Synthetic Protocells Interact with Viral Nanomachinery and Inactivate Pathogenic Human Virus

We present a new antiviral strategy and research tool that could be applied to a wide range of enveloped viruses that infect human beings via membrane fusion. We test this strategy on two emerging zoonotic henipaviruses that cause fatal encephalitis in humans, Nipah (NiV) and Hendra (HeV) viruses. In the new approach, artificial cell-like particles (protocells) presenting membrane receptors in a biomimetic manner were developed and found to attract and inactivate henipavirus envelope glycoprotein pseudovirus particles, preventing infection. The protocells do not accumulate virus during the inactivation process. The use of protocells that interact with, but do not accumulate, viruses may provide significant advantages over current antiviral drugs, and this general approach may have wide potential for antiviral development

Antiviral activity of gliotoxin, gentian violet and brilliant green against Nipah and Hendra virus in vitro

Background: Using a recently described monolayer assay amenable to high throughput screening format for the identification of potential Nipah virus and Hendra virus antivirals, we have partially screened a low molecular weight compound library (8,000 compounds) directly against live virus infection and identified twenty eight promising lead molecules. Initial single blind screens were conducted with 10 M compound in triplicate with a minimum efficacy of 90% required for lead selection. Lead compounds were then further characterised to determine the median efficacy (IC), cytotoxicity (CC) and the in vitro therapeutic index in live virus and pseudotype assay formats. Results: While a number of leads were identified, the current work describes three commercially available compounds: brilliant green, gentian violet and gliotoxin, identified as having potent antiviral activity against Nipah and Hendra virus. Similar efficacy was observed against pseudotyped Nipah and Hendra virus, vesicular stomatitis virus and human parainfluenza virus type 3 while only gliotoxin inhibited an influenza A virus suggesting a non-specific, broad spectrum activity for this compound. Conclusion: All three of these compounds have been used previously for various aspects of anti-bacterial and anti-fungal therapy and the current results suggest that while unsuitable for internal administration, they may be amenable to topical antiviral applications, or as disinfectants and provide excellent positive controls for future studies

Inhibition of Nipah Virus Infection In Vivo: Targeting an Early Stage of Paramyxovirus Fusion Activation during Viral Entry

In the paramyxovirus cell entry process, receptor binding triggers conformational changes in the fusion protein (F) leading to viral and cellular membrane fusion. Peptides derived from C-terminal heptad repeat (HRC) regions in F have been shown to inhibit fusion by preventing formation of the fusogenic six-helix bundle. We recently showed that the addition of a cholesterol group to HRC peptides active against Nipah virus targets these peptides to the membrane where fusion occurs, dramatically increasing their antiviral effect. In this work, we report that unlike the untagged HRC peptides, which bind to the postulated extended intermediate state bridging the viral and cell membranes, the cholesterol tagged HRC-derived peptides interact with F before the fusion peptide inserts into the target cell membrane, thus capturing an earlier stage in the F-activation process. Furthermore, we show that cholesterol tagging renders these peptides active in vivo: the cholesterol-tagged peptides cross the blood brain barrier, and effectively prevent and treat in an established animal model what would otherwise be fatal Nipah virus encephalitis. The in vivo efficacy of cholesterol-tagged peptides, and in particular their ability to penetrate the CNS, suggests that they are promising candidates for the prevention or therapy of infection by Nipah and other lethal paramyxoviruses

A General Strategy to Endow Natural Fusion-protein-Derived Peptides with Potent Antiviral Activity

Fusion between the viral and target cell membranes is an obligatory step for the infectivity of all enveloped virus, and blocking this process is a clinically validated therapeutic strategy

Absence of COVID-19-associated changes in plasma coagulation proteins and pulmonary thrombosis in the ferret model

