The Coronavirus Disease 2019 (COVID-19) pandemic caused by the Severe Acute Respiratory Syndrome Related Coronavirus 2 (SARS-CoV-2) is a global health emergency. As only very limited therapeutic ...options are clinically available, there is an urgent need for the rapid development of safe, effective, and globally available pharmaceuticals that inhibit SARS-CoV-2 entry and ameliorate COVID-19 severity. In this study, we explored the use of small compounds acting on the homeostasis of the endolysosomal host-pathogen interface, to fight SARS-CoV-2 infection. We find that fluoxetine, a widely used antidepressant and a functional inhibitor of acid sphingomyelinase (FIASMA), efficiently inhibited the entry and propagation of SARS-CoV-2 in the cell culture model without cytotoxic effects and also exerted potent antiviral activity against two currently circulating influenza A virus subtypes, an effect which was also observed upon treatment with the FIASMAs amiodarone and imipramine. Mechanistically, fluoxetine induced both impaired endolysosomal acidification and the accumulation of cholesterol within the endosomes. As the FIASMA group consists of a large number of small compounds that are well-tolerated and widely used for a broad range of clinical applications, exploring these licensed pharmaceuticals may offer a variety of promising antivirals for host-directed therapy to counteract enveloped viruses, including SARS-CoV-2.
Infection of mammals by avian influenza viruses requires adaptive mutations to achieve high-level replication in the new host. However, the basic mechanism underlying this adaptation process is still ...unknown. Here we show that avian polymerases, lacking the human signature PB2-E627K, are incapable of generating usable complementary RNA templates in cultured human cells and therefore require adaptation. Characterization of the highly pathogenic human H5N1 isolate A/Thailand/1(KAN-1)/2004 that retained the avian PB2-E627 reveals that the defect in RNA replication is only partially compensated by mutations in the polymerase. Instead, mutations in the nuclear export protein are required for efficient polymerase activity. We demonstrate that adaptive mutations in nuclear export proteins of several human isolates enhance the polymerase activity of avian polymerases in human cultured cells. In conclusion, when crossing the species barrier, avian influenza viruses acquire adaptive mutations in nuclear export protein to escape restricted viral genome replication in mammalian cells.
Interferon-stimulated genes (ISGs) form the backbone of the innate immune system and are important for limiting intra- and intercellular viral replication and spread. We conducted a ...mass-spectrometry-based survey to understand the fundamental organization of the innate immune system and to explore the molecular functions of individual ISGs. We identified interactions between 104 ISGs and 1,401 cellular binding partners engaging in 2,734 high-confidence interactions. 90% of these interactions are unreported so far, and our survey therefore illuminates a far wider activity spectrum of ISGs than is currently known. Integration of the resulting ISG-interaction network with published datasets and functional studies allowed us to identify regulators of immunity and processes related to the immune system. Given the extraordinary robustness of the innate immune system, this ISG network may serve as a blueprint for therapeutic targeting of cellular systems to efficiently fight viral infections.
In influenza A virus-infected cells, replication and transcription of the viral genome occurs in the nucleus. To be packaged into viral particles at the plasma membrane, encapsidated viral genomes ...must be exported from the nucleus. Intriguingly, the nuclear export protein (NEP) is involved in both processes. Although NEP stimulates viral RNA synthesis by binding to the viral polymerase, its function during nuclear export implicates interaction with viral ribonucleoprotein (vRNP)-associated M1. The observation that both interactions are mediated by the C-terminal moiety of NEP raised the question whether these two features of NEP are linked functionally. Here we provide evidence that the interaction between M1 and the vRNP depends on the NEP C terminus and its polymerase activity-enhancing property for the nuclear export of vRNPs. This suggests that these features of NEP are linked functionally. Furthermore, our data suggest that the N-terminal domain of NEP interferes with the stability of the vRNP-M1-NEP nuclear export complex, probably mediated by its highly flexible intramolecular interaction with the NEP C terminus. On the basis of our data, we propose a new model for the assembly of the nuclear export complex of Influenza A vRNPs.
Background: NEP stimulates viral RNA synthesis and nuclear vRNP export.
Results: NEP is required to stabilize M1-vRNP binding for nuclear export. Deletion of the last three amino acids of NEP abrogates nuclear export and the polymerase-enhancing function of NEP.
Conclusion: The polymerase-enhancing function and the nuclear export function of NEP are linked functionally.
Significance: This study provides new insights into the assembly of the nuclear export complex.
Coronavirus disease 2019 (COVID-19), the illness caused by a novel coronavirus now called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to more than 260 million confirmed ...infections and 5 million deaths to date. While vaccination is a powerful tool to control pandemic spread, medication to relieve COVID-19-associated symptoms and alleviate disease progression especially in high-risk patients is still lacking. In this study, we explore the suitability of the rapid accelerated fibrosarcoma/mitogen-activated protein kinase/extracellular signal-regulated kinase (Raf/MEK/ERK) pathway as a druggable target in the treatment of SARS-CoV-2 infections. We find that SARS-CoV-2 transiently activates Raf/MEK/ERK signaling in the very early infection phase and that ERK1/2 knockdown limits virus replication in cell culture models. We demonstrate that ATR-002, a specific inhibitor of the upstream MEK1/2 kinases which is currently evaluated in clinical trials as an anti-influenza drug, displays strong anti-SARS-CoV-2 activity in cell lines as well as in primary air–liquid-interphase epithelial cell (ALI) cultures, with a safe and selective treatment window. We also observe that ATR-002 treatment impairs the SARS-CoV-2-induced expression of pro-inflammatory cytokines, and thus might prevent COVID-19-associated hyperinflammation, a key player in COVID-19 progression. Thus, our data suggest that the Raf/MEK/ERK signaling cascade may represent a target for therapeutic intervention strategies against SARS-CoV-2 infections and that ATR-002 is a promising candidate for further drug evaluation.
