Matrix
Protein VP40
Sarah Cleeton '09 and Rebecca Cleeton '09
Contents:
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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