ABSTRACT
Objective: Apart from the respiratory system, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can potentially infect multiple other organs including podocytes in the kidney. The latter play a crucial role in glomerular filtration. Podocytes can be damaged by increased fluid flow shear stress (FFSS) of the ultrafiltrate in Bowman's space in the setting of glomerular hyperfiltration that occurs in disease states such as hypertension, diabetes or in several forms of chronic kidney disease. These conditions are associated with an increased risk of a more severe course of coronavirus disease 2019 (COVID-19) and mortality. Design and method: To assess the susceptibility of human podocytes (hPC) for SARS-CoV-2 infection in the context of hyperfiltration in vitro, we used a recently established model system (Streamer Shear Stress Device)) to mimic hyperfiltration by exposing hPC to increased FFSS of 1 dyne/cm2 for 2 h. In this setting we nalysed the effects of FFSS on mRNA expression of angiotensin I-converting enzyme 2 (ACE2) as the pivotal entry receptor for SARS-CoV-2 infection in hPC. Moreover, other potential critical host cell factors including transmembrane serine protease 2 (TMPRSS2), furin (FURIN), and neuropilin 1 (NRP1) were also assessed in parallel with changes of the F-actin fiber structure, i.e. an important cytoskeletal marker in hPC. Results: Under control conditions, hPC displayed long, parallel F-actin fibers crossing the entire cell body. After FFSS, an enrichment of cells that express F-actin in a cortically condensed pattern near the cell membrane was observed. FFSS induced a significant upregulation of ACE2 expression (about twofold) and of all other nalysed SARS-CoV-2 entry factors in hPC (p < 0.05, respectively compared to control conditions, Figure 1 with data plotted as log2fold change [FC]). Conclusions: Our data support a potential link between glomerular hyperfiltration, podocyte damage and renal tropism of SARS-CoV-2 that may contribute to kidney damage including albuminuria development in COVID-19 patients.
ABSTRACT
Background: Human immunodeficiency virus (HIV) and Influenza A virus (IAV) remain a global health concern. Further, emergence of novel coronavirus SARS-CoV-2, which rapidly became global pandemic, increases the concern in biomedical research field for antiviral treatment. To develop new antiviral therapy, we must need to understand the molecular and cellular mechanisms involved in assembly and replication. It is known for some viruses (HIV and IAV) that the host actin cytoskeleton has been involved in various stages of the virus life cycle. Regulation of actin cytoskeleton requires several actin binding proteins, which organize the actin filaments (F-actin) into higher order structures such as actin bundles, branches, filopodia and microvilli, for further assistance in viral particle production. Thus, our objective for this work is to understand the role of these actin regulator proteins, like cofilin and one of its cofactor WDR1, in viral particle assembly and release. Methods: Here we used a combination of different experimental methods like RNA interference, immunoblot, immunoprecipitation, immunofluorescence coupled to confocal and STED fluorescence microscopy. In order to study only virus release, and bypass viral entry, we set up a minimal system for virus-like particles production in transfected cells, giving HIV-1 Gag-VLP, Influenza M1-VLP and SARS-CoV-2 MNE-VLP (developed by D. Muriaux lab). For image analysis, we used Image J software. Statistical analysis was performed with non-parametric t-tests or one-way Anova test. Results: Using siRNA strategy, we have shown that upon knock down of actin protein cofilin or WDR1, HIV-1 and IAV particles production increases in contrario to SARS-CoV-2 VLP release. Further, using immunoprecipitation, we report that HIV-1 Gag is able to form an intracellular complex with WDR1 and cofilin. Similarly, IAV-M1, which like HIV Gag-MA binds with plasma membrane phospholipids, is able to form an intracellular complex with cofilin. These results suggested that virus budding from the host cell plasma membrane seemed restricted by the cofilin/WDR1 complex. Finally, using confocal/STED microscopy on cell producing VLP, we observed actin fibers rearrangement with cell protrusions, suggesting a role for actin in viral particles assembly and release. Conclusion: In conclusion, regulators of actin dynamic are involved in HIV-1 Gag, IAV-M1 and SARS-CoV-2 VLP production but play a differential role in assembly and release of these RNA enveloped viruses.
ABSTRACT
Acidosis, such as respiratory acidosis and metabolic acidosis, can be induced by coronavirus disease 2019 (COVID-19) infection and is associated with increased mortality in critically ill COVID-19 patients. It remains unclear whether acidosis further promotes SARS-CoV-2 infection in patients, making virus removal difficult. For antacid therapy, sodium bicarbonate poses great risks caused by sodium overload, bicarbonate side effects, and hypocalcemia. Therefore, new antacid antidote is urgently needed. Our study showed that an acidosis-related pH of 6.8 increases SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) expression on the cell membrane by regulating intracellular microfilament polymerization, promoting SARS-CoV-2 pseudovirus infection. Based on this, we synthesized polyglutamic acid-PEG materials, used complexation of calcium ions and carboxyl groups to form the core, and adopted biomineralization methods to form a calcium carbonate nanoparticles (CaCO3-NPs) nanoantidote to neutralize excess hydrogen ions (H+), and restored the pH from 6.8 to approximately 7.4 (normal blood pH). CaCO3-NPs effectively prevented the heightened SARS-CoV-2 infection efficiency due to pH 6.8. Our study reveals that acidosis-related pH promotes SARS-CoV-2 infection, which suggests the existence of a positive feedback loop in which SARS-CoV-2 infection-induced acidosis enhances SARS-CoV-2 infection. Therefore, antacid therapy for acidosis COVID-19 patients is necessary. CaCO3-NPs may become an effective antacid nanoantidote superior to sodium bicarbonate.
ABSTRACT
Middle East respiratory syndrome coronavirus (MERS-CoV), a pathogen causing severe respiratory disease in humans that emerged in June 2012, is a novel beta coronavirus similar to severe acute respiratory syndrome coronavirus (SARS-CoV). In this study, immunoprecipitation and proximity ligation assays revealed that the nucleocapsid (N) protein of MERS-CoV interacted with human translation elongation factor 1A (EF1A), an essential component of the translation system with important roles in protein translation, cytokinesis, and filamentous actin (F-actin) bundling. The C-terminal motif (residues 359-363) of the N protein was the crucial domain involved in this interaction. The interaction between the MERS-CoV N protein and EF1A resulted in cytokinesis inhibition due to the formation of inactive F-actin bundles, as observed in an in vitro actin polymerization assay and in MERS-CoV-infected cells. Furthermore, the translation of a CoV-like reporter mRNA carrying the MERS-CoV 5'UTR was significantly potentiated by the N protein, indicating that a similar process may contribute to EF1A-associated viral protein translation. This study highlights the crucial role of EF1A in MERS-CoV infection and provides new insights into the pathogenesis of coronavirus infections.