Polyamines mediate enterovirus attachment directly and indirectly through cellular heparan sulfate synthesis.

Productive viral infection begins with attachment to a susceptible cell, and viruses have evolved complex mechanisms to attach to and subsequently enter cells. Prior to engagement with a cellular receptor, viruses frequently interact with nonspecific attachment factors that can facilitate virus-receptor interactions and viral entry. Polyamines, small positively-charged molecules abundant in mammalian cells, mediate viral attachment, though the mechanism was not fully understood. Using the Coxsackievirus B3 (CVB3) enterovirus model system, we show that polyamines mediate viral attachment both directly and indirectly. The polyamine putrescine specifically enhances viral attachment to cells depleted of polyamines. Putrescine's positive charge mediates its ability to enhance viral attachment, and polyamine analogs are less efficient at mediating viral attachment. In addition to this direct role of polyamines in attachment, polyamines facilitate the cellular expression of heparan sulfates, negatively-charged molecules found on the cell surface. In polyamine-depleted cells, heparan sulfates are depleted from the surface of cells, resulting in reduced viral attachment. We find that this is due to polyamines' role in the process of hypusination of eukaryotic initiation factor 5A, which facilitates cellular translation. These data highlight the important role of polyamines in mediating cellular attachment, as well as their function in facilitating cellular heparan sulfate synthesis.

LRRC15 mediates an accessory interaction with the SARS-CoV-2 spike protein

The interactions between severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and human host factors enable the virus to propagate infections that lead to COVID-19. The spike protein is the largest structural component of the virus and mediates interactions essential for infection, including with the primary ACE2 receptor. We performed two independent cell-based systematic screens to determine whether there are additional proteins by which the spike protein of SARS-CoV-2 can interact with human cells. We discovered that in addition to ACE2, expression of LRRC15 also causes spike protein binding. This interaction is distinct from other known spike attachment mechanisms such as heparan sulfates or lectin receptors. Measurements of orthologous coronavirus spike proteins implied the interaction was restricted to SARS-CoV-2, suggesting LRRC15 represents a novel class of spike binding interaction. We localized the interaction to the C-terminus of the S1 domain, and showed that LRRC15 shares recognition of the ACE2 receptor binding domain. From analyzing proteomics and single-cell transcriptomics, we identify LRRC15 expression as being common in human lung vasculature cells and fibroblasts. Although infection assays demonstrated that LRRC15 alone is not sufficient to permit viral entry, we present evidence it can modulate infection of human cells. This unexpected interaction merits further investigation to determine how SARS-CoV-2 exploits host LRRC15 and whether it could account for any of the distinctive features of COVID-19.

A case series of 13 patients with COVID-19 associated respiratory symptoms treated with Nebulized OTR4120 (Cacipliq20®)

We report a series of 13 patients with COVID-19 treated with Cacipliq20®, an heparan sulfate mimetic approved for the treatment of hard to heal cutaneous ulcers. Heparan sulfates play important roles in tissue repair and possess antiviral activity. Cacipliq20® was administered through nebulization at a dose of 45 mg twice a day for 5.5 consecutive days. All patients presented respiratory symptoms with some dyspnea and in most cases pulmonary abnormalities on chest CT-Scan. Eight patients presented with a moderate form of the disease, three patients with a severe form, one with a mild form, and one with a critical form. In all patients the treatment was added to the standard of care. Ten patients were treated during the acute stage of the disease (<4 weeks from symptoms onset) while 3 patients were in the post-acute stage (>4 weeks from symptoms onset). A second treatment was administered for another 5.5 days in 6 patients. All patients showed clinically improvement after treatment. The time to first improvement ranged from 2 to 4 days after first treatment onset with a median of 3 days. Time to full clinical recovery ranged between 6 to 27 days from treatment onset with a median of 6 days. Lung CT scans followed clinical impression and showed a clear improvement of the lesions in most cases. The treatment was well tolerated in all patients. These preliminary observations should justify further evaluation through a well-designed placebo-controlled therapeutic trial.

Cell entry inhibitor with sulfonated colloid gold as new potent broad-spectrum virucides

We recently developed nontoxic, broad-spectrum virucidal gold nanoparticles,less than 10nm sized, modified with sulfonic acids (mesilate) that mimic heparan sulfates. Camostat, a serine protease inhibitor can introduce gold nanoparticles to the influenza virus via ionic bonds. In this study, we examined the ability of a novel sulfonated colloid gold to inhibit the virus in vivo. Our data showed that fully protected the mice from lethal infection and significantly decreased viral titers in the lungs of infected animals. Thus, camostat-colloid gold is a promising candidate for the development of antiviral drugs to prevent and treat influenza infection.

Shape and Function of Interstitial Chemokine CCL21 Gradients Are Independent of Heparan Sulfates Produced by Lymphatic Endothelium

Gradients of chemokines and growth factors guide migrating cells and morphogenetic processes. Migration of antigen-presenting dendritic cells from the interstitium into the lymphatic system is dependent on chemokine CCL21, which is secreted by endothelial cells of the lymphatic capillary, binds heparan sulfates and forms gradients decaying into the interstitium. Despite the importance of CCL21 gradients, and chemokine gradients in general, the mechanisms of gradient formation are unclear. Studies on fibroblast growth factors have shown that limited diffusion is crucial for gradient formation. Here, we used the mouse dermis as a model tissue to address the necessity of CCL21 anchoring to lymphatic capillary heparan sulfates in the formation of interstitial CCL21 gradients. Surprisingly, the absence of lymphatic endothelial heparan sulfates resulted only in a modest decrease of CCL21 levels at the lymphatic capillaries and did neither affect interstitial CCL21 gradient shape nor dendritic cell migration toward lymphatic capillaries. Thus, heparan sulfates at the level of the lymphatic endothelium are dispensable for the formation of a functional CCL21 gradient.

Intermedilysin cytolytic activity depends on heparan sulfates and membrane composition

Cholesterol-dependent cytolysins (CDCs), of which intermedilysin (ILY) is an archetypal member, are a group of pore-forming toxins secreted by a large variety of pathogenic bacteria. These toxins, secreted as soluble monomers, oligomerize upon interaction with cholesterol in the target membrane and transect it as pores of diameters of up to 100 to 300 Å. These pores disrupt cell membranes and result in cell lysis. The immune receptor CD59 is a well-established cellular factor required for intermedilysin pore formation. In this study, we applied genome-wide CRISPR-Cas9 knock-out screening to reveal additional cellular co-factors essential for ILY-mediated cell lysis. We discovered a plethora of genes previously not associated with ILY, many of which are important for membrane constitution. We show that heparan sulfates facilitate ILY activity, which can be inhibited by heparin. Furthermore, we identified hits in both protein and lipid glycosylation pathways and show a role for glucosylceramide, demonstrating that membrane organization is important for ILY activity. We also cross-validated identified genes with vaginolysin and pneumolysin and found that pneumolysin’s cytolytic activity strongly depends on the asymmetric distribution of membrane phospholipids. This study shows that membrane-targeting toxins combined with genetic screening can identify genes involved in biological membrane composition and metabolism.