Citation: Lok S-M, Costin JM, Hrobowski YM, Hoffmann AR, Rowe DK, et al. (2012) Release of Dengue Virus Genome Induced by a Peptide Inhibitor. Editor: Young-Min Lee, Utah State University, United States of America Received January 29, 2012; Accepted October 30, 2012; Published November 30, 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: The work was supported by Defense Threat Reduction Agency awards HDTRA1-08-1-0003, HDTRA1-09-1-0004, and HDTRA1-10-1-0009 to SI and SFM, by National Institutes of Health Grants R01 AI76331 to MGR, AI64617 to RFG and SFM, GM60000 to WCW, and by NRF fellowship award R-913301-015-281 to SML. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Tulane University has applied for patents covering the peptide described in this work with RFG and SFM as inventors (7,416,733 issued 8/ 26/2008, 7,854,937 issued 12/21/2010, application 20110130328 filed 8/22/2008). This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: [email protected] �a Current address: Operations and Tactics Division, Center for Naval Analyses, Alexandria, Virginia, United States of America �b Current address: Cleveland Center for Membrane and Structural Biology, Case Western Reserve University, Cleveland, Ohio, United States of America �c Current address: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida, United States of America �d Current address: Department of Microbiology, University of Washington, Seattle, Washington, United States of America �e Current address: NIAID Integrated Research Facility, Ft. Detrick, Frederick, Maryland, United States of America . These authors contributed equally to this work.
Introduction
The four dengue virus serotypes, dengue virus types 1, 2, 3 and 4, are major mosquito-transmitted, human pathogens. Currently there are no available vaccines or therapeutics. Dengue is a positive-sense RNA virus, encapsulated by a lipid membrane [1,2]. The surface of the mature virus particle is composed of 180 envelope (E) glycoprotein molecules and an equal number of membrane (M) protein molecules that assemble at endoplasmic reticulum-derived membranes [1,2]. The ectodomains of the E glycoproteins are arranged in a herringbone pattern on the surface of the lipid membrane that facilitates binding of the virus to host cells [3] and fusion of the virus with the host membrane after receptor-mediated endocytosis [4,5,6]. Each E monomer consists of three domains: DI, DII and DIII [7,8,9,10]. The C-terminal portion of the E protein consists of the stem and membraneanchor regions. The stem region is highly conserved among flaviviruses and is folded into amphipathic helices H1 and H2 that lie underneath the E ectodomain, partially embedded in the lipid envelope (Figure 1A, B) [2]. Ligands that mimic the structure of viral envelope components can sometimes interfere with the normal infection process and, thus, have potential as antiviral agents. For example, the T20 peptide, which is approved for treatment of HIV [11], has a sequence that mimics part of the C-terminal region of the HIV gp41 glycoprotein, and inhibits fusion with host cells [12]. Similarly, DIII of dengue virus E can prevent fusion of virions to host cells [13]. Furthermore, peptides that mimic other regions of E have also been shown to inhibit infection [14,15]. Some of these peptides bind to E and appear to cause changes in the organization of the glycoproteins on the viral surface [15]. Figure 1. The DN59 peptide inhibits dengue virus infectivity. (A) Sequence comparison of the DN59 amino acid sequence, representing the dengue virus 2 E stem region (residues 412?44), with the stem region of other flaviviruses. YFV – yellow fever virus, RSSEV – Russian spring-summer encephalitis virus, CEEV – Central European encephalitis virus. Non-identical residues are colored in grey. The % amino acid divergence from dengue 2 ?and IC50 values against other flaviviruses are also shown. (B) The C-a backbone of the E protein of dengue 2 as fitted into the 9A resolution cryoEM map of the mature virus [2]. The region mimicked by DN59 is shown in black outline. Grey bars indicate the lipid bilayer membrane. Part of the stem region helix 2 (H2) interacts with the outer lipid layer of the membrane. (C) FFU reduction assay showing dose response inhibition of infection of dengue virus serotypes 1-4, in mammalian epithelial cells. (D) FFU reduction assay showing dose response inhibition of infection of dengue virus 2 in mosquito cells.the E protein stem region causes the release of the genome from the virus particle.
Results and Discussion
A 33 amino acid peptide, known as DN59, mimics the dengue virus type 2 E stem region (residues 412 to 444). This peptide was previously shown to inhibit the infectivity of dengue 2 virus and West Nile virus, but activity against other flaviviruses and the mechanism of action were unknown [14]. In Figure 1C, we now show that at concentrations of 2-5 mM, the DN59 peptide reduced the infectivity of all four dengue virus serotypes by 50% (IC50) in a FFU infection assay using mammalian epithelial cells. The infectivity of other flaviviruses (yellow fever virus, Central European encephalitis virus, and Russian spring-summer encephalitis virus) was inhibited at higher DN59 concentrations (Figure S1A). Cryo-electron (cryoEM) microscopy of dengue virus type 2 particles incubated at 37uC for 30 minutes with 100 mM DN59 in 1% (v/v) DMSO in a 5:1 molar ratio of peptide to E protein on the virus had lost most of their RNA genomes whereas control virus particles in the presence of 1% (v/v) DMSO showed novisible loss of RNA genome (Figure 2A). Additional images showing larger numbers of control and treated particles are shown in Figure S2. The release of RNA presumably accounted for an increase of viscosity of the virus solution as well as a rather electron dense background on the cryoEM micrographs. Although treatment with peptide may disrupt the symmetry of the virus particle, a three-dimensional icosahedral reconstruction of a small number of particles supported the absence of RNA and suggested the formation of holes at the five-fold vertices through which the RNA might exit (Figure 2B and Figure S3). The release of viral RNA from the particles was consistent with the results of a genome sensitivity assay conducted by exposing peptide-treated virus particles to RNase digestion, followed by quantitative reverse transcription PCR to determine the amount of protected viral RNA. The RNA genomes of untreated particles were protected from RNase digestion, whereas the genomes of particles co-incubated with increasing concentrations of DN59 were susceptible to digestion in a doseresponsive manner (Figure 2C, D).
