Synthetic Protocells Interact with Viral Nanomachinery and Inactivate Pathogenic Human Virus
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Synthetic Protocells Interact with Viral Nanomachinery and Inactivate Pathogenic Human Virus Matteo Porotto1., Feng Yi2., Anne Moscona1*, David A. LaVan2* 1 Departments of Pediatrics and of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, United States of America, 2 Material Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland, United States of America Abstract 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. Citation: Porotto M, Yi F, Moscona A, LaVan DA (2011) Synthetic Protocells Interact with Viral Nanomachinery and Inactivate Pathogenic Human Virus. PLoS ONE 6(3): e16874. doi:10.1371/journal.pone.0016874 Editor: Meni Wanunu, University of Pennsylvania, United States of America Received September 29, 2010; Accepted January 16, 2011; Published March 1, 2011 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Funding: This work was supported by Public Health Service grants AI076335 from National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (NIAID) to AM, EB011707 from the National Institute of Biomedical Imaging and Bioengineering to MP, NIH (NIAID) Northeast Center of Excellence for Biodefense, subcontracts to AM and to DL from the National Center for Design of Biomimetic Nanoconductors, funded by Grant Number PHS 2 PN2 EY016570B from the National Institutes of Health through the NIH Roadmap for Medical Research (PI: E. Jakobsson), Emerging Infectious Disease Research U54AI057158 grants to AM and MP (PI of Center of Excellence grant: W.I. Lipkin), by March of Dimes Research Grant 06-322 to AM, and by a Grand Challenges Exploration grant from the Bill and Melinda Gates Foundation to AM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have read the journal’s policy and have the following conflicts: AM serves on advisory boards or as a consultant for Medimmune, GlaxoSmithKline, and Roche, and has received minor funding in partial support of a project from NexBio. MP has received minor funding in partial support of a project from NexBio. DL and FY have no competing interests to report. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: [email protected] (AM); [email protected] (DL) . These authors contributed equally to this work. Introduction pathogens in the paramyxovirus family include parainfluenza viruses, respiratory syncytial virus (RSV), mumps and measles, all Nipah (NiV) and Hendra (HeV) viruses are emerging zoonotic causes of significant human diseases affecting millions of children paramyxoviruses of the henipavirus family that cause fatal yearly. encephalitis in humans, with fatality rates of up to 75% [1–3]. The first steps in infection with paramyxoviruses are binding to Emerging zoonotic viruses are recently identified agents that can the target cells, via interaction of the viral receptor-binding be transmitted from animals to humans and person-to-person; molecule (G for HeV or NiV) with the specific receptor molecules they often spread widely through animal hosts before infecting on the cell surface –EFNB2 (Ephrin-B2) for HeV, and both humans. Paramyxoviruses are single stranded RNA viruses that EFNB2 and EFNB3 for NiV, followed by the fusion of the viral include a number of human pathogens. Henipaviruses share envelope with the plasma membrane of the cell, a process common structures such as a lipid envelope, receptor binding mediated by the viral fusion protein (F). The receptor binding proteins (referred to as ‘‘G’’) and fusion proteins (referred to as protein G, when engaged with receptor, ‘‘triggers’’ F to assume its ‘‘F’’). Direct transmission of NiV from its fruit bat (flying fox) fusion-ready conformation with its fusion domain exposed [8]. mammalian reservoir to humans, and person-to-person transmis- While F-activation is key for entry, the correct timing is critical. sion, allows infection to bypass the pig host which served to We suggest[9] that this timing of activation represents a target for transmit the virus as it emerged into the human population[4]. intervention with infection, and that with the correct receptor HeV, also via a bat host, has caused disease in horses with mimic presented in the proper biological context, it is possible to transmission to horse-handlers and veterinarians, and since 1995 induce triggering and to inactivate F, rendering the virus has caused sporadic illness in Australia [5]. NiV, in addition to noninfectious. acute infection, may cause asymptomatic infection in up to 60% of Artificial cell-like particles, or protocells, bearing receptor exposed people, and may lead to late-onset disease or relapse of mimics to attract and inactivate henipavirus envelope glycoprotein encephalitis years after initial infection [6]. The vast geographic pseudotypes offer a new approach to preventing infection. To range of the fruit bat mammalian reservoir raises the possibility of assess this strategy, we used quantitative assays for inhibitors of wide spread of these human diseases, which presently have no henipavirus infection that are based on envelope glycoprotein approved clinical treatment or vaccine [7]. Other related human pseudotypes [10]. The pseudotypes bear the henipavirus entry PLoS ONE | www.plosone.org 1 March 2011 | Volume 6 | Issue 3 | e16874 Synthetic Protocells Inactivate Pathogenic Virus molecules on a BSL2 agent (vesicular stomatitis virus - VSV) and Protocells inactivate pseudotyped viruses use the identical entry mechanism as live henipaviruses[10]. Protocells were incubated with pseudotyped viruses for one Agents found to be inhibitory in this assay may either be those that hour at 37uC and then the viral titer (as determined by viral entry block binding, or interfere with processing of F, or interfere with F on cultured human cells) was assessed and compared to the titer of activation and fusion. This strategy was used here to assess the untreated pseudotyped viruses. Individual protocells contained inhibitory potential of this novel approach to anti-infectives: to either EFNB1, EFNB2 or mixtures of the two; EFNB2 presenting determine whether synthetic protocells that bear the entry protocells effectively inhibited entry by HeV pseudotyped virions, receptor for henipaviruses inactivate pseudotyped viruses. causing a dramatic decrease in viral titer after incubation with the protocells. The inhibitory effect of the protocells is specific for Development of artificial cells presenting membrane henipaviruses. Incubation with paramyxovirus human parainflu- bound receptors (‘‘protocells’’) enza virus type 3, and with the rhabdovirus VSV, each of which Protocells have great potential to fight human disease [11] and uses a different entry receptor, did not result in a significant serve as a platform to apply nanobiotechnology to treat disease decrease in titer. In these experiments, pseudovirus infection was [12,13]. The protocells described here allow for biomimetic reduced 100% using the EFNB2 bearing protocells but VSV presentation of surface receptors in lipid bilayers that are infection was reduced 11% when exposed to EFNB2 protocells supported on highly hydrophilic nanoporous silica particles. The (relative standard deviation is 13% for these measurements), construction of the protocells bearing henipavirus entry receptors confirming the specificity of inhibition. is diagrammed in figure 1. Test protocells were prepared (shown in The inhibitory activity of EFNB2-bearing protocells is temper- figure 1a) using 3 mm diameter nanoporous SiO2 particles ature dependent; infection of cultured cells is specifically inhibited following the methods of Brinker et al [14,15]. Previous work after incubation at 37uC but not at 4uC (figure 2). In these has shown that lipid bilayers can be conveniently assembled over experiments, pseudotyped viruses (hereafter called pseudovirus) nanoporous silica surfaces and support the lipid bilayer in a fluid were incubated with EFNB2 or EFNB1-bearing protocells at 4uC state analogous to the lipid bilayer supported on the actin or 37uC, and then introduced to a culture with mammalian cells. cytoskeleton; nanoporous silica particles were selected to form the In parallel, pseudoviruses were incubated with EFNB2-bearing cell-like particles [16,17] (Movie S1 shows confocal results of cell- sepharose beads at 4uCor37uC.