A Novel Small Molecule Inhibitor of Influenza A Viruses that Targets Polymerase Function and Indirectly Induces Interferon

Influenza viruses continue to pose a major public health threat worldwide and options for antiviral therapy are limited by the emergence of drug-resistant virus strains. The antiviral cytokine, interferon (IFN) is an essential mediator of the innate immune response and influenza viruses, like many viruses, have evolved strategies to evade this response, resulting in increased replication and enhanced pathogenicity. A cell-based assay that monitors IFN production was developed and applied in a high-throughput compound screen to identify molecules that restore the IFN response to influenza virus infected cells. We report the identification of compound ASN2, which induces IFN only in the presence of influenza virus infection. ASN2 preferentially inhibits the growth of influenza A viruses, including the 1918 H1N1, 1968 H3N2 and 2009 H1N1 pandemic strains and avian H5N1 virus. In vivo, ASN2 partially protects mice challenged with a lethal dose of influenza A virus. Surprisingly, we found that the antiviral activity of ASN2 is not dependent on IFN production and signaling. Rather, its IFN-inducing property appears to be an indirect effect resulting from ASN2-mediated inhibition of viral polymerase function, and subsequent loss of the expression of the viral IFN antagonist, NS1. Moreover, we identified a single amino acid mutation at position 499 of the influenza virus PB1 protein that confers resistance to ASN2, suggesting that PB1 is the direct target. This two-pronged antiviral mechanism, consisting of direct inhibition of virus replication and simultaneous activation of the host innate immune response, is a unique property not previously described for any single antiviral molecule

Poly-ADP Ribosyl Polymerase 1 (PARP1) Regulates Influenza A Virus Polymerase

Influenza A viruses (IAV) are evolutionarily successful pathogens, capable of infecting a number of avian and mammalian species and responsible for pandemic and seasonal epidemic disease in humans. To infect new species, IAV typically must overcome a number of species barriers to entry, replication, and egress, even while virus replication is counteracted by antiviral host factors and innate immune mechanisms. A number of host factors have been found to regulate the replication of IAV by interacting with the viral RNA-dependent RNA polymerase (RdRP). The host factor PARP1, a poly-ADP ribosyl polymerase, was required for optimal functions of human, swine, and avian influenza RdRP in human 293T cells. In IAV infection, PARP1 was required for efficient synthesis of viral nucleoprotein (NP) in human lung A549 cells. Intriguingly, pharmacological inhibition of PARP1 enzymatic activity (PARylation) by 4-amino-1,8-naphthalimide led to a 4-fold increase in RdRP activity, and a 2.3-fold increase in virus titer. Exogenous expression of the natural PARylation inhibitor PARG also enhanced RdRP activity. These data suggest a virus-host interaction dynamic where PARP1 protein itself is required, but cellular PARylation has a distinct suppressive modality, on influenza A viral polymerase activity in human cells

Newcastle Disease Virus Expressing a Dendritic Cell-Targeted HIV Gag Protein Induces a Potent Gag-Specific Immune Response in Mice ▿

Viral vaccine vectors have emerged as an attractive strategy for the development of a human immunodeficiency virus (HIV) vaccine. Recombinant Newcastle disease virus (rNDV) stands out as a vaccine vector since it has a proven safety profile in humans, it is a potent inducer of both alpha interferon (IFN-α) and IFN-β) production, and it is a potent inducer of dendritic cell (DC) maturation. Our group has previously generated an rNDV vector expressing a codon-optimized HIV Gag protein and demonstrated its ability to induce a Gag-specific CD8+ T cell response in mice. In this report we demonstrate that the Gag-specific immune response can be further enhanced by the targeting of the rNDV-encoded HIV Gag antigen to DCs. Targeting of the HIV Gag antigen was achieved by the addition of a single-chain Fv (scFv) antibody specific for the DC-restricted antigen uptake receptor DEC205 such that the DEC205 scFv-Gag molecule was encoded for expression as a fusion protein. The vaccination of mice with rNDV coding for the DC-targeted Gag antigen induced an enhanced Gag-specific CD8+ T cell response and enhanced numbers of CD4+ T cells and CD8+ T cells in the spleen relative to vaccination with rNDV coding for a nontargeted Gag antigen. Importantly, mice vaccinated with the DEC205-targeted vaccine were better protected from challenge with a recombinant vaccinia virus expressing the HIV Gag protein. Here we demonstrate that the targeting of the HIV Gag antigen to DCs via the DEC205 receptor enhances the ability of an rNDV vector to induce a potent antigen-specific immune response

ASN2 inhibits the replication of influenza A virus and displays <i>in vivo</i> antiviral activity.

