1 within a species (mutations frequent lead to

1  
}          State of the Art

 

 

1.1.
Fusion Peptides
(FPs)

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Viruses are infectious agents that replicates only
within the cells of living hosts, mainly bacteria, plants, and animals. They’re
usually composed of an RNA or DNA core, a nuclear membrane, and, in more
complex types, a surrounding envelope (plasma membrane). Enveloped viruses
(e.g. influenza, HIV, Dengue) have viral envelopes covering their genetic
material and typically derive from portions of the host cell membranes
(phospholipids and proteins), including some viral glycoproteins. Glycoproteins
on the surface of the envelope help identifying and binding to receptor sites
on the host’s membrane, inducing membrane fusion, allowing the viral genome to
enter and infect the host. This process is collectively known as “viral entry”1, 2 and although it seems like it, this mechanism is not simple and
there are significant differences among different viruses2.Despite glycoproteins induce membrane fusion, researchers believe
that it’s a small portion that triggers the whole mechanism – the fusion peptide.
This peptide segment has membrane -perturbing activity and cause fusion
proteins’ irreversible conformational changes during viral fusion 3.

All FPs share common characteristics, which are
determinant for their function: they are hydrophobic, rich in Gly and Ala
residues, contain aromatic residues and are usually conserved within a species
(mutations frequent lead to a loss of function).4, 5 Apart from these general characteristics, FPs from virus belonging
to different families can be quite diverse 4. Some FPs (e.g. Influenza and HIV) are located at the N-terminal
tip of the fusion protein, whereas the peptides of other viruses (e.g. dengue
and Ebola) are internal fusion loops 4. The peptides from different families are also quite different at
the sequence level and structure levels. The influenza FP is helical in lipidic
environments 6, 7, whereas the HIV FP tends to adopt ?-sheet structures8, although it can become helical depending on membrane composition9. Other FPs, such as the one from dengue virus, which only has 14
residues, do not have a defined secondary structure10. It is not clear how peptides with such distinct characteristics
play a common role in membrane fusion.

 

1.2. Viral Entry Explained

Many fusion proteins are C-terminal fragments of a
larger precursor (eg: HA2 fragment of influenza virus hemagglutinin; gp41
fragment of HIV Env)3 and the mechanism by which fusion proteins mediate
membrane fusion is a complex process that involves several segments of these
proteins. First the fusion protein opens up and forms a bridge between the two
bilayer membranes. Usually a C-terminal transmembrane region holds the fusion protein
in the membrane for the fusion peptide (located ate the N-terminal fragment of
the fusion protein or internally – fusion loops – depending on the virus) could
connect and interact with the viral membrane. This interaction possibly makes
the fusion protein undergo many conformational changes until the bridge
col-lapses resulting in a junction of the fusion peptide and the C-terminal
fragment, creating a fusion pore.3

Figure 1 Schematic
representation of the sequence of events in membrane fusion promoted by a viral
fusion protein.

(a) The protein in the pre-fusion conformation, with
its fusion peptide or loop (light green) held. Some features of specific
proteins are not represented (eg: displacement of the N-terminal fragment of
proteins that are cleaved from a precursor or the dimer-to-trimer rearrangement
on the surface of flaviviruses).

(b) The protein opens up, extending the fusion peptide
or loop to interact with the target bilayer. The part of the protein that bears
the fusion peptide forms a trimer cluster.

(c) A C-terminal segment of the protein folds back
along the outside of the trimer core. The segments from the three subunits fold
back independently, so that at any point in the process they can extend to
different distances along the trimer axis, and the entire trimer can bow
outward, away from the deforming membrane.

(d) When collapse of 
the intermediate has brought the two bilayers into contact, proximal
leaflets merge into a hemi-fusion stalk.

(e) As the hemifused bilayers open into a fusion pore,
the final zipping up of the C-terminal segments breaks the refolded trimer into
its fully symmetric, post-fusion conformation, preventing the pore from
resealing 3.

 

There are at least four distinct
mechanisms (eg.: pH, binding to another surface protein, temperature or fusion
protein cleavage) by which viral fusion proteins can be triggered to undergo
fusion inducing conformational changes 11. Despite this
diversity, all characterized viral fusion proteins convert from a
fusion-competent state (dimers or trimers, depending on the class) to a
membrane-embedded homotrimeric prehairpin, and then to a trimer-of-hairpins.
Additionally, all fusion proteins contain a fusion peptide (FP), which inserts
into the host membrane during fusion.

Three distinct classes of viral fusion
proteins have been identified based on structural criteria.

