Page 15 - Avian Virology: Current Research and Future Trends

8 | Perez et al.

trimers. The cleavage of the immature HA0 in LPAIVs occurs at NP is the most abundant structural protein in the vRNPs.
a single arginine residue by trypsin-like proteases present in the NP’s main function is the encapsidation of vRNA through
lumen of the intestinal and/or respiratory tract in birds and in the the interaction between the positive charges on NP residues
respiratory tract of mammals. The HA0 of the HPAIV H5 and and the negatively charged phosphate backbone on the vRNA
H7 subtypes contains a stretch of polybasic amino acids that are (reviewed in Shaw and Palese, 2013b). No specific motifs on
recognized by furin and other subtilisin-like proteases present in NP have been found to be involved in this interaction, sug-
the Golgi apparatus (Fig. 1.1). Early maturation of the H5 and gesting that most of the protein is able to interact with vRNA.
H7 HAs in the intracellular compartment is thought to favour A reconstructed image based on electron microscopy analysis
cell-to-cell spread and systemic infection of HPAIVs, particularly show the NP as an elongated structure with a minor and a major
in galliformes. curvature (Martín-Benito et al., 2001) (Ortega et al., 2000)
The HA1 subunit, with a globular shape, contains the receptor- that allows ≈ 24 nts of RNA to wrap around the protein. NP
binding site (RBS), a shallow pocket at the membrane distal tip of binding does not protect the vRNA from RNase degradation.
each monomer. The RBS participates in binding of glycan struc- NP undergoes oligomerization by interaction of two regions
tures terminating in sialic acid (SA, N-acetylneuraminic acid). denominated as NP-1 and NP-2. Based on a binding assay, NP–
The binding to SAs involves four structural features in the RBS: NP interactions have estimated to have a K of approximately
d
The 130-loop, the 190 alpha-helix, the 220-loop and a set of highly 200 nM (Elton et al., 1999), and are essential to maintain the
conserved amino acids that form the base of the site (residues structure of vRNPs. NP also binds to +ssRNA, namely cRNA
98, 153, 183, and 195, numbering based on the matured H3 HA that is synthesized during genome replication. Another impor-
subtype). In nature, the SA is linked to the penultimate galactose tant role of this protein is to facilitate vRNPs nuclear import
(Gal) in two major conformations: alpha2,3SA or alpha2,6SA. by its nuclear localization signal (NLS); although PB2, PB1
Small amino acid changes in the RBS as well as differences in the and PA have NLS, the NLS from NP appears to be the one
type of SA linkage expressed in different host species are major driving the translocation of the complex. The NLS on NP is
determinants of the host range of IAVs as discussed below. located at amino acid positions 327 to 345 (Davey et al., 1985).
The HA2, known as the stem, extends approximately 76 Å from Typically, NLSs are arginine- or lysine-rich; however, NLS on
the membrane and forms the triple-stranded coiled coil of alpha- NP is a non-conventional NLS peptide (not arginine- or lysine-
helices in the trimer. The N-terminal 20–24 amino acids of the rich) (Wang et al., 1997). Interestingly, evidence that IAV NP
HA2 subunit correspond to the ‘fusion peptide’ region necessary may have more peptides involved in intracellular trafficking
for the fusion of the lipid bilayer of the target membrane with the was made available a few years later (Neumann et al., 1997).
viral membrane. X-ray crystallography studies have revealed the It was found that a motif located at amino acid positions 1
HA conformational changes involved in fusion (reviewed in Cross to 38 had a role on nuclear import, export, and cytoplasmic
et al., 2009). On the virus’ surface the HA trimer is a metastable accumulation of NP by developing mutagenic NPs lacking the
structure with the fusion peptide buried in a charged pocket in the aforementioned region and/or the NLS at amino acid positions
HA2 subunit. Low-pH activation results in extrusion of the fusion 327 to 345. In addition, those results suggested that there may
peptide towards the fusion target membrane as the N-terminus be other regions on NP related to trafficking when mutants
of a newly formed trimeric coiled coil and repositioning of the lacking both the 1–38 and the 327–345 fragments were trans-
C-terminal membrane anchor near the fusion peptide at the located into the nucleus (Neumann et al., 1997). In order to
same end of a rod-shaped molecule (Tamm, 2003). The fusion achieve nuclear translocation, karyopherin α (importin α) binds
step normally occurs between pH 5.0 and 6.0, although strains directly to the NLS on NP and then recruits karyopherin β
with higher or lower pH than this have been identified (Cross et (O’Neill et al., 1995; O’Neill and Palese, 1995; Wang et al.,
al., 2009). Different studies have shown that the fusion peptide 1997). Some reports have suggested that differential interac-
region is a virulence marker (DuBois et al., 2011). Among pro- tion with karyopherin α isoforms may be a determinant on
totypical strains of HPAIV H5N1 of Asian origin, those whose host range (Gabriel et al., 2008, 2011). The aforementioned
fusion is activated at pH ≤ 5.4 are better adapted to infect in mam- vRNPs are formed by the interaction between the polymerase
mals (Zaraket et al., 2013). Towards the C-terminus, a stretch of complexes (PB2-PB1-PA) and the encapsidated vRNA. It has
26–27 amino acids span the transmembrane domain followed been shown that NP directly interacts with PB2 and PB1 in
by 10–11 amino acids in the cytoplasmic tail. The cytoplasmic both human and avian species (Biswas et al., 1998; Naffakh et
tail, modified by palmitoylation, is highly conserved among HA al., 2000; Martín-Benito et al., 2001). NP also interacts with the
subtypes and can modulate virion assembly (Veit and Schmidt, M1 viral protein forming the vRNP–M1 complexes, a step on
1993; Jin et al., 1994; Ponimaskin and Schmidt, 1998; Chen et the nuclear export process of vRNPs for further virion assembly
al., 2005). (Martin and Helenius, 1991a; Noton et al., 2007). Moreover,
NP is able to interact with other host cell proteins such as
Segment 5: nucleoprotein F-actin and Crm1. Interactions with F-actin take place during
NP is an arginine-rich protein, positively charged (+ 14 at pH of late infection, presumably to facilitate accumulation of vRNPs
6.5) and phosphorylated (Winter and Fields, 1981). Although in the cytoplasm in preparation for virion assembly (reviewed
there is a predominance of basic amino acids, the C-terminus is in Portela and Digard, 2002). Crm1 (also known as exportin-1)
predominantly acidic (reviewed in Portela and Digard, 2002). is responsible for vRNP nuclear export. Previous research has