Obernolte, H. | Search bioRxiv

bioRxiv2024

AMPK/ULK1 activation downregulates TXNIP, Rab5, and Rab7 and inhibits endocytosis-mediated entry of human pathogenic viruses

Cellular metabolism must adapt rapidly to environmental alterations and adjust nutrient uptake. Low glucose availability activates the AMP-dependent kinase (AMPK) pathway. We demonstrate that activation of AMPK or the downstream Unc-51-like autophagy-activating kinase (ULK1) inhibits receptor-mediated endocytosis. Beyond limiting dextran-uptake, this activation prevents endocytic uptake of human pathogenic enveloped and non-enveloped, positive and negative-stranded RNA viruses, such as yellow fever, dengue, tick-borne encephalitis, chikungunya, polio, rubella, rabies lyssavirus and SARS-CoV-2 not only in mammalian and insect cells but in precision-cut lung slices and neuronal organoids. However, receptor presentation at the cytoplasmic membrane was unaffected, indicating that receptor-binding remained unaltered and later steps of endocytosis were targeted. Indeed, AMPK pathway activation reduced early endocytic factors TXNIP, Rab5 and the late endosomal marker Rab7 amounts. Furthermore, AMPK activation impaired SARS-CoV-2 late-replication steps by reducing viral RNAs and proteins and the endo-lysosomal markers LAMP1 and GRP78, suggesting a reduction of early and late endosomes and lysosomes. Inhibition of the PI3K and mTORC2 pathways, which sense amino acids and growth factor availability, promotes AMPK activity and blocks viral entry. Our results indicate that AMPK and ULK1 emerge as restriction factors of cellular endocytosis, impeding the receptor-mediated endocytic entry of enveloped and non-enveloped RNA viruses.

microbiology↗

bioRxiv2024

N-acylethanolamine acid amidase inhibition reduces SARS-CoV-2 infection in Human Precision cut-lung slices and downregulates NF-kappaB signalling.

Like other positive-sense RNA viruses, SARS-CoV-2 manipulates host lipid metabolism to facilitate its replication by enhancing lipogenesis and lipid droplet formation. In doing so, SARS-CoV-2 infection perturbs bioactive lipid levels associated with the inflammatory response. One of these, Palmitoylethanolamide (PEA) is suppressed during SARS-CoV-2 infection since it activates the Peroxisome Proliferator-Activated Receptor- (PPAR-), a transcription factor that suppresses the nuclear factor-B (NF-{kappa}B), which is mandatory to sustain SARS-CoV-2 replication. PEA levels are regulated by N-acylethanolamine acid amidase (NAAA), a lysosomal enzyme responsible for catalysing the breakdown of PEA. We hypothesized that NAAA inhibition might interfere with SARS-CoV-2 replication since it will lead PEA to accumulate, activating PPAR- and, consequently, suppressing NF-{kappa}B. Our results reveal that genetic or chemical ablation of NAAA significantly suppresses SARS-CoV-2 replication by three log10 in human-derived precision-cut lung slices. Therefore, we investigated whether inhibiting NAAA could influence NF-{kappa}B activation through the activation of PPAR-. We observed PPAR- increased expression in NAAA-/-cells, while PPAR- expression remained low in infected parental cells. As expected, the elevated PPAR- expression correlated with a parallel reduction in NF-{kappa}B activation when NAAA is ablated. These findings underscore NAAA as an essential host factor for SARS-CoV-2 replication and propose a potential mechanism of action rooted in the attenuation of NF-{kappa}B activation during viral replication. Author summaryOver the past three years, COVID-19 has claimed nearly 7 million lives worldwide, prompting extensive efforts to find effective treatments. While RNA-based vaccines have been developed rapidly, they alone have not completely halted the spread of the virus, making the search for antiviral therapies crucial. One promising approach targets the anti-inflammatory lipid PEA, which has shown some success in COVID-19 clinical trials. PEA is quickly degraded by the enzyme NAAA. Researchers have found that inhibiting NAAA can enhance and prolong PEA anti-inflammatory effects. NAAA inhibitors have already shown effectiveness in reducing chronic pain and lung inflammation in animal models and have also been effective against Zika virus replication. Our research focused on testing the NAAA inhibitor ARN726 against SARS-CoV-2. In human lung cells and lung tissue samples, ARN726 significantly reduced SARS-CoV-2 replication and inflammation. We discovered that this inhibition suppresses the NF-{kappa}B pathway, which the virus uses to fuel its replication and sustain Cytokine storm. Overall, our findings suggest that NAAA inhibitors like ARN726 could be repurposed to combat COVID-19 and potentially other coronaviruses, offering a novel and effective antiviral strategy.

microbiology↗

bioRxiv2022

Acetylsalicylic acid and Salicylic acid inhibit SARS-CoV-2 replication in precision-cut lung slices

Aspirin, with its active compound acetylsalicylic acid (ASA), shows antiviral activity against rhino- and influenza viruses and at high concentrations. We sought to investigate whether ASA and its metabolite salicylic acid (SA) inhibit SARS-CoV-2 since it might use similar pathways to influenza viruses. The compound-treated cells were infected with SARS-CoV-2. Viral replication was analyzed by RTqPCR. The compounds suppressed SARS-CoV-2 replication in cell culture cells and in a patient-near replication system using human precision-cut lung slices by two orders of magnitude. The compounds did not interfere with viral entry but led to lower viral RNA expression after 24 h.

microbiology↗