and subgenomic vRNA are shown on the right, and the ethidium bromide-stained 28S rRNA band is shown as a loading control. Representative results from one of three independent experiments are shown.</p></div

MALT1 Protease Activation Triggers Acute Disruption of Endothelial Barrier Integrity via CYLD Cleavage

Microvascular endothelial cells maintain a tight barrier to prevent passage of plasma and circulating immune cells into the extravascular tissue compartment, yet endothelial cells respond rapidly to vasoactive substances, including thrombin, allowing transient paracellular permeability. This response is a cornerstone of acute inflammation, but the mechanisms responsible are still incompletely understood. Here, we demonstrate that thrombin triggers MALT1 to proteolytically cleave cylindromatosis (CYLD). Fragmentation of CYLD results in microtubule disruption and a cascade of events leading to endothelial cell retraction and an acute permeability response. This finding reveals an unexpected role for the MALT1 protease, which previously has been viewed mostly as a driver of pro-inflammatory NF-κB signaling in lymphocytes. Thus, MALT1 not only promotes immune cell activation but also acutely regulates endothelial cell biology, actions that together facilitate tissue inflammation. Pharmacologic inhibition of MALT1 may therefore have synergistic impact by targeting multiple disparate steps in the overall inflammatory response