Page 62 - Avian Virology: Current Research and Future Trends

Newcastle Disease Virus | 55

119 341 433 481 508 538

NH - CT TM STALK DOMAIN GLOBULAR HEAD DOMAIN -COOH
2

(1-26) (27-48) Topological domain (48-577)

Figure 2.7 Schematic diagram representing different domains of the Newcastle disease virus attachment protein haemagglutinin-
neuraminidase (HN). The positions of the cytoplasmic tail (CT) domain, the transmembrane (TM) domain, and the stalk and globular head
domains of NDV HN protein are indicated in the parenthesis. Lollipop represents the sites used for addition of N-linked carbohydrate.

and a 444- to 490-residue globular head region (Fig. 2.7). The HN is a glycosylated protein. Glycosylation is important for
predicted molecular weight of the HN protein is 74 kDa. proper folding, intracellular-transport, stability, and receptor
Analysis of the HN sequences revealed 12 different HN binding. The HN protein contains six potential N-linked glyco-
Figure 7 sylation sites, N119, N341, N433, N481, N508, and N538 (Fig.
protein lengths, with each length representing a viral lineage
2.7). Five of these sites are relatively conserved among NDV
and virulence phenotype, indicating evolutionary conservation
of HN length. The HN protein length diversity arise from strains, except site N508 which is absent in some strains. Stud-
differences in the position of the stop codon of HN ORF ies have shown that sites N119, N341, N433, and N481 are used
(Gould et al., 2003; Murulitharan et al., 2013; Zhang et al., (McGinnes and Morrison, 1995; Panda et al., 2004b). Site N538
2014). Most virulent NDV strains have HN protein of 571 aa, is not used because it is not accessible for glycosylation (Pitt et
while most low virulence strains have HN proteins of 577 aa al., 2000). Glycomic studies of the glycan structure revealed
and 616 aa. Only in the case of the HN protein with 616 aa two main classes of oligosaccharides, complex-type and high
(HN ) post-translational cleavage is required for activation of mannose-type (Pegg et al., 2017). Removal of N-glycans from
0
the HN protein, but not for proteins of other lengths (Nagai the HN protein of NDV had a significant effect on its exocytic
et al., 1976). It was found that the C-terminal extension in transport, surface expression and pathogenicity (McGinnes and
616 aa HN protein engages a secondary sialic acid binding site Morrison, 1995; Panda et al., 2004b). In addition, an O-linked
present in NDV HN protein that most likely blocks its attach- glycosylation site (T71) in the stalk region has been identified
ment function, leading to an auto-inhibited state of HN (Yuan (Pegg et al., 2017). Although T71 is highly conserved among
et al., 2012). The C-terminal extension region of HN contains NDV strains, it is not present in some strains. The biological sig-
0
a cysteine residue at position 596, which regulates HN activa- nificance of O-glycosylation is not known.
tion and forms an inter-subunit disulfide bond. It was shown The crystal structures for both the head and the stalk domains
that introduction of several different C-terminal extensions to of NDV HN have been obtained (Crennell et al., 2000; Zaitsev
the HN protein of a virulent 571 aa HN length strain resulted et al., 2004; Yuan et al., 2011). The globular head consists of four
in mutants with variously decreased levels of pathogenicity and six-bladed β-sheet propeller fold monomers, each harbouring a
replication in vivo (Kim et al., 2014a). A greater reduction in centrally located sialic acid binding site (site I) that also has NA
HN function and pathogenicity was achieved by introduction activity (Crennell et al., 2000). However, a second sialic acid bind-
of cysteine 596 residue, indicating an important role for these ing site (site II) was later discovered at the dimer interface in the
extended sequences in modulating NDV pathogenesis (Kim et globular head (Zaitsev et al., 2004). The second site is made up
al., 2014a). These results suggest that the shorter HN proteins of hydrophobic residues from both the monomers and interacts
arose evolutionarily from a longer ancestral HN protein by with sialic acid but lacks NA activity. Mutations in site I abolish
introduction of translational stop codons and the natural HN receptor binding and NA (Iorio et al., 2001; Li et al., 2004); while
length is optimal for that strain. mutations at site II abolish receptor binding and fusion promo-
The HN protein of NDV contains 14 cysteine residues, which tion without affecting NA (Bousse et al., 2004). Although, the role
are well conserved among strains; except for cysteine 123, which of the second sialic acid binding site is still not fully understood,
is replaced by tryptophan in some strains (McGinnes et al., 1987). studies suggest that binding of site I with the receptor leads to the
Cysteine 123 has been shown to be involved in disulfide-linked activation of site II. The activated site II has higher receptor avid-
dimer formation (McGinnnes and Morrison, 1994). However, ity than site I (Porotto et al., 2006). Receptor binding to site II
this cysteine residue is not conserved in all NDV strains, but efficiently transmits the fusion signal to the stalk region. The stalk
the protein still forms a dimer and tetramer, suggesting that this region then interacts with the F protein to promote membrane
disulfide linkage only stabilizes the oligomeric structure and may fusion (Porotto et al., 2012). The high avidity of site II maintains
not be involved in the function of HN (McGinnes and Morrison, receptor binding which is essential throughout the fusion process
1994). Mutation in different cysteine residues can block folding (Mahon et al., 2011). It has been shown that mutation of certain
at different stages of maturation of the HN protein, indicating that key residues in site I can attenuate the virulence of NDV (Khattar
they are important for the structural integrity of the HN protein et al., 2009). The globular head is connected to the stalk through
(McGinnes and Morrison, 1994). a short flexible unstructured linker that allows the globular head