PARN Activity Assay and Kinetic Analysis
The enzymatic activity was determined by the spectrophotometric methylene blue assay as described before [70]. Deadenylation rates as a function of time were determined with time-course assays. The reactions were performed using 0.01?.02 mM recombinant PARN and the substrate concentration [poly (A)] varied from 0.1 to 0.6 mM [16]. DNP-poly(A) concentrations varied from 0.082 to 3 mM.calculated values that were used in our statistical analysis. Indicated with red colour are the inhibitors most highly correlated with Ki based on R2 and C coefficients. (DOCX)
Table S3 Drug likeness properties of our previously reported nucleoside analog inhibitors of PARN, including the consensus score of drug likeness, a toxicity measure and an ease-of-synthesis approximation. (DOCX)
Release of Dengue Virus Genome Induced by a Peptide Inhibitor
Shee-Mei Lok1,6., Joshua M. Costin2., Yancey M. Hrobowski2,3�a, Andrew R. Hoffmann4, Dawne K. Rowe2, Petra Kukkaro6, Heather Holdaway1�b, Paul Chipman1�c, Krystal A. Fontaine2�d, Michael R. Holbrook5�e, Robert F. Garry3, Victor Kostyuchenko6, William C. Wimley4, Sharon Isern2, Michael G. Rossmann1, Scott F. Michael2*
1 Department of Biological Sciences, Purdue University, West Lafayette, Indiana, United States of America, 2 Department of Biological Sciences, Florida Gulf Coast University, Fort Myers, Florida, United States of America, 3 Department of Microbiology and Immunology and Graduate Program in Cellular and Molecular Biology, Tulane University Health Sciences Center, New Orleans, Louisiana, United States of America, 4 Department of Biochemistry, Tulane University Health Sciences Center, New Orleans, Louisiana, United States of America, 5 Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America, 6 Emerging Infectious Diseases, DukeUS, Department of Biological Sciences, National University of Singapore, Singapore, Singapore
Abstract
Dengue virus infects approximately 100 million people annually, but there is no available therapeutic treatment. The mimetic peptide, DN59, consists of residues corresponding to the membrane interacting, amphipathic stem region of the dengue virus envelope (E) glycoprotein. This peptide is inhibitory to all four serotypes of dengue virus, as well as other flaviviruses. Cryo-electron microscopy image reconstruction of dengue virus particles incubated with DN59 showed that the virus particles were largely empty, concurrent with the formation of holes at the five-fold vertices. The release of RNA from the viral particle following incubation with DN59 was confirmed by increased sensitivity of the RNA genome to exogenous RNase and separation of the genome from the E protein in a tartrate density gradient. DN59 interacted strongly with synthetic lipid vesicles and caused membrane disruptions, but was found to be non-toxic to mammalian and insect cells. Thus DN59 inhibits flavivirus infectivity by interacting directly with virus particles resulting in release of the genomic RNA.
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. 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 membrane anchor 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].