The Plasma Membrane Induces the Membrane Penetration of the Ebola Virus Matrix Protein: A Key Step in VP40 Oligomerization and Viral Egress (2013)

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 8, pp. 5779 –5789, February 22, 2013 © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. The Ebola Virus Matrix Protein Penetrates into the Plasma Membrane A KEY STEP IN VIRAL PROTEIN 40 (VP40) OLIGOMERIZATION AND VIRAL EGRESS * Received for publication, December 11, 2012, and in revised form, January 3, 2013 Published, JBC Papers in Press, January 6, 2013, DOI 10.1074/jbc.M112.443960 Emmanuel Adu-Gyamfi ‡1 , Smita P. Soni § , Yi Xue § , Michelle A. Digman ¶2 , Enrico Gratton ¶2 , and Robert V. Stahelin ‡§¶3 From the § Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, South Bend, Indiana 46617, the ‡ Department of Chemistry and Biochemistry, the Eck Institute for Global Health, and the Center for Rare and Neglected Diseases, University of Notre Dame, South Bend, Indiana 46556, and the ¶ Department of Biomedical Engineering, University of California, Irvine, California 92697 Ebola, a fatal virus in humans and non-human primates, has no Food and Drug Administration-approved vaccines or thera- peutics. The virus from the Filoviridae family causes hemor- rhagic fever, which rapidly progresses and in some cases has a fatality rate near 90%. The Ebola genome encodes seven genes, the most abundantly expressed of which is viral protein 40 (VP40), the major Ebola matrix protein that regulates assembly and egress of the virus. It is well established that VP40 assembles on the inner leaflet of the plasma membrane; however, the mechanistic details of plasma membrane association by VP40 are not well understood. In this study, we used an array of bio- physical experiments and cellular assays along with mutagenesis of VP40 to investigate the role of membrane penetration in VP40 assembly and egress. Here we demonstrate that VP40 is able to penetrate specifically into the plasma membrane through an interface enriched in hydrophobic residues in its C-terminal domain. Mutagenesis of this hydrophobic region consisting of Leu 213 , Ile 293 , Leu 295 , and Val 298 demonstrated that membrane penetration is critical to plasma membrane localization, VP40 oligomerization, and viral particle egress. Taken together, VP40 membrane penetration is an important step in the plasma membrane localization of the matrix protein where oligomerization and budding are defective in the absence of key hydrophobic interactions with the membrane. * This work was supported, in whole or in part, by National Institutes of Health Grants AI081077 (to R. V. S.) and P41-RRO3155 and P50-GM076516 (to E. G. and M. A. D.). This work was also supported by the Notre Dame Center for Rare and Neglected Diseases, the Indiana University School of Medicine- South Bend Imaging and Flow Cytometry Core Facility, and the Notre Dame Integrated Imaging Facility (to R. V. S.). Supported by a fellowship from the Eck Institute for Global Health. Supported by Keck Foundation Grant 44769549507. To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Indiana University School of Medicine, 143 Raclin-Car- michael Hall, 1234 Notre Dame Ave., South Bend, IN 46617. Tel.: 574-631- 5054; Fax: 574-631-7821; E-mail: rstaheli@iupui.edu. FEBRUARY 22, 2013 • VOLUME 288 • NUMBER 8 Viral hemorrhagic fevers such as that caused by the Ebola virus present a serious health threat in central and eastern Africa with fatality rates as high as 90% (1, 2). The Ebola virus from the Filoviridae family uses a negative sense RNA genome encoding seven proteins to replicate in the host cell (3, 4). The nucleoprotein, VP24, 4 VP30, VP35, and L protein constitute the nucleocapsid, which is critical for transcription and viral replication (4). The glycoprotein is rooted in the lipid envelope of the virus and is responsible for entry of virions (5) in an NPC1-dependent manner (6). VP40 and the enigmatic VP24 are the viral matrix proteins and are important for budding as well as virus structure and stability (7–10). VP40 is the major matrix protein, is the most abundantly expressed protein of the virus, and plays a central role in the budding of Ebola from the plasma membrane (PM). For example, expression of VP40 alone in mammalian cells is sufficient to form virus-like parti- cles (VLPs) that have similar characteristics to the actual Ebola virus (11, 12). Additionally, studies have shown that in the absence of VP40 the nucleocapsid is not effectively transported to the PM, the site of assembly and budding, where it is incor- porated into the virions (13). Therefore, understanding how VP40 regulates assembly of VLPs both in vitro and in live cells is crucial for understanding the viral life cycle and could have a significant impact in identifying potential therapeutic targets. The assembly of VLPs by Ebola VP40 also represents an attrac- tive model for studying the assembly of the virus in a biosafety level 2 setting because the VLPs are noninfectious. VP40 associates with the PM (14) where it initiates assembly, oligomerization (15, 16), and recruitment of the nucleoprotein. In addition to membrane association, VP40 has been shown to The abbreviations used are: VP, viral protein; BCA, bicinchoninic acid; NM, nuclear membrane; PC, phosphatidylcholine; POPC, 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine; POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine; POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-phos- phatidylserine; PM, plasma membrane; PS, phosphatidylserine; VLP, virus- like particle; TIRF, total internal reflection; NB EGFP, enhanced GFP; mN, millinewtons. JOURNAL OF BIOLOGICAL CHEMISTRY Downloaded from http://www.jbc.org/ at Univ of California - Irvine on August 25, 2016 Background: The Ebola virus matrix protein (VP40) regulates the plasma membrane assembly and egress of the Ebola virus. Results: The plasma membrane induces membrane penetration of the VP40 C-terminal domain. Conclusion: Membrane penetration by VP40 is important for VP40 cellular localization, oligomerization, and viral budding. Significance: A better understanding of VP40-membrane interactions will help us to understand Ebola virus assembly and budding.