Class I fusion proteins, observed in influenza
virus, HIV and SARS virus, are characterized by a trimeric assembly of ?-helical coiled coil hairpins in the post-fusion state
Figure 2.2

These fusion proteins usually require proteolytic
processing into two subunits (e.g., influenza HA, paramyxovirus F), for some
viruses (e.g., Ebola virus GP) processing into the two subunits occurs for the
wt protein, but is not essential for infection 12. Some coronavirus S precursors
are (e.g., MHV), whereas others (e.g., SARS) are not, proteolytically processed
during biosynthesis. These latter coronaviruses S proteins as well as for Ebola
virus GP and Hendra and Nipah virus F, may be substituted by post synthetic
cleavage by extracellular or intracellular (e.g., endosomal cathepsins)
proteases 13

They are characterized for being metastable on the
virion and perpendicular (project as a spike) to the viral membrane. The major
secondary structure of the native fusion subunit is ?-helical, and the oligomeric structure is a trimer as well as the oligomeric
structure of fusion-active form (membrane-embedded prehairpin and bundles),
however, the structure of the post-fusion form is a trimer-of-hairpins (central
?-helical coiled-coil, 6HB in figure1 iv).
In class I native fusion proteins the fusion peptide is buried in the subunit
interface, whereas in the primary sequence this fusion peptide is located at or
near the N-terminus 14.

On
the other hand, class II fusion
proteins, found in flaviviruses and alphaviruses, are categorized as trimers of
hairpins composed of ?-sheets in the post
fusion state Figure 3.  15

 

 

These class II fusion proteins consist primarily of ?-sheet structure with internal fusion peptides formed
as loops at the tips of ?-strands. They are
associated with a chaperone protein (p62 for SFV E1 and prM for TBEV E), which
is cleaved during or soon after viral assembly so that the fusion protein
generates a competent form. Similarly to class I fusion proteins, class II
FProt are also metastable on the virion, however, unlike class I, these class
II orientation is parallel (close to) the viral membrane. Their native fusion
protein oligomeric structure are dimers, whereas the oligomeric structure of
the fusion-active form (membrane – embedded prehairpin and bundles) is a
trimer, and the structure of the post-fusion form is a trimer of hairpins
(mainly ?-structure).
In the native fusion protein, the FP is masked in the trimer interface, at the
tip of the extended ?-strands, and its
location in a primary sequence is internally.14

 

 

 

A class III fusion protein, found in vesicular
stomatitis virus and herpes simplex virus, is also characterized by trimers of
hairpins although formed by the helical coiled-coil and ?-sheets structures. 2 The pre-fusion form of
VSV G is a trimer, but the trimer interface is small. In contrast to those in
the pre-fusion conformations of all other fusion proteins known to date, the
fusion loops (red) are located on the outside of the structure, not protected
at an interface. Upon acidification, a series of conformational changes occur
in VSV G that reposition the fusion loops (red) into the vicinity of the target
membrane. A second series of conformational changes then bend the protein back,
reorienting the C-terminal portion anti-parallel to the N-terminal segment,
thereby bringing the viral and target membranes together. (see Figure 4).

 

Figure 4 Crystal
structures of the neutral (i and ii) and low pH (iii and iv) forms of the VSV G
ectodomain.

In the first step, conformational
changes occur in two regions, Ex1 and Ex2 (orange in i, ii, and iii). Each region has two parts, one
is an unstructured linker, the other has helical structure. During the
conformational change the unstructured linker of Ex1 becomes helical and the
helical residues become unstructured, resulting in movement of the fusion
domain (DIV) approximately 90°. The motion of DIV is completed by changes in
Ex2, in which linkers between DII (blue-gray in ii) and DIII (cyan), become helical, extending each of the two DII helices (blue-gray
and orange in iii). The result is
the rotation of both DIII and DIV such thatthe fusion loops (red) are now near the target membrane. Finally,
inversion of the C-terminal stem is accomplished by additional structural rearrangements
in Domain II. In particular, an unstructured loop (Inv, green
in ii) becomes an ?-helix, which we refer to in Figure 6D as the “C-helix” (green in iii),
that is oriented antiparallel to the core structure. Consequently, the
“C-helix” packs against the now elongated helix of Domain II (blue-gray
and orange in iii), bringing the
C-terminus and viral membrane into proximity with the target membrane, thereby
facilitating fusion.14

 

Class III G fusion proteins’ conformational changes of
some rhabdovirus are hypothesized to be reversible (Gb fusion protein it’s
still unknow) since the pre-fusion and post-fusion states are in thermodynamic
equilibrium, with the equilibrium shifted towards the post fusion state at low
pH 16, unlike the majority
of other viral fusion proteins, which are metastable and irreversibly
inactivated (lose the capacity to mediate fusion with a subsequently presented
target membrane) if triggered in the absence of a target membrane. Also, these
fusion proteins don’t require proteolytic processing to generate a fusion able
form, and are perpendicularly orientated towards the viral membrane. The
oligomeric structure of the fusion protein’s native form it’s a trimer as well
as its fusion-active form (membrane-embedded prehairpin and bundles), while the
post-fusion structure is a trimer of hairpins (central ?-helical coiledcoil and significant ?-structure). Regarding the fusion peptide’s location, in the native form
it’s exposed at the tips of the extended ?-strands, whereas in primary sequence it’s located internally, containing
two loops found at the tips of two neighboring ?-strands.

The fusion peptide (FP) is one of the most relevant
players in the fusion process, 11 this segment of the
fusion protein inserts in the host membrane and has an active role in promoting
fusion. The FP is a very promising drug target, since it is conserved within a
viral species and is vital for the infection process (e.g. antibodies against
dengue virus target this region 4, 17).

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