<p>(A) Virus titers from A549 cells infected with influenza A/WSN/33 virus (MOI = 0.01) and treated with increasing concentrations of ASN2 for 48 hours (orange curve). Cell viability analysis of A549 cells treated with increasing concentrations of ASN2 for 48 hours (black curve). Curves represent means of triplicate values ± standard deviation. (B) Bodyweight and survival curves of BALB/c mice (groups of 9) infected with influenza A/WSN/33 virus (5LD₅₀) and treated with 100 mg/kg of ASN2 every 8 hours for 8 days. Compound was delivered intraperitoneally beginning 8 hr prior to infection. Three mice from each group were sacrificed on days 3 and 8 post infection to determine viral lung titers (data not shown). Curves represent means ± standard deviation, *p<0.001. Mice that fell below 75% of their initial weight were sacrificed in accordance with our animal protocol.</p

Antiviral activity of ASN2 is independent of interferon production and NS1.

<p>(A) Virus titers from A549 and VERO cells infected with influenza A/WSN/33 virus (MOI = 0.01) and treated with increasing concentrations of ASN2 (6.25–50 µM) for 48 hours. (B) Plaque-reduction analysis of VERO cells infected with the indicated viruses in the presence of either DMSO or ASN2 (50 or 25 µM). Plaques were immunostained using an NP antibody. (C) Western blot analysis of cellular extracts from A549 cells infected with influenza A/WSN/33 virus (MOI = 1) and treated with ASN2 (50 µM) for 24 hours. Specific antibodies were used for each of the indicated proteins. (D) Reporter assay analysis of A549 cells transfected with IFNβ-luciferase and pRL-TK reporters, empty plasmid (-) or decreasing concentrations of NS1 (325, 32.5, and 3.25 ng) for 24 hours prior to infection with influenza A/WSN/33 virus (MOI = 1). Cells were treated with either ASN2 (50 µM) or DMSO and luciferase activity was determined 24 hours post infection. Values were normalized to <i>Renilla</i> luciferase activity for each sample and are represented as fold induction over uninfected DMSO-treated sample (mock). Bars represent means of triplicate values ± standard deviation.</p

ASN2 inhibits viral RNA synthesis.

<p>(A) Influenza A virus mini-genome activity in A549 cells transfected with the influenza virus firefly luciferase mini-genome reporter, pRL-TK control reporter, PB1, PB2, PA, and NP protein expressing plasmids. Treatment with DMSO or increasing concentrations of ASN2 (6.25–50 µM) was performed during transfection. Luciferase activity was assayed 24 hours post transfection. Firefly and Renilla luciferase values are represented as % activity from the mock (minigenome without NP expression) relative to the DMSO control. Error bars reflect the standard deviation of % activity. (B) Primer extension analysis of influenza virus mRNA and vRNA from A549 cells infected with influenza A/WSN/33 virus (MOI = 5) and treated with DMSO or decreasing concentrations of ASN2 (50–12.5 µM) for 6 hours. PB2, NA, and NS segments were analyzed. Ribavirin (100 µM) was used as a positive control and 5S rRNA was used as a loading control. Quantification of mRNA and vRNA expression, normalized to 5S rRNA, is shown on the right.</p

ASN2 induces an antiviral response during influenza A virus infection.

<p>(A) qRT-PCR analysis of IFNβ, ISG56 and IP10 transcripts in A549 cells infected with influenza A/WSN/33 virus (MOI = 1) and treated with ASN2 (50 µM) for 24 hours. Values were normalized to α-tubulin for each sample and are represented as fold induction over uninfected DMSO-treated sample (MOCK). Error bars reflect standard deviation of fold change. p<0.05, **p<0.005, ***p<0.0005 (B) Antiviral bioassay analysis of A549 cells infected with influenza A virus and treated with ASN2 for 18 hours. Supernatants from these cells were collected and UV-inactivated prior to overlaying them onto freshly plated VERO cells for a 24 hour incubation period prior to infection with NDV-GFP. Graphs represent percent inhibition of NDV-GFP induced by the supernatants collected from the indicated treatments. Percentage inhibition induced by supernatants from Sendai virus (SeV) infection was set to 100%. Bars represent mean of triplicate values ± standard deviation. (C) Deep sequencing analysis of mRNA from A549 cells infected with A/WSN/33 (MOI = 1) and treated with DMSO or ASN2 (50 µM) for 24 hours. The total number of reads obtained for the indicated genes for DMSO and ASN2 treatments are shown. *p<0.05.</p

Concept for high-throughput compound screen and identification of ASN2.

<p>(A) MDCK cells stably expressing IFNβ-luciferase reporter are not responsive to wild type (wt) influenza A virus infection due to the presence of a fully functional NS1 protein (left panel). Infection with a mutant virus expressing a truncated NS1 protein (rPR8 NS1-113), which is unable to antagonize the IFNβ production pathway, can induce the IFNβ-luciferase reporter (middle panel). The HTS assay consisted of infecting the reporter cells with wt influenza A virus in the presence of small molecular weight compounds. The aim was to identify compounds that are able to restore IFNβ in the presence of wt influenza A virus (right panel). (B) Chemical structure of ASN2 with its molecular weight (MW) and chemical formula. (C) Reporter assays of MDCK IFNβ-luciferase cells treated with increasing concentrations of ASN2 for 2 hours prior to mock infection or infection with influenza A/PR/8/34 virus and VSV-GFP. Luciferase activity was assayed 18 hours post infection. Curves represent the mean of triplicate values ± standard deviation.</p