VP40 is a dimer; the dimer structure is critical for trafficking and matrix assembly

Proteins, particularly viral proteins, can be multifunctional, but the mechanisms behind multifunctionality are not fully understood. Here, we illustrate through multiple crystal structures, biochemistry, and cellular microscopy that VP40 rearranges into different structures, each with a distinct function required for the ebolavirus life cycle. A butterfly-shaped VP40 dimer traffics to the cellular membrane. Once there, electrostatic interactions trigger rearrangement of the polypeptide into a linear hexamer. These hexamers construct a multilayered, filamentous matrix structure that is critical for budding and resembles tomograms of authentic virions. A third structure of VP40, formed by a different rearrangement, is not involved in virus assembly but instead uniquely binds RNA to regulate viral transcription inside infected cells. These results provide a functional model for ebolavirus matrix assembly and the other roles of VP40 in the virus life cycle and demonstrate how a single wild-type, unmodified polypeptide can assemble into different structures for different functions.

Introduction

Gomis-Rüth et al., 2003 Gomis-Rüth F.X.

Dessen A.

Timmins J.

Bracher A.

Kolesnikowa L.

Becker S.

Klenk H.D.

Weissenhorn W. The matrix protein VP40 from Ebola virus octamerizes into pore-like structures with specific RNA binding properties. Gomis-Rüth et al., 2003 Gomis-Rüth F.X.

Dessen A.

Timmins J.

Bracher A.

Kolesnikowa L.

Becker S.

Klenk H.D.

Weissenhorn W. The matrix protein VP40 from Ebola virus octamerizes into pore-like structures with specific RNA binding properties. Hoenen et al., 2010a Hoenen T.

Biedenkopf N.

Zielecki F.

Jung S.

Groseth A.

Feldmann H.

Becker S. Oligomerization of Ebola virus VP40 is essential for particle morphogenesis and regulation of viral transcription. Currently, the VP40 ring and the interfaces by which it assembles provide the only known structural model of how VP40 might assemble the ebolavirus matrix. However, these rings are observed only in infected cells, not in mature purified ebolaviruses (), suggesting the ring structure is not involved in matrix assembly. Instead, the RNA-binding VP40 rings likely play a critical, but currently undefined role inside the infected cell ().

Hoenen et al., 2005 Hoenen T.

Volchkov V.

Kolesnikova L.

Mittler E.

Timmins J.

Ottmann M.

Reynard O.

Becker S.

Weissenhorn W. VP40 octamers are essential for Ebola virus replication. Despite the wealth of structural and biochemical data, no cohesive model exists for how the ebolavirus matrix is assembled. It is currently unclear which structure of VP40 migrates to the cell membrane and how that structure is triggered to further oligomerize into the viral matrix. Although RNA binding by VP40 is critical to the viral life cycle (), the exact role of the VP40 RNA-binding ring has remained elusive. In addition and perhaps most significantly, the structural arrangement of VP40 within the ebolavirus matrix remains unknown. Here, we set out to provide a structurally and biologically supported model of VP40-driven matrix assembly and elucidate the different roles played by VP40 in the ebolavirus life cycle.

We first purified VP40 and determined that it is a dimer in solution, not a monomer. We then determined multiple crystal structures of dimeric VP40, from Ebola virus and Sudan virus, revealing a conserved dimeric interface. Targeted mutagenesis of the dimer interface abolishes trafficking of VP40 to the cell membrane and budding of VLPs, suggesting that the VP40 dimer is a critical precursor of the viral matrix. We next analyzed a conserved CTD-to-CTD interface by which VP40 dimers are linked together in crystals. Structure-based mutagenesis of the CTD-to-CTD interface abolishes assembly and budding of virus-like particles, but not trafficking to or interactions with the membrane. Thus, assembly of VP40 dimers via CTD interactions likely represents a critical step in matrix assembly.

Adu-Gyamfi et al., 2012 Adu-Gyamfi E.

Digman M.A.

Gratton E.

Stahelin R.V. Investigation of Ebola VP40 assembly and oligomerization in live cells using number and brightness analysis. Ruigrok et al., 2000 Ruigrok R.W.

Schoehn G.

Dessen A.

Forest E.

Volchkov V.

Dolnik O.

Klenk H.D.

Weissenhorn W. Structural characterization and membrane binding properties of the matrix protein VP40 of Ebola virus. Scianimanico et al., 2000 Scianimanico S.

Schoehn G.

Timmins J.

Ruigrok R.H.W.

Klenk H.D.

Weissenhorn W. Membrane association induces a conformational change in the Ebola virus matrix protein. Beniac et al., 2012 Beniac D.R.

Melito P.L.

Devarennes S.L.

Hiebert S.L.

Rabb M.J.

Lamboo L.L.

Jones S.M.

Booth T.F. The organisation of Ebola virus reveals a capacity for extensive, modular polyploidy. Bharat et al., 2011 Bharat T.A.

Riches J.D.

Kolesnikova L.

Welsch S.

Krähling V.

Davey N.

Parsy M.L.

Becker S.

Briggs J.A. Cryo-electron tomography of Marburg virus particles and their morphogenesis within infected cells. We next turned our attention to understanding how the VP40 dimer interacts with the membrane and conformational changes believed to occur at the membrane and be important to matrix assembly. Previous work has demonstrated that electrostatic interactions between the VP40 CTD and the membrane are likely important to matrix assembly (). Through targeted mutagenesis, we identified a conserved basic patch in the CTD that mediates both membrane interaction and subsequent matrix assembly events. An electrostatic mimic of the membrane was found to trigger conformational rearrangement of VP40 dimers into a linear hexameric assembly. In the crystal structure of this assembly, VP40 hexamers also connect into continuous filaments via the conserved CTD interactions. We propose that the VP40 hexamer structure represents a building block of the flexible, filamentous ebolavirus matrix. Our hexameric VP40 structure suggests that VP40 assembles into a multilayered matrix along the membrane, which is consistent with recent electron tomographic analysis of filoviruses in scale, dimension, and layered construction ().

We also determined that the VP40 RNA-binding ring structure, although derived from the VP40 dimer, is not involved in or required for matrix assembly and budding. Instead, we show that the VP40 ring plays a distinct and critical role in regulation of viral transcription inside infected cells and that this function is dependent on its unique RNA-binding capability.

Taken together, our results illustrate that the highly plastic VP40 polypeptide is able to rearrange itself into distinct structural assemblies. Each of these distinct structures is assembled by the unmodified wild-type polypeptide, and each structure is required for a separate and essential function in the virus life cycle: a dimeric precursor critical for cellular trafficking, a hexameric structural component for the viral matrix assembly, and a nonstructural RNA-binding ring structure essential for regulating viral transcription. Thus, the physical plasticity inherent in VP40 demonstrates how a structural rearrangement can expand the functional repertoire of a single viral gene.