BACKGROUND: Many patients who are diagnosed with coronavirus disease 2019 (COVID-19) suffer from venous thromboembolic complications despite the use of stringent anticoagulant prophylaxis. Studies on the exact mechanism(s) underlying thrombosis in COVID-19 are limited as animal models commonly used to study venous thrombosis pathophysiology (i.e. rats and mice) are naturally not susceptible to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Ferrets are susceptible to SARS-CoV-2 infection, successfully used to study virus transmission, and have been previously used to study activation of coagulation and thrombosis during influenza virus infection. OBJECTIVES: This study aimed to explore the use of (heat-inactivated) plasma and lung material from SARS-CoV-2-inoculated ferrets studying COVID-19-associated changes in coagulation and thrombosis. MATERIAL AND METHODS: Histology and longitudinal plasma profiling using mass spectrometry-based proteomics approach was performed. RESULTS: Lungs of ferrets inoculated intranasally with SARS-CoV-2 demonstrated alveolar septa that were mildly expanded by macrophages, and diffuse interstitial histiocytic pneumonia. However, no macroscopical or microscopical evidence of vascular thrombosis in the lungs of SARS-CoV-2-inoculated ferrets was found. Longitudinal plasma profiling revealed minor differences in plasma protein profiles in SARS-CoV-2-inoculated ferrets up to 2 weeks post-infection. The majority of plasma coagulation factors were stable and demonstrated a low coefficient of variation. CONCLUSIONS: We conclude that while ferrets are an essential and well-suited animal model to study SARS-CoV-2 transmission, their use to study SARS-CoV-2-related changes relevant to thrombotic disease is limited

Biophysical properties and antiviral activities of measles fusion protein derived peptide conjugated with 25-Hydroxycholesterol

© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).Measles virus (MV) infection is re-emerging, despite the availability of an effective vaccine. The mechanism of MV entry into a target cell relies on coordinated action between the MV hemagglutinin (H) receptor binding protein and the fusion envelope glycoprotein (F) which mediates fusion between the viral and cell membranes. Peptides derived from the C-terminal heptad repeat (HRC) of F can interfere with this process, blocking MV infection. As previously described, biophysical properties of HRC-derived peptides modulate their antiviral potency. In this work, we characterized a MV peptide fusion inhibitor conjugated to 25-hydroxycholesterol (25HC), a cholesterol derivative with intrinsic antiviral activity, and evaluated its interaction with membrane model systems and human blood cells. The peptide (MV–HC) has a 90% inhibitory concentration (IC90) several logs more advantageous than the equivalent peptide bearing a polyethylene glycol (PEG)-cholesterol moiety. In membrane interaction studies, MV–HC shows a preference for pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) monolayers and membranes rich in sphingomyelin, and interacts less with POPC:cholesterol membranes. MV–HC tends to self-aggregate in aqueous solution, in a concentration-dependent manner. Our results suggest that increased membrane interaction dynamicity results from 25HC conjugation, with a concomitant increase in peptide antiviral efficacy.This work was supported by Fundação para a Ciência e Tecnologia—Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES, Portugal) grant PTDC/BBB-BQB/3494/2014, and NIH AI119762, AI121349, and NS091263 to M.P. B.G. also acknowledges FCT-MCTES fellowship SPRH/BD/52431/2013.info:eu-repo/semantics/publishedVersio

Measles virus fusion machinery activated by sialic acid binding globular domain

Paramyxoviruses, including the human pathogen measles virus (MV) and the avian Newcastle disease virus (NDV), enter host cells through fusion of the viral envelope with the target cell membrane. This fusion is driven by the concerted action of two viral envelope glycoproteins: the receptor binding protein and the fusion protein (F). TheMV receptor binding protein (hemagglutinin [H]) attaches to proteinaceous receptors on host cells, while the receptor binding protein ofNDV(hemagglutinin-neuraminidase [HN]) interacts with sialic acid-containing receptors. The receptor-bound HN/H triggers F to undergo conformational changes that render it competent to mediate fusion of the viral and cellular membranes. The mechanism of fusion activation has been proposed to be different for sialic acid-binding viruses and proteinaceous receptor-binding viruses.Wereport that a chimeric protein containing theNDVHNreceptor binding region and theMVHstalk domain can activateMVF to fuse, suggesting that the signal to the stalk of a protein-binding receptor binding molecule can be transmitted from a sialic acid binding domain. By engineering theNDVHNglobular domain to interact with a proteinaceous receptor, the fusion activation signal was preserved. Our findings are consistent with a unified mechanism of fusion activation, at least for the Paramyxovirinae subfamily, in which the receptor binding domains of the receptor binding proteins are interchangeable and the stalk determines the specificity of F activation. © 2013, American Society for Microbiology

