Dynamin recruitment and membrane scission at the neck of a clathrin-coated pit

Dynamin, the GTPase required for clathrin-mediated endocytosis, is recruited to clathrin-coated pits in two sequential phases. The first is associated with coated pit maturation; the second, with fission of the membrane neck of a coated pit. Using gene-edited cells that express dynamin2-EGFP instead of dynamin2 and live-cell TIRF imaging with single-molecule EGFP sensitivity and high temporal resolution, we detected the arrival of dynamin at coated pits and defined dynamin dimers as the preferred assembly unit. We also used live-cell spinning-disk confocal microscopy calibrated by single-molecule EGFP detection to determine the number of dynamins recruited to the coated pits. A large fraction of budding coated pits recruit between 26 and 40 dynamins (between 1 and 1.5 helical turns of a dynamin collar) during the recruitment phase associated with neck fission; 26 are enough for coated vesicle release in cells partially depleted of dynamin by RNA interference. We discuss how these results restrict models for the mechanism of dynamin-mediated membrane scission

Synergistic block of SARS-CoV-2 infection by combined drug inhibition of the host entry factors PIKfyve kinase and TMPRSS2 protease

Repurposing FDA-approved inhibitors able to prevent infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could provide a rapid path to establish new therapeutic options to mitigate the effects of coronavirus disease 2019 (COVID-19). Proteolytic cleavages of the spike (S) protein of SARS-CoV-2, mediated by the host cell proteases cathepsin and TMPRSS2, alone or in combination, are key early activation steps required for efficient infection. The PIKfyve kinase inhibitor apilimod interferes with late endosomal viral traffic and through an ill-defined mechanism prevents in vitro infection through late endosomes mediated by cathepsin. Similarly, inhibition of TMPRSS2 protease activity by camostat mesylate or nafamostat mesylate prevents infection mediated by the TMPRSS2-dependent and cathepsin-independent pathway. Here, we combined the use of apilimod with camostat mesylate or nafamostat mesylate and found an unexpected ∼5- to 10-fold increase in their effectiveness to prevent SARS-CoV-2 infection in different cell types. Comparable synergism was observed using both a chimeric vesicular stomatitis virus (VSV) containing S of SARS-CoV-2 (VSV-SARS-CoV-2) and SARS-CoV-2. The substantial ∼5-fold or higher decrease of the half-maximal effective concentrations (EC50s) suggests a plausible treatment strategy based on the combined use of these inhibitors.Peer reviewe

Complete Protection from SARS-CoV-2 Lung Infection in Mice Through Combined Intranasal Delivery of PIKfyve Kinase and TMPRSS2 Protease Inhibitors

Emerging variants of concern of SARS-CoV-2 can significantly reduce the prophylactic and therapeutic efficacy of vaccines and neutralizing antibodies due to mutations in the viral genome. Targeting cell host factors required for infection provides a complementary strategy to overcome this problem since the host genome is less susceptible to variation during the life span of infection. The enzymatic activities of the endosomal PIKfyve phosphoinositide kinase and the serine protease TMPRSS2 are essential to meditate infection in two complementary viral entry pathways. Simultaneous inhibition in cultured cells of their enzymatic activities with the small molecule inhibitors apilimod dimesylate and nafamostat mesylate synergistically prevent viral entry and infection of native SARS-CoV-2 and vesicular stomatitis virus (VSV)-SARS-CoV-2 chimeras expressing the SARS-CoV-2 surface spike (S) protein and of variants of concern. We now report prophylactic prevention of lung infection in mice intranasally infected with SARS-CoV-2 beta by combined intranasal delivery of very low doses of apilimod dimesylate and nafamostat mesylate, in a formulation that is stable for over 3 months at room temperature. Administration of these drugs up to 6 hours post infection did not inhibit infection of the lungs but substantially reduced death of infected airway epithelial cells. The efficiency and simplicity of formulation of the drug combination suggests its suitability as prophylactic or therapeutic treatment against SARS-CoV-2 infection in households, point of care facilities, and under conditions where refrigeration would not be readily available

Cisternal Organization of the Endoplasmic Reticulum during Mitosis

The endoplasmic reticulum (ER) of animal cells is a single, dynamic, and continuous membrane network of interconnected cisternae and tubules spread out throughout the cytosol in direct contact with the nuclear envelope. During mitosis, the nuclear envelope undergoes a major rearrangement, as it rapidly partitions its membrane-bound contents into the ER. It is therefore of great interest to determine whether any major transformation in the architecture of the ER also occurs during cell division. We present structural evidence, from rapid, live-cell, three-dimensional imaging with confirmation from high-resolution electron microscopy tomography of samples preserved by high-pressure freezing and freeze substitution, unambiguously showing that from prometaphase to telophase of mammalian cells, most of the ER is organized as extended cisternae, with a very small fraction remaining organized as tubules. In contrast, during interphase, the ER displays the familiar reticular network of convolved cisternae linked to tubules

Molecularly Distinct Clathrin-Coated Pits Differentially Impact EGFR Fate and Signaling

International audienc

Nanomolar inhibition of SARS-CoV-2 infection by an unmodified peptide targeting the prehairpin intermediate of the spike protein

Publisher Copyright: © 2022 National Academy of Sciences. All rights reserved.Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available coronavirus disease 2019 vaccines and monoclonal antibody therapies through epitope change on the receptor binding domain of the viral spike glycoprotein. Hence, there is a specific urgent need for alternative antivirals that target processes less likely to be affected by mutation, such as the membrane fusion step of viral entry into the host cell. One such antiviral class includes peptide inhibitors, which block formation of the so-called heptad repeat 1 and 2 (HR1HR2) six-helix bundle of the SARS-CoV-2 spike (S) protein and thus interfere with viral membrane fusion. We performed structural studies of the HR1HR2 bundle, revealing an extended, well-folded N-terminal region of HR2 that interacts with the HR1 triple helix. Based on this structure, we designed an extended HR2 peptide that achieves single-digit nanomolar inhibition of SARS-CoV-2 in cell-based and virus-based assays without the need for modifications such as lipidation or chemical stapling. The peptide also strongly inhibits all major SARS-CoV-2 variants to date. This extended peptide is ∼100-fold more potent than all previously published short, unmodified HR2 peptides, and it has a very long inhibition lifetime after washout in virus infection assays, suggesting that it targets a prehairpin intermediate of the SARS-CoV-2 S protein. Together, these results suggest that regions outside the HR2 helical region may offer new opportunities for potent peptide-derived therapeutics for SARS-CoV-2 and its variants, and even more distantly related viruses, and provide further support for the prehairpin intermediate of the S protein.Peer reviewe

SARS-CoV-2 requires acidic pH to infect cells

Publisher Copyright: Copyright © 2022 the Author(s). Published by PNAS.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell entry starts with membrane attachment and ends with spike (S) protein–catalyzed membrane fusion depending on two cleavage steps, namely, one usually by furin in producing cells and the second by TMPRSS2 on target cells. Endosomal cathepsins can carry out both. Using real-time three-dimensional single-virion tracking, we show that fusion and genome penetration require virion exposure to an acidic milieu of pH 6.2 to 6.8, even when furin and TMPRSS2 cleavages have occurred. We detect the sequential steps of S1-fragment dissociation, fusion, and content release from the cell surface in TMPRRS2-overexpressing cells only when exposed to acidic pH. We define a key role of an acidic environment for successful infection, found in endosomal compartments and at the surface of TMPRSS2-expressing cells in the acidic milieu of the nasal cavity.Peer reviewe