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Matrix Protein VP40

Sarah Cleeton '09 and Rebecca Cleeton '09


I. Introduction

The matrix protein VP40 is an Ebola virus membrane-associated protein that is thought to be necessary for the assembly and budding of new virus particles. The Ebola virus is responsible for a mostly lethal hemorrhagic fever in humans characterized by bleeding and coagulation abnormalities. Outbreaks of this disease occur occasionally in parts of central Africa. The Ebola virus is a filovirus, negative-stranded nonsegmented RNA viruses that enter cells using their surface glycoproteins in an infectious process of receptor-mediated endocytosis. So far, only two members of this virus family have been identified: the Marburg and Ebola viruses. While the structure of the Marburg virus is almost identical to that of the Ebola virus, it triggers different antibodies in infected organisms. (Scianimanico, 2000). While several regions of VP40 from the Ebola virus and the Marburg virus are highly conserved, a sequence alignment of both proteins shows only a 29% similarity. Both the Marburg and Ebola viruses are thought to have been transmitted to humans via other animals like bats, for example, who can replicate filoviriade-like viruses. While transmission of these diseases from person to person occurs through contact with bodily fluids, the path of transmission from other organisms to humans is not yet clear. (Dessen, 2000).

VP40 is the most abundant protein in virus particle, and it is located directly under the viral lipid bilayer to ensure structural support. The Ebola virus replicates in the cytoplasm, and the assembly of new virus particles and budding occurs at the plasma membrane. During the process of budding, VP40 associates with cellular membranes and they are brought together through interactions with the C-terminal ends of viral glycoproteins.(Dessen, 2000). The matrix protein VP40 forms an octameric disc-shaped ring structure in solution. It is composed of 4 antiparallel homodimers, and each subunit binds an RNA tribonucleotide with the sequence UGA at the inner pore surface. This selective RNA-protein interaction helps to stabilize the molecule’s ring-like structure. (Gomis-Ruth, 2003).  

II. General Structure

In solution the matrix protein VP40 forms an octamer resembling a ring, with a central pore. The biological multimer was created from the monomer form with the help of the PQS website. The ring-like structure is 80 Å across, 42 Å wide and is composed of 8 monomers or 4 dimers which each have antiparallel β-sandwich domains. A β-sandwich is a tertiary structure formed by two antiparallel β-sheets stacked on top of each other. (Gomis-Ruth, 2003). Protein folds, which consist mainly of β-sheets, often exhibit an antiparallel arrangement. Each elongated monomer is composed of 140 amino acids, has a molecular weight of 15,323, and has the dimensions of 40x50x25 Å. (Dessen, 2000).

In the VP40 molecule, each domain consists of 6 antiparallel β-strands, forming 2 β-sheets and 3 α-helices that pack against the sheets laterally. See secondary structure. In each monomer, the C-terminal end crosses over and makes contact with the neighboring monomer. This arrangement positions the C-terminal end on the outside of the ring above the β-sandwich. In this dimer the N-terminal domains are also positioned pointing outside the ring, and they are close to the C-terminal ends. Since both the N and C-terminal domains fold into similar structures, it is possible that they may have evolved via gene duplication. (Dessen, 2000). At the dimer-dimer interface , each monomer binds a short strand of RNA. The RNA is a tribonucleotide with the sequence UGA, and it forms a ring at the inner pore surface. (Gomis-Ruth, 2003).  

III. Subunit Interactions

Monomer-monomer interactions: Polar interactions between Trp95 and Gln184 and salt bridges formed between Glu160, Arg148, and Arg151 assist in stabilization of the dimer. The hydrophobic core interactions between Trp95, Pro97, Phe161, Ile74, and Ile182 further stabilize the dimer. Residues 189 – 194 interact on the outside of the ring in an extended conformation with the neighboring molecule.

Dimer-dimer interactions: Hydrogen bonding occurs both between the carbonyl of Tyr171 and the amide of Gly141, and the oxygen of Thr173 and the amide of Gly139. A weak hydrophobic core on both ends of the β-sandwich structures are created when the neighboring monomers interact. Also a selective binding pocket for a particular tribonucleotide sequence is formed at the dimer-dimer interface (Gomis-Ruth, 2003).

