Cholesterol 25-hydroxylase (CH25H) is an interferon (IFN)-stimulated gene that shows broad antiviral activities against a wide range of enveloped viruses. Here, using an IFN-stimulated gene screen against vesicular stomatitis virus (VSV)-SARS-CoV and VSV-SARS-CoV-2 chimeric viruses, we identified CH25H and its enzymatic product 25-hydroxycholesterol (25HC) as potent inhibitors of SARS-CoV-2 replication. Internalized 25HC accumulates in the late endosomes and potentially restricts SARS-CoV-2 spike protein catalyzed membrane fusion via blockade of cholesterol export. Our results highlight one of the possible antiviral mechanisms of 25HC and provide the molecular basis for its therapeutic development.

Type 3 secretion systems (T3SSs) of bacterial pathogens translocate bacterial effector proteins that mediate disease into the eukaryotic cytosol. Effectors traverse the plasma membrane through a translocon pore formed by T3SS proteins. In a genome-wide selection, we identified the intermediate filament vimentin as required for infection by the T3SS-dependent pathogen Shigella flexneri. We found that vimentin is required for efficient T3SS translocation of effectors by S. flexneri and other pathogens that use T3SS, Salmonella Typhimurium and Yersinia pseudotuberculosis. Vimentin and the intestinal epithelial intermediate filament keratin 18 interact with the C-terminus of the Shigella translocon pore protein IpaC. Vimentin and its interaction with IpaC are dispensable for pore formation, but are required for stable docking of S. flexneri to cells; moreover, stable docking triggers effector secretion. These findings establish that stable docking of the bacterium specifically requires intermediate filaments, is a process distinct from pore formation, and is a prerequisite for effector secretion.

Paramyxovirus entry into cells requires the fusion protein (F) and a receptor binding protein (hemagglutinin-neuraminidase [HN], H, or G

The Paramyxoviridae family of enveloped viruses enters cells through the concerted action of two viral glycoproteins. The receptor-binding protein, hemagglutinin-neuraminidase (HN), H, or G, binds its cellular receptor and activates the fusion protein, F, which, through an extensive refolding event, brings viral and cellular membranes together, mediating virus-cell fusion. However, the underlying mechanism of F activation on receptor engagement remains unclear. Current hypotheses propose conformational changes in HN, H, or G propagating from the receptor-binding site in the HN, H, or G globular head to the F-interacting stalk region. We provide evidence that the receptor-binding globular head domain of the paramyxovirus parainfluenza virus 5 HN protein is entirely dispensable for F activation. Considering together the crystal structures of HN from different paramyxoviruses, varying energy requirements for fusion activation, F activation involving the parainfluenza virus 5 HN stalk domain, and properties of a chimeric paramyxovirus HN protein, we propose a simple model for the activation of paramyxovirus fusion. Paramyxovirus-mediated fusion depends on the concerted actions of two glycoproteins, an attachment protein [hemagglutinin-neuraminidase (HN), H, or G] and its cognate fusion (F) protein, the latter initially folding into a metastable form. The attachment protein is thought to trigger the fusion protein in a receptor-dependent manner (1-5). This triggering of the metastable F (6) by the receptor-binding protein couples receptor binding of HN, H, or G to lowering the activation energy barrier of F so that F refolds into a highly stable postfusion form (7). In the process, F undergoes a series of structural rearrangements involving several intermediates and brings about membrane merger (8).HN proteins bind sialic acid as their receptor and also have neuraminidase (receptor-destroying) activity. The PIV5 HN protein comprises 565 residues and has a short N-terminal cytoplasmic tail followed by a single transmembrane domain and a large ectodomain (residues 37-565). The protein consists of a stalk region (residues 1-117) that supports a large globular head domain (residues 118-565) containing the receptor-binding and neuraminidase-active site. X-ray crystal structures of the globular head domain of PIV5, NDV, Nipah virus, Hendra virus, measles virus, and human parainfluenza virus 3 (hPIV3) attachment proteins have been obtained (9-16). The PIV5 HN globular head structure (16) reveals a neuraminidase-like fold with a six-bladed β-propeller structure that is a common feature of the other paramyxovirus HN/H/G head domains, regardless of receptor specificity. The sialic acid-binding site is placed centrally within the β-propeller. PIV5 HN exists as a pair of disulfide-linked dimers with the disulfide bond at cysteine 111 (16,17). These dimers are associated noncovalently to form a dimer-of-dimer oligomer (16,18). The PIV5 HN dimer-of-dimer structure showed the dimers arranged at an approximately 90°angle to each oth...