Biology subjects

Brunotte, L.

Publications and source records attributed to Brunotte, L..

7 recordsLinked to original sources

bioRxiv2022

Glycolytic interference blocks influenza A virus propagation by impairing viral polymerase-driven synthesis of genomic vRNA

Influenza A virus (IAV), like any other virus, provokes considerable modifications of its host cells metabolism. This includes a substantial increase in the uptake as well as the metabolization of glucose. Although it is known for quite some time that suppression of glucose metabolism restricts virus replication, the exact molecular impact on the viral life cycle remained enigmatic so far. Using 2-deoxy-D-glucose (2-DG) we examined how well inhibition of glycolysis is tolerated by host cells and which step of the IAV life cycle is affected. We observed that effects induced by 2-DG are reversible and that cells can cope with relatively high concentrations of the inhibitor by compensating the loss of glycolytic activity by upregulating other metabolic pathways. Moreover, mass spectrometry data provided information on various metabolic modifications induced by either the virus or agents interfering with glycolysis. In the presence of 2-DG viral titers were significantly reduced in a dose-dependent manner. The supplementation of direct or indirect glycolysis metabolites led to a partial or almost complete reversion of the inhibitory effect of 2-DG on viral growth and demonstrated that indeed the inhibition of glycolysis and not of N-linked glycosylation was responsible for the observed phenotype. Importantly, we could show via conventional and strand-specific qPCR that the treatment with 2-DG led to a prolonged phase of viral mRNA synthesis while the accumulation of genomic vRNA was strongly reduced. At the same time, minigenome assays showed no signs of a general reduction of replicative capacity of the viral polymerase. Therefore, our data suggest that the significant reduction in IAV replication by glycolytic interference occurs mainly due to an impairment of the dynamic regulation of the viral polymerase which conveys the transition of the enzymes function from transcription to replication. Author SummaryUpon infection the influenza A virus alters the metabolism of infected cells. Among others, this includes a pronounced increase in glucose metabolism. We aimed to get a better understanding of these metabolic virus-host interactions and to unravel the mechanism by which glycolytic inhibition impairs the viral life cycle. On the one hand, we observed a virus-induced upregulation of many glycolysis metabolites which could often be reversed by the administration of a glycolysis inhibitor. On the other hand, our data suggested that the inhibitor treatment severely impaired viral propagation by interfering with the regulation of the viral polymerase. This manifested in an extended phase of transcription, while replication was strongly reduced. Additionally, we assessed the safety and tolerability of the used drug in immortalized and primary cells. Our study sheds more light on metabolic virus-host interactions and provides a better understanding of metabolic interference as a potential host-targeted antiviral approach, which does not bear the risk of creating resistances.

microbiology↗

bioRxiv2021

SARS-CoV-2 infects and replicates in photoreceptor and retinal ganglion cells of human retinal organoids

Several studies have pointed to retinal involvement in COVID-19 disease, yet many questions remain regarding the ability of SARS-CoV-2 to infect and replicate in retinal cells and its effects on the retina. Here we have used human stem cell-derived retinal organoids to study retinal infection by the SARS-CoV-2 virus. Indeed, SARS-CoV-2 can infect and replicate in retinal organoids, as it is shown to infect different retinal lineages, such as retinal ganglion cells and photoreceptors. SARS-CoV-2 infection of retinal organoids also induces the expression of several inflammatory genes, such as interleukin 33, a gene associated with acute COVID-19 disease and retinal degeneration. Finally, we show that the use of antibodies to block the ACE2 receptor significantly reduces SARS-CoV-2 infection of retinal organoids, indicating that SARS-CoV-2 infects retinal cells in an ACE2-dependent manner. These results suggest a retinal involvement in COVID-19 and emphasize the need to monitor retinal pathologies as potential sequelae of "long COVID".

molecular biology↗

bioRxiv2021

Differential interferon-α subtype immune signatures suppress SARS-CoV-2 infection

Type I interferons (IFN-I) exert pleiotropic biological effects during viral infections, balancing virus control versus immune-mediated pathologies and have been successfully employed for the treatment of viral diseases. Humans express twelve IFN-alpha () subtypes, which activate downstream signalling cascades and result in distinct patterns of immune responses and differential antiviral responses. Inborn errors in type I IFN immunity and the presence of anti-IFN autoantibodies account for very severe courses of COVID-19, therefore, early administration of type I IFNs may be protective against life-threatening disease. Here we comprehensively analysed the antiviral activity of all IFN subtypes against SARS-CoV-2 to identify the underlying immune signatures and explore their therapeutic potential. Prophylaxis of primary human airway epithelial cells (hAEC) with different IFN subtypes during SARS-CoV-2 infection uncovered distinct functional classes with high, intermediate and low antiviral IFNs. In particular IFN5 showed superior antiviral activity against SARS-CoV-2 infection. Dose-dependency studies further displayed additive effects upon co-administered with the broad antiviral drug remdesivir in cell culture. Transcriptomics of IFN-treated hAEC revealed different transcriptional signatures, uncovering distinct, intersecting and prototypical genes of individual IFN subtypes. Global proteomic analyses systematically assessed the abundance of specific antiviral key effector molecules which are involved in type I IFN signalling pathways, negative regulation of viral processes and immune effector processes for the potent antiviral IFN5. Taken together, our data provide a systemic, multi-modular definition of antiviral host responses mediated by defined type I IFNs. This knowledge shall support the development of novel therapeutic approaches against SARS-CoV-2.