IV. Protein-RNA Interactions

While the crystal structure contains short RNA segments, in vivo the strucutre is believed to include part of the Ebola genome wound inside the protein. The dimer-dimer interface is stabilized by interactions with viral ssRNA inside of the central cavity of the ring. Both termini of the RNA oligonucleotide point to the interior of the pore. The RNA is composed of an adenosine phosphate (Ade3R) at the 3’ end and a guanosine phosphate (Gua2R) followed by a uridine residue (Uri1R) at the 5’-phosphate-depleted end. Here, the base moiety is positioned in syn conformation by internal hydrogen bonding. Strong π interactions result from the parallel positioning of the aromatic base ring to Uri1R. Gua2R is attached to Uri1R by a phosphate-linker. This base is located in a pocket at the inner pore surface and at a protein subunit interface. After Gua2R the sugar-phosphate backbone folds to interact with Ade3R perpendicularly. This adenine interacts in an anti conformation with the preceding phosphate group. C3’-endo sugar puckering enables interaction with the preceding sugar residues. The phosphate backbone of Gua2R is linked to Arg134 by a double-headed salt bridge while its sugar interacts with the backbone of the protein.

A central guanosine phosphate is mostly responsible for the overall binding specificity of the RNA. This guanine base interacts with Arg134 and Phe125 and is located in a deep pocket at the interface of two protein subunits. The interaction with these two amino acids is characterized by parallel ring stacking between aromatic plants of one of the protein chains. Not many contacts exist between the protein and the flanking residues of the oligonucleotide. Nucleoside Uri7R, for example, is not involved in any polar protein interactions. The aromatic base, however, establishes hydrophobic forces with the side chains of Leu158 and Leu132. 3’-terminal Ade3R sits in a shallow cavity formed by the side chains of Ile152, Tyr171, and Asn154 and by Gly126 and His123 from the protein main chain. The base of Ade3R forms a hydrogen bond with Asn154 and its sugar forms a second hydrogen bond with the protein as well. See stereo view of RNA-protein interactions.

RNA-protein interactions are both sequence and RNA specific. Each polar residue involved in these specific interactions is conserved between Marburg and Ebola VP40 sequences. Van der Waals interactions, however, are in some cases different. (Gomis-Ruth, 2003).  

V. Medical Implications

The filovirus Ebola causes hemorrhagic fever in humans with high mortality rates. While natural outbreaks of this disease in central Africa are rare, they have risen in recent years. In fact, there is currently an Ebola outbreak in Uganda. Because this disease is so deadly (up to 90% mortality rates in some epidemics), it is feared that the Ebola virus could be used as an agent of bioterrorism. (Panchal, 2003). For these reasons, filoviruses are a serious threat to global public health and steps should be taken to better understand the mechanisms by which they replicate and spread.

While there is a vaccine that is 100% effective in preventing Ebola in monkeys, an effective vaccine has yet to be developed for humans. Since the matrix protein VP40 is essential for the assembly and budding of new virus particles, one would assume that if a drug could inactivate or inhibit production of this protein then perhaps new viruses would be unable to form, and that is why understanding the structure of VP40 may prove to be key in the search for a cure for Ebola.

VI. References

Dessen, A., O. Dolnik, H. Klenk, V. Volchkov, W. Weissenhorn. 2000. Crystal structure of the matrix protein VP40 from Ebola virus. The EMBO Journal 19: 4228-4236.

Gomis-Ruth, X. F., S. Becker, A. Bracher, A. Dessen, H. Klenk, L. Kolesnikowa, J. Timmins, W. Weissenhorn. 2003. The matrix proteinVP40 from Ebola virus octamerizes into pore-like structures with specific RNA binding properties. Structure 11: 423-433.

Panchal, R. G., M. J. Aman, S. Badie, S. Bavari, G. H. Kallstrom, T. A. Kenny, D. Lane, L. Li, G. Ruthel. 2003. In vivo oligomerization and raft localization of Ebola virus protein VP40 during vesicular budding. Proclaimed National Academy of Sciences 100: 15936-15941.

Scianimanico, S., H. Klenk, R. H. W. Ruigrok, G. Schoehn, J. Timmins, W. Weissenhorn. 2000. Membrane association induces a conformational change in the Ebola virus matrix protein. The EMBO Journal 19: 6732-6741.

Timmins, J., R. W. H. Ruigrok, W. Weissenhorn. 2004. Structural studies on the Ebola virus matrix protein VP40 indicate that matrix proteins of enveloped RNA viruses are analogues but not homologues. FEMS Microbiology Letters 233: 179-186.

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