Discovery and synthesis of novel benzofurazan derivatives as inhibitors of influenza A virus

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Characterization of a Novel STAT 2 Knock Out Hamster Model of Crimean Congo Hemorrhagic Fever Virus Pathogenesis

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne pathogen causing a febrile illness in humans, which can progress to hemorrhagic manifestations, multi-organ failure, and death. Current mouse models of CCHFV infection reliably succumb to virus challenge but vary in their ability to reflect signs of disease similar to humans. In this study, we established a signal transducer and activator of transcription 2 (STAT2) knockout hamster model to expand the repertoire of animal models of CCHFV pathogenesis that can be used for therapeutic development. These hamsters demonstrated a systemic and lethal disease in response to infection. Hallmarks of human disease were observed including petechial rash, blood coagulation dysfunction, and various biochemistry and blood cell count abnormalities. Furthermore, we also demonstrated the utility of this model for anti-CCHFV therapeutic evaluation. The STAT2 knock-out hamster model of CCHFV infection may provide some further insights into clinical disease, viral pathogenesis, and pave the way for testing of potential drug and vaccine candidates

Identification of a PA-Binding Peptide with Inhibitory Activity against Influenza A and B Virus Replication

There is an urgent need for new drugs against influenza type A and B viruses due to incomplete protection by vaccines and the emergence of resistance to current antivirals. The influenza virus polymerase complex, consisting of the PB1, PB2 and PA subunits, represents a promising target for the development of new drugs. We have previously demonstrated the feasibility of targeting the protein-protein interaction domain between the PB1 and PA subunits of the polymerase complex of influenza A virus using a small peptide derived from the PA-binding domain of PB1. However, this influenza A virus-derived peptide did not affect influenza B virus polymerase activity. Here we report that the PA-binding domain of the polymerase subunit PB1 of influenza A and B viruses is highly conserved and that mutual amino acid exchange shows that they cannot be functionally exchanged with each other. Based on phylogenetic analysis and a novel biochemical ELISA-based screening approach, we were able to identify an influenza A-derived peptide with a single influenza B-specific amino acid substitution which efficiently binds to PA of both virus types. This dual-binding peptide blocked the viral polymerase activity and growth of both virus types. Our findings provide proof of principle that protein-protein interaction inhibitors can be generated against influenza A and B viruses. Furthermore, this dual-binding peptide, combined with our novel screening method, is a promising platform to identify new antiviral lead compounds

Comprehending a Killer: The Akt/mTOR Signaling Pathways Are Temporally High-Jacked by the Highly Pathogenic 1918 Influenza Virus

Previous transcriptomic analyses suggested that the 1918 influenza A virus (IAV1918), one of the most devastating pandemic viruses of the 20th century, induces a dysfunctional cytokine storm and affects other innate immune response patterns. Because all viruses are obligate parasites that require host cells for replication, we globally assessed how IAV1918 induces host protein dysregulation. We performed quantitative mass spectrometry of IAV1918-infected cells to measure host protein dysregulation. Selected proteins were validated by immunoblotting and phosphorylation levels of members of the PI3K/AKT/mTOR pathway were assessed. Compared to mock-infected controls, >170 proteins in the IAV1918-infected cells were dysregulated. Proteins mapped to amino sugar metabolism, purine metabolism, steroid biosynthesis, transmembrane receptors, phosphatases and transcription regulation. Immunoblotting demonstrated that IAV1918 induced a slight up-regulation of the lamin B receptor whereas all other tested virus strains induced a significant down-regulation. IAV1918 also strongly induced Rab5b expression whereas all other tested viruses induced minor up-regulation or down-regulation. IAV1918 showed early reduced phosphorylation of PI3K/AKT/mTOR pathway members and was especially sensitive to rapamycin. These results suggest the 1918 strain requires mTORC1 activity in early replication events, and may explain the unique pathogenicity of this virus. Keywords: RNA virus, Virus infection, Host cell alterations, Mass spectrometry, Liquid chromatography, Bioinformatic

Discovery and synthesis of novel benzofurazan derivatives as inhibitors of influenza A virus

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Identification of High-Affinity PB1-Derived Peptides with Enhanced Affinity to the PA Protein of Influenza A Virus Polymerase ▿