Influenza viruses (IV) exploit a variety of signaling pathways. Previous studies showed that the rapidly accelerated fibrosarcoma/mitogen-activated protein kinase/extracellular signal-regulated ...kinase (Raf/MEK/ERK) pathway is functionally linked to nuclear export of viral ribonucleoprotein (vRNP) complexes, suggesting that vRNP export is a signaling-induced event. However, the underlying mechanism remained completely enigmatic. Here we have dissected the unknown molecular steps of signaling-driven vRNP export. We identified kinases RSK1/2 as downstream targets of virus-activated ERK signaling. While RSK2 displays an antiviral role, we demonstrate a virus-supportive function of RSK1, migrating to the nucleus to phosphorylate nucleoprotein (NP), the major constituent of vRNPs. This drives association with viral matrix protein 1 (M1) at the chromatin, important for vRNP export. Inhibition or knockdown of MEK, ERK or RSK1 caused impaired vRNP export and reduced progeny virus titers. This work not only expedites the development of anti-influenza strategies, but in addition demonstrates converse actions of different RSK isoforms.
Since November 2019 the SARS-CoV-2 pandemic has caused nearly 200 million infection and more than 4 million deaths globally (Updated information from the World Health Organization, as on 2
nd
Aug ...2021). Within only one year into the pandemic, several vaccines were designed and reached approval for the immunization of the world population. The remarkable protective effects of the manufactured vaccines are demonstrated in countries with high vaccination rates, such as Israel and UK. However, limited production capacities, poor distribution infrastructures and political hesitations still hamper the availability of vaccines in many countries. In addition, due to the emergency of SARS-CoV-2 variants with immune escape properties towards the vaccines the global numbers of new infections as well as patients developing severe COVID-19, remains high. New studies reported that about 8% of infected individuals develop long term symptoms with strong personal restrictions on private as well as professional level, which contributes to the long socioeconomic problems caused by this pandemic. Until today, emergency use-approved treatment options for COVID-19 are limited to the antiviral Remdesivir, a nucleoside analogue targeting the viral polymerase, the glucocorticosteroide Dexamethasone as well as neutralizing antibodies. The therapeutic benefits of these treatments are under ongoing debate and clinical studies assessing the efficiency of these treatments are still underway. To identify new therapeutic treatments for COVID-19, now and by the post-pandemic era, diverse experimental approaches are under scientific evaluation in companies and scientific research teams all over the world. To accelerate clinical translation of promising candidates, repurposing approaches of known approved drugs are specifically fostered but also novel technologies are being developed and are under investigation. This review summarizes the recent developments from the lab bench as well as the clinical status of emerging therapeutic candidates and discusses possible therapeutic entry points for the treatment strategies with regard to the biology of SARS-CoV-2 and the clinical course of COVID-19.
The ongoing SARS-CoV-2 pandemic requires efficient and safe antiviral treatment strategies. Drug repurposing represents a fast and low-cost approach to the development of new medical treatment ...options. The direct antiviral agent remdesivir has been reported to exert antiviral activity against SARS-CoV-2. Whereas remdesivir only has a very short half-life time and a bioactivation, which relies on pro-drug activating enzymes, its plasma metabolite GS-441524 can be activated through various kinases including the adenosine kinase (ADK) that is moderately expressed in all tissues. The pharmacokinetics of GS-441524 argue for a suitable antiviral drug that can be given to patients with COVID-19. Here, we analyzed the antiviral property of a combined treatment with the remdesivir metabolite GS-441524 and the antidepressant fluoxetine in a polarized Calu-3 cell culture model against SARS-CoV-2. The combined treatment with GS-441524 and fluoxetine were well-tolerated and displayed synergistic antiviral effects against three circulating SARS-CoV-2 variants in vitro in the commonly used reference models for drug interaction. Thus, combinatory treatment with the virus-targeting GS-441524 and the host-directed drug fluoxetine might offer a suitable therapeutic treatment option for SARS-CoV-2 infections.
Human infection with highly pathogenic avian influenza viruses (HPAIV) is often associated with severe tissue damage due to hyperinduction of interferons and proinflammatory cytokines. The reasons ...for this excessive cytokine expression are still incompletely understood, which has hampered the development of efficient immunomodulatory treatment options. The host protein TRIM28 associates to the promoter regions of over 13,000 genes and is recognized as a genomic corepressor and negative immune regulator. TRIM28 corepressor activity is regulated by post-translational modifications, specifically phosphorylation of S473, which modulates binding of TRIM28 to the heterochromatin-binding protein HP1. Here, we identified TRIM28 as a key immune regulator leading to increased IFN-β and proinflammatory cytokine levels during infection with HPAIV. Using influenza A virus strains of the subtype H1N1 as well as HPAIV of subtypes H7N7, H7N9, and H5N1, we could demonstrate that strain-specific phosphorylation of TRIM28 S473 is induced by a signaling cascade constituted of PKR, p38 MAPK, and MSK1 in response to RIG-I independent sensing of viral RNA. Furthermore, using chemical inhibitors as well as knockout cell lines, our results suggest that phosphorylation of S473 facilitates a functional switch leading to increased levels of IFN-β, IL-6, and IL-8. In summary, we have identified TRIM28 as a critical factor controlling excessive expression of type I IFNs as well as proinflammatory cytokines during infection with H5N1, H7N7, and H7N9 HPAIV. In addition, our data indicate a novel mechanism of PKR-mediated IFN-β expression, which could lay the ground for novel treatment options aiming at rebalancing dysregulated immune responses during severe HPAIV infection.
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