Antiviral activity of gliotoxin, gentian violet and brilliant green against Nipah and Hendra virus in vitro

Background: Using a recently described monolayer assay amenable to high throughput screening format for the identification of potential Nipah virus and Hendra virus antivirals, we have partially screened a low molecular weight compound library (8,000 compounds) directly against live virus infection and identified twenty eight promising lead molecules. Initial single blind screens were conducted with 10 M compound in triplicate with a minimum efficacy of 90% required for lead selection. Lead compounds were then further characterised to determine the median efficacy (IC), cytotoxicity (CC) and the in vitro therapeutic index in live virus and pseudotype assay formats. Results: While a number of leads were identified, the current work describes three commercially available compounds: brilliant green, gentian violet and gliotoxin, identified as having potent antiviral activity against Nipah and Hendra virus. Similar efficacy was observed against pseudotyped Nipah and Hendra virus, vesicular stomatitis virus and human parainfluenza virus type 3 while only gliotoxin inhibited an influenza A virus suggesting a non-specific, broad spectrum activity for this compound. Conclusion: All three of these compounds have been used previously for various aspects of anti-bacterial and anti-fungal therapy and the current results suggest that while unsuitable for internal administration, they may be amenable to topical antiviral applications, or as disinfectants and provide excellent positive controls for future studies

Connexin-Dependent Transfer of cGAMP to Phagocytes Modulates Antiviral Responses

Recent studies suggest that extracellular cGAMP can be taken up by macrophages to engage STING through several mechanisms. Our work demonstrates that connexin-dependent communication between epithelial cells and macrophages plays a significant role in the amplification of antiviral responses mediated by cGAMP and suggests that pharmacological strategies aimed at modulating connexins may have therapeutic applications to control antiviral responses in humans.Activation of cyclic GMP-AMP (cGAMP) synthase (cGAS) plays a critical role in antiviral responses to many DNA viruses. Sensing of cytosolic DNA by cGAS results in synthesis of the endogenous second messenger cGAMP that activates stimulator of interferon genes (STING) in infected cells. Critically, cGAMP can also propagate antiviral responses to uninfected cells through intercellular transfer, although the modalities of this transfer between epithelial and immune cells remain poorly defined. We demonstrate here that cGAMP-producing epithelial cells can transactivate STING in cocultured macrophages through direct cGAMP transfer. cGAMP transfer was reliant upon connexin expression by epithelial cells and pharmacological inhibition of connexins blunted STING-dependent transactivation of the macrophage compartment. Macrophage transactivation by cGAMP contributed to a positive-feedback loop amplifying antiviral responses, significantly protecting uninfected epithelial cells against viral infection. Collectively, our findings constitute the first direct evidence of a connexin-dependent cGAMP transfer to macrophages by epithelial cells, to amplify antiviral responses

Genome-wide siRNA Screening at Biosafety Level 4 Reveals a Crucial Role for Fibrillarin in Henipavirus Infection.

Hendra and Nipah viruses (genus Henipavirus, family Paramyxoviridae) are highly pathogenic bat-borne viruses. The need for high biocontainment when studying henipaviruses has hindered the development of therapeutics and knowledge of the viral infection cycle. We have performed a genome-wide siRNA screen at biosafety level 4 that identified 585 human proteins required for henipavirus infection. The host protein with the largest impact was fibrillarin, a nucleolar methyltransferase that was also required by measles, mumps and respiratory syncytial viruses for infection. While not required for cell entry, henipavirus RNA and protein syntheses were greatly impaired in cells lacking fibrillarin, indicating a crucial role in the RNA replication phase of infection. During infection, the Hendra virus matrix protein co-localized with fibrillarin in cell nucleoli, and co-associated as a complex in pulldown studies, while its nuclear import was unaffected in fibrillarin-depleted cells. Mutagenesis studies showed that the methyltransferase activity of fibrillarin was required for henipavirus infection, suggesting that this enzyme could be targeted therapeutically to combat henipavirus infections

Dual microRNA Screens Reveal That the Immune-Responsive miR-181 Promotes Henipavirus Entry and Cell-Cell Fusion

<div><p>Hendra and Nipah viruses (family <i>Paramyxoviridae</i>, genus <i>Henipavirus</i>) are bat-borne viruses that cause fatal disease in humans and a range of other mammalian species. Gaining a deeper understanding of host pathways exploited by henipaviruses for infection may identify targets for new anti-viral therapies. Here we have performed genome-wide high-throughput agonist and antagonist screens at biosafety level 4 to identify host-encoded microRNAs (miRNAs) impacting henipavirus infection in human cells. Members of the miR-181 and miR-17~93 families strongly promoted Hendra virus infection. miR-181 also promoted Nipah virus infection, but did not affect infection by paramyxoviruses from other genera, indicating specificity in the virus-host interaction. Infection promotion was primarily mediated via the ability of miR-181 to significantly enhance henipavirus-induced membrane fusion. Cell signalling receptors of ephrins, namely EphA5 and EphA7, were identified as novel negative regulators of henipavirus fusion. The expression of these receptors, as well as EphB4, were suppressed by miR-181 overexpression, suggesting that simultaneous inhibition of several Ephs by the miRNA contributes to enhanced infection and fusion. Immune-responsive miR-181 levels was also up-regulated in the biofluids of ferrets and horses infected with Hendra virus, suggesting that the host innate immune response may promote henipavirus spread and exacerbate disease severity. This study is the first genome-wide screen of miRNAs influencing infection by a clinically significant mononegavirus and nominates select miRNAs as targets for future anti-viral therapy development.</p></div

miR-181 significantly enhances HeV RNA synthesis and F- and G-mediated cell-cell fusion.

