Page 61 - Avian Virology: Current Research and Future Trends

54 | Samal

glycosylation sites at residues 85, 191, 366, 447, 471 and 541 host cell. The host cell receptor specificity of the HN protein
that are conserved in all strains (de Leeuw and Peeters, 1999; ensures that the cleaved F protein is activated at ‘the right time
Paldurai et al., 2010). Reverse genetic analysis showed that a and right place’. The NDV HN atomic structure indicates that
live virus could not be rescued when the putative glycosylation the head domain can adopt either a ‘heads down’ form contact-
site at residue 541 was removed, indicating that the presence of ing the stalk or a ‘heads up’ form where the stalk is exposed
carbohydrate at this site is essential for virus viability. Removal (Yuan et al., 2012; Welch et al., 2013). NDV HN upon binding
of carbohydrate at each of the remaining five glycosylation sites to sialic acid undergo a conformational change from the inac-
did not affect the cleavage of F protein or cell surface expression. tive ‘four heads down’ state to the active ‘four heads up’ state
Loss of carbohydrate at residues 85, 119, 366, and 471 did not (Bose et al., 2012; Yuan et al., 2012; Welch et al., 2013). This
have any effect on syncytia formation but the mutant viruses movement exposes the F-activating region of the HN stalk for
were attenuated; whereas, loss of carbohydrate at residue 447 physical interaction with the F protein to trigger membrane
increased syncytia formation and the mutant virus was slightly fusion. This model has been termed as the provocateur or stalk
more virulent. The sites at residues 191 and 471 are present in exposure model (Fig. 2.6B). Hydrophobic regions in both the
heptad repeat domains HRA and HRB, respectively. It was shown head and stalk of F have been implicated in interaction with
that removal of carbohydrate at both these sites resulted in a virus HN (Tsurudome et al., 1998; Lee et al., 2008). After triggering
that replicated more efficiently in vitro and was more virulent in of the F protein, the HRA adjacent to the fusion peptide under-
eggs and chicks, suggesting that the N-glycans in HRA and HRB goes a major conformational change that extends the fusion
co-ordinately down-regulate viral fusion and virulence (Samal et peptide outward for insertion into the target membrane. This
al., 2012). process leads to a prehairpin intermediate form. The prehairpin
The cysteine residues in viral proteins are involved in disulfide is a unique structure in which the F protein is simultaneously
bond formation and thus play important role in structure and anchored into two different membranes. After prehairpin for-
function of the protein. The F protein of NDV contains 13 mation, the HRB adjacent to the TM domain translocates to
cysteine residues of which 11 are conserved among the F proteins bind HRA in an anti-parallel way, forming a stable six-helix
of other paramyxoviruses. The conservation in the position of bundle. This process draws the two membranes close enough
the cysteine residues suggests that folding of the molecule and to merge (Lamb and Parks, 2013).
intramolecular disulphide bonds are crucial to the function of
F protein. The NDV F protein undergoes major conformational The haemagglutinin-neuraminidase protein
changes soon after its synthesis and these changes are achieved by The HN protein of NDV is a multifunctional protein. HN has
rearrangement of intramolecular disulphide bonds. three distinct functions: (i) it is responsible for attachment of
the virion to sialic acid-containing cell surface receptors; (ii) it
The atomic structure of F protein and the promotes the fusion activity of the F protein, thereby allowing
mechanisms of membrane fusion the virus to penetrate the host cell; and (iii) it has neuraminidase
The NDV F protein, like other class I viral fusion proteins, exists activity (NA) that cleaves sialic acid from sugar side chains,
in two different conformational states in infected cells (Swanson thereby releasing progeny virions from surface of infected cells.
et al., 2010). The newly synthesized F monomers are folded in the The NA also removes sialic acid from progeny virions to prevent
ER into a metastable, high energy ‘prefusion’ trimer conforma- self-aggregation (Lamb and Parks, 2013). In addition, HN is one
tion. Upon activation by the HN protein, metastable prefusion of the major protective antigens of the virus and plays an impor-
F undergoes ATP-independent major conformational changes to tant role in the pathogenesis and immunogenicity of the virus
a highly stable, low energy ‘postfusion’ conformation. The trigger (Huang, Z. et al., 2004a; de Leeuw et al., 2005; Kim et al., 2013).
for this major refolding of F protein is thought to be binding of The HN protein is also a crucial determinant of NDV thermosta-
the HN protein to host cell receptor. The energy released during bility (Wen et al., 2016).
F refolding is believed to drive the membrane fusion (Lamb and The HN protein of NDV is a type II integral membrane pro-
Parks, 2013). tein that contains an uncleaved signal/anchor sequence located
The atomic structure for postfusion NDV F is available (Swan- near the N-terminal cytoplasmic tail and a large extracellular
son et al., 2010). Although an atomic structure for prefusion NDV C-terminal domain, connected by a hydrophobic transmembrane
F is not available, the prefusion F from other paramyxoviruses domain (Fig. 2.7). The HN exists on the surface of virions and in
suggest that the structure is highly conserved among paramyxo- virus infected cells as a homotetramer composed of two disulfide
viruses (Yin et al., 2006; McLellan et al., 2013). The atomic linked dimers (Crennell et al., 2000; Zaitsev et al., 2004). HN
structures indicate that the prefusion F trimer is characterized by translation, oligomerization and glycosylation occur in the ER.
a globular head and trihelical coiled-coil stalk that extends into the The HN ectodomain is a mushroom like structure consisting of a
viral membrane, whereas the postfusion F trimer is characterized long stalk that supports a terminal globular head. The attachment,
by a compacted globular head and stable membrane-proximal six- NA and all known antibody binding sites reside in the globular
helix bundle (Swanson et al., 2010). head (Mirza et al., 1993). The stalk mediates interaction with the
The concerted activities of homotypic HN and F proteins F protein (Deng et al., 1995; Mirza and Iorio, 2013). The NDV
mediate fusion of the viral membrane with the host cell mem- HN is composed of a 26-residue cytoplasmic tail, a 23-residue
brane which creates a pore for transfer of viral RNP into the transmembrane domain, a 77-residue ectodomain stalk region