cell biology↗

bioRxiv2020

Drug synergy of combinatory treatment with remdesivir and the repurposed drugs fluoxetine and itraconazole effectively impairs SARS-CoV-2 infection in vitro

The SARS-COV-2 pandemic and the global spread of coronavirus disease 2019 (COVID-19) urgently calls for efficient and safe antiviral treatment strategies. A straightforward approach to speed up drug development at lower costs is drug repurposing. Here we investigated the therapeutic potential of targeting the host- SARS-CoV-2 interface via repurposing of clinically licensed drugs and evaluated their use in combinatory treatments with virus- and host-directed drugs. We tested the antiviral potential of repurposing the antifungal itraconazole and the antidepressant fluoxetine on the production of infectious SARS-CoV-2 particles in the polarized Calu-3 cell culture model and evaluated the added benefit of a combinatory use of these host-directed drugs with remdesivir, an inhibitor of viral RNA polymerase. Drug treatments were well-tolerated and potent impaired viral replication was observed with all drug treatments. Importantly, both itraconazole-remdesivir and fluoxetine-remdesivir combinations inhibited the production of infectious SARS-CoV-2 particles > 90% and displayed synergistic effects in commonly used reference models for drug interaction. Itraconazole-Remdesivir and Fluoxetine-Remdesivir combinations are promising therapeutic options to control SARS-CoV-2 infection and severe progression of COVID-19.

pharmacology and toxicology↗

bioRxiv2020

SARS-CoV-2 Quasispecies Mediate Rapid Virus Evolution and Adaptation

Since the pandemic spread of SARS-CoV-2, the virus has exhibited remarkable genome stability, but recent emergence of novel variants show virus evolution potential. Here we show that SARS-CoV-2 rapidly adapts to Vero E6 cells that leads to loss of furin cleavage motif in spike protein. The adaptation is achieved by asymptotic expansion of minor virus subpopulations to dominant genotype, but wildtype sequence is maintained at low percentage in the virus swarm, and mediate reverse adaptation once the virus is passaged on human lung cells. The Vero E6-adapted virus show defected cell entry in human lung cells and the mutated spike variants cannot be processed by furin or TMPRSS2. However, the mutated S1/S2 site is cleaved by cathepsins with higher efficiency. Our data show that SARS-CoV-2 can rapidly adapt spike protein to available proteases and advocate for deep sequence surveillance to identify virus adaptation potential and novel variant emergence. Significance StatementRecently emerging SARS-CoV-2 variants B1.1.1.7 (UK), B.1.351 (South Africa) and B.1.1.248 (Brazil) harbor spike mutation and have been linked to increased virus pathogenesis. The emergence of these novel variants highlight coronavirus adaptation and evolution potential, despite the stable consensus genotype of clinical isolates. We show that subdominant variants maintained in the virus population enable the virus to rapidly adapt upon selection pressure. Although these adaptations lead to genotype change, the change is not absolute and genome with original genotype are maintained in virus swarm. Thus, our results imply that the relative stability of SARS-CoV-2 in numerous independent clinical isolates belies its potential for rapid adaptation to new conditions.

microbiology↗

bioRxiv2020

Targeting the endolysosomal host-SARS-CoV-2 interface by the clinically licensed antidepressant fluoxetine

The Corona Virus 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. 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 and COVID 19.

microbiology↗

bioRxiv2020

SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface

COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a novel betacoronavirus discovered in December 2019 and closely related to the SARS coronavirus (CoV). Both viruses use the human ACE2 receptor for cell entry, recognizing it with the Receptor Binding Domain (RBD) of the S1 subunit of the viral spike (S) protein. The S2 domain mediates viral fusion with the host cell membrane. Experience with SARS and MERS coronaviruses has shown that potent monoclonal neutralizing antibodies against the RBD can inhibit the interaction with the virus cellular receptor (ACE2 for SARS) and block the virus cell entry. Assuming that a similar strategy would be successful against SARS-CoV-2, we used phage display to select from the human naive universal antibody gene libraries HAL9/10 anti-SARS-CoV-2 spike antibodies capable of inhibiting interaction with ACE2. 309 unique fully human antibodies against S1 were identified. 17 showed more than 75% inhibition of spike binding to cells expressing ACE2 in the scFv-Fc format, assessed by flow cytometry and several antibodies showed even an 50% inhibition at a molar ratio of the antibody to spike protein or RBD of 1:1. All 17 scFv-Fc were able to bind the isolated RBD, four of them with sub-nanomolar EC50. Furthermore, these scFv-Fc neutralized active SARS-CoV-2 virus infection of VeroE6 cells. In a final step, the antibodies neutralizing best as scFv-Fc were converted into the IgG format. The antibody STE73-2E9 showed neutralization of active SARS-CoV-2 with an IC50 0.43 nM and is binding to the ACE2-RBD interface. Universal libraries from healthy human donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovered patients in a pandemic situation.

biochemistry↗