<p>(A) qRT-PCR measurements of intracellular viral RNA copy number in HeLa cells infected with HeV (MOI 5). ***p≤0.001; *p≤0.05. HeV RNA values were normalised to cellular 18S levels. (B) Cell-to-cell fusion of HeV-F and HeV-G-expressing HEK-293T effector cells to HeLa cells treated with indicated siRNA or miRNA agonists. Syncytia were imaged using automated fluorescence microscopy. Nuclei are shown in blue (DAPI), effector cells in green (HeV-G staining) and target cells red (DiO lipid dye). (f1) and (f2) are images of cells transfected with miR-181d from two different microscopy fields. (C) Quantification of fusion events by counting all nuclei present in all syncytia. Values are normalised as a percentage to siNEG or miNEG. *p ≤ 0.05, ***p ≤0.001 compared to respective negative controls.</p

Select Eph receptors inhibit HeV infection and cell-cell-fusion and are miR-181 target genes.

<p>(A) Relative mRNA levels of indicated target genes in HeLa cells 72 h post-transfection with siRNAs (40 nM). ***p<0.001 compared to siNEG (B) Relative percentage of cells infected with HeV (24 h, MOI 0.5), after 72 h transfection with siRNAs targeting indicated molecules. *p<0.05, ***p<0.001 compared to siNEG. (C) Relative mRNA levels of Eph receptors A4, A5, A7 and B4 in HeLa cells, 72 h post transfection with miRNA agonists (25 nM). N.s. not significant; **p<0.01, ***p≤0.001, compared to control agonist. (D) Cell-cell fusion of HeV-F and–G expressing HEK-293T cells to HeLa cells treated with indicated siRNAs. Values are normalised as a percentage to siNEG or control agonist.</p

Expression levels of miR-181 in biofluids of animals infected with HeV are increased.

<p>Sixteen ferrets were infected with HeV at BSL-4. At predetermined time-points, ten different tissues were harvested and analysed for viral RNA loads by qRT-PCR (A). (B) qRT-PCR analysis of miR-181d levels in the serum samples of the ferrets. Values were normalized to the U6 RNA. *p≤0.05 compared to day 0. (C) qRT-PCR analysis of miR-181d in blood of horses from a published HeV infection study [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005974#ppat.1005974.ref052" target="_blank">52</a>].</p

miR-181d promotes henipavirus infection specifically.

<p>Infectivity assays were applied to assess changes in virus production or virus infection of HeLa cells infected with NiV, MeV, MuV, RSV or influenza A/WSN/33 virus for 24 h. Cells were previously transfected with miR-181d or negative control agonists (miNEG) for 72 h. **p≤0.01; n.s. not significant.</p

miR-17 promotes henipavirus infection but does not enhance HeV F- and G-mediated cell-cell fusion.

<p>(A) Percentage of cells infected with HeV or RSV (24 h, MOI 1), after 72 h transfection with miR-17, miR-93 or control agonists. ***p≤0.001, **p≤0.01, *p≤0.05 compared to control agonist. (B) TCID₅₀ virus titres of supernatants derived from HeLa cells infected with HeV for 24 h (MOI 1), at 72 h post-transfection with agonists. *p≤0.05 compared to control agonist. (C) Cell-cell fusion of HeV-F and -G expressing HEK-293T cells to HeLa cells treated with indicated siRNA or miRNA agonists. Syncytia were imaged using automated fluorescence microscopy. Nuclei are shown in blue (DAPI) and effector cells in green (HeV-G staining). (D) Quantification of fusion events by counting of all nuclei present in all syncytia. Values are normalised as a percentage to siNEG or control agonist. n.s. not significant; *p≤0.05, **p≤0.01; ***p≤0.001 compared to respective negative controls.</p

Genome-wide complementary agonist and antagonist screens of host-encoded miRNAs impacting henipavirus infection at BSL-4.

<p>(A) Schematic of optimized protocol for performing functional RNAi screens with an infectious BSL-4 virus. Two sets of daughter plates were generated from library plates consisting of 1,239 miRNA agonists (grey plates) and 1,225 antagonists (black plates). HeLa cells were added to both plate sets for reverse transfection of the miRNA agonists and antagonists. 72 h post transfection, one set of plates were processed at BSL-2 for cell viability analysis by DAPI staining. The other set was then transferred into BSL-4 (red box), infected with luciferase-expressing HeV, and lysed for luminescence reading at 24 hours post-infection (h.p.i.) in BSL-4. (B and C) Results from the miRNA agonist (B) and antagonist (C) screens, with miRNAs ranked using a robust Z-score approach, from lowest (decreased virus infection) to highest (increased virus infection). Dotted horizontal lines represent the threshold of hit identification (Z ≥ 2 or ≤ -2). The number of miRNA hits above this threshold is shown. (D) Venn diagram identifying pro- and anti-viral miRNAs from both screens.</p