The influenza A virus polymerase complex, consisting of the subunits PB1, PB2, and PA, represents a promising target for the development of new antiviral drugs. We have previously demonstrated the feasibility of targeting the protein-protein interaction domain between PA and PB1 using peptides derived from the extreme N terminus of PB1 (amino acids [aa] 1 to 15), comprising the PA-binding domain of PB1. To increase the binding affinity of these peptides, we performed a systematic structure-affinity relationship analysis. Alanine and aspartic acid scans revealed that almost all amino acids in the core binding region (aa 5 to 11) are indispensable for PA binding. Using a library of immobilized peptides representing all possible single amino acid substitutions, we were able to identify amino acid positions outside the core PA-binding region (aa 1, 3, 12, 14, and 15) that are variable and can be replaced by affinity-enhancing residues. Surface plasmon resonance binding studies revealed that combination of several affinity-enhancing mutations led to an additive effect. Thus, the feasibility to enhance the PA-binding affinity presents an intriguing possibility to increase antiviral activity of the PB1-derived peptide and one step forward in the development of an antiviral drug against influenza A viruses

Limited Effects of Type I Interferons on Kyasanur Forest Disease Virus in Cell Culture.

BACKGROUND:The tick-borne flavivirus, Kyasanur Forest disease virus (KFDV) causes seasonal infections and periodic outbreaks in south-west India. The current vaccine offers poor protection with reported issues of coverage and immunogenicity. Since there are no approved prophylactic therapeutics for KFDV, type I IFN-α/β subtypes were assessed for antiviral potency against KFDV in cell culture. METHODOLOGY/PRINCIPAL FINDINGS:The continued passage of KFDV-infected cells with re-administered IFN-α2a treatment did not eliminate KFDV and had little effect on infectious particle production whereas the IFN-sensitive, green fluorescent protein-expressing vesicular stomatitis virus (VSV-GFP) infection was controlled. Further evaluation of the other IFN-α/β subtypes versus KFDV infection indicated that single treatments of either IFN-αWA and IFN-αΚ appeared to be more effective than IFN-α2a at reducing KFDV titres. Concentration-dependent analysis of these IFN-α/β subtypes revealed that regardless of subtype, low concentrations of IFN were able to limit cytopathic effects (CPE), while significantly higher concentrations were needed for inhibition of virion release. Furthermore, expression of the KFDV NS5 in cell culture before IFN addition enabled VSV-GFP to overcome the effects of IFN-α/β signalling, producing a robust infection. CONCLUSIONS/SIGNIFICANCE:Treatment of cell culture with IFN does not appear to be suitable for KFDV eradication and the assay used for such studies should be carefully considered. Further, it appears that the NS5 protein is sufficient to permit KFDV to bypass the antiviral properties of IFN. We suggest that other prophylactic therapeutics should be evaluated in place of IFN for treatment of individuals with KFDV disease

Antiviral activity of interferon-α/β against the cytopathology of KFDV and VSV-GFP.

<p>Antiviral activity of interferon-α/β against the cytopathology of KFDV and VSV-GFP.</p

Screening interferon-α/β subtypes against KFDV.

<p>Cultures of A549 cells were pre-treated (grey bars) 24 hours prior to infection or post-treated (black bars) 1 hour after infection with 1, 000 U/mL of IFN-α (B2, C, D, F, G, H2, I, J1, K, WA, 2a, 2b or 4b), IFN-β (beta-1) and a recombinant IFN-α (Universal) subtypes and infected with KFDV at a MOI of 1. Supernatants were harvested for each treatment after 72 hours of incubation and quantified (expressed in log₁₀ scale TCID₅₀/mL) on BHK-21 (ATCC) when the mock-treated control cells displayed CPE near 100%. Pre-infection treatment experiments were assayed in two biological replicates and post-infection treatment experiments were assayed in three biological replicates; the resulting averages and standard deviations are presented. Mock, Mock-treated with IFN. UI, Un-infected control. IFN-α2b was excluded from 24-hour pre-infection treatment. * Significant compared to mock-treated samples (P < 0.1). *** Significant compared to mock-treated samples (P < 0.01).</p

IFN-α2a does not clear KFDV infection in A549 cells.

<p>A549 cells were infected with a 11 TCID₅₀ units (MOI of 0.00001) of the indicated virus, and either treated or mock-treated with 2, 000 U/mL of IFN-α2a (designated as P0). Monolayers were passaged when untreated controls reached CPE of 90%, 96 and 48 hpi for KFDV and VSV-GFP, respectively and 2, 000 U/mL of IFN-α2a was either added or omitted (P1) and after 72 hpi for KFDV and 48 hpi for VSV-GFP. This procedure was repeated again for passage 2 (P2). (A) Before each passage, supernatants were harvested for titration by TCID₅₀ assay determination on BHK-21 cells. The averages and standard deviation from three biological replicates are shown graphically and expressed in log₁₀ scale TCID₅₀/mL. Statistical significance is denoted as * P < 0.1, ** P < 0.05, *** P < 0.01. (B) Cell monolayers were visualized with light microscopy prior to passaging. Mock, non-IFN treated/infected controls. UI, Un-infected controls.</p