Triggering of Human Parainfluenza Virus 3 Fusion Protein (F) by the Hemagglutinin-Neuraminidase (HN) Protein: an HN Mutation Diminishes the Rate of F Activation and Fusion

For human parainfluenza virus type 3 and many other paramyxoviruses, membrane fusion mediated by the fusion protein (F) has a stringent requirement for the presence of the homotypic hemagglutinin-neuraminidase protein (HN). With the goal of gaining further insight into the role of HN in the fusion process, we developed a simple method for quantitative comparison of the ability of wild-type and variant HNs to activate F. In this method, HN/F-coexpressing cells with red blood cells (RBC) bound to them at 4°C are transferred to 22°C, and at different times after transfer 4-guanidino-neu5Ac2en (4-GU-DANA) is added; this inhibitor of the HN-receptor interaction then releases all reversibly bound RBC but not those in which F insertion in the target membrane or fusion has occurred. Thus, the amount of irreversibly bound (nonreleased) RBC provides a measure of F activation, and the use of fluorescently labeled RBC permits microscopic assessment of the extent to which F insertion has progressed to fusion. We studied two neuraminidase-deficient HN variants, C28a, which has two mutations, P111S and D216N, and C28, which possesses the D216N mutation only. C28a but not C28 exhibits a slow fusion phenotype, although determination of the HNs' receptor-binding avidity (with our sensitive method, employing RBC with different degrees of receptor depletion) showed that the receptor-binding avidity of C28a or C28 HN was not lower than that of the wild type. The F activation assay, however, revealed fusion-triggering defects in C28a HN. After 10 and also 20 min at 22°C, irreversible RBC binding was significantly less for cells coexpressing wild-type F with C28a HN than for cells coexpressing wild-type F with wild-type HN. In addition, F insertion progressed to fusion more slowly in the case of C28a HN-expressing cells than of wild-type HN-expressing cells. Identical defects were found for P111S HN, whereas for C28 HN, representing the 216 mutation of C28a, F activation and fusion were as rapid as for wild-type HN. The diminished fusion promotion capacity of C28a HN is therefore attributable to P111S, a mutation in the stalk region of the molecule that causes no decrease in receptor-binding avidity. C28a HN is the first parainfluenza virus variant found so far to be specifically defective in HN′s F-triggering and fusion promotion functions and may contribute to our understanding of transmission of the activating signal from HN to F

A Second Receptor Binding Site on Human Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase Contributes to Activation of the Fusion Mechanism

The hemagglutinin-neuraminidase (HN) protein of paramyxoviruses carries out three discrete activities that each affect the ability of HN to promote viral fusion and entry: receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein. The interrelationship between the receptor binding and fusion-triggering functions of HN has not been clear. For human parainfluenza type 3 (HPIV3), one bifunctional site on HN can carry out both receptor binding and neuraminidase activities, and this site's receptor binding can be inhibited by the small receptor analog zanamivir. We now report experimental evidence, complemented by computational data, for a second receptor binding site near the HPIV3 HN dimer interface. This second binding site can mediate receptor binding even in the presence of zanamivir, and it differs from the second receptor binding site of the paramyxovirus Newcastle disease virus in its function and its relationship to the primary binding site. This second binding site of HPIV3 HN is involved in triggering F. We suggest that the two receptor binding sites on HPIV3 HN each contribute in distinct ways to virus-cell interaction; one is the multifunctional site that contains both binding and neuraminidase activities, and the other contains binding activity and also is involved in fusion promotion