Page 17 - Avian Virology: Current Research and Future Trends

10 | Perez et al.

2000; Akarsu et al., 2003; Shaw and Palese, 2013b). This interac- give origin to M1 and M2, respectively; mRNA3 that represses
tion involves an expose tryptophan at position 78, surrounded protein synthesis during early stages of infection; and mRNA4
by a cluster of glutamate residues on NEP, and the NLS on M1 which gives origin to M42 (Inglis and Brown, 1981; Lamb and
(Akarsu et al., 2003). Binding of NS2/NEP at the NLS on M1 Choppin, 1981; Lamb et al., 1981; Shih et al., 1998). M42 is an
may have a potential role in preventing vRNP–M1–NS2/NEP alternative M2 protein that has an antigenically distinct ectodo-
complexes from being translocated back into the nucleus. NS2/ main. Experiments with mutants lacking M2 expression have
NEP binds other cellular proteins, such as Crm1 to complete the shown an up-regulation of mRNA4 and synthesis of M42, which
export (see below for NS protein function and interactions). reconstitutes ion channel activity in vivo and in vitro. Its ability to
Segment 7 also encodes for the M2 protein by translation of an functionally replace M2 did not appeared to be affected by M42
spliced mRNA originated from an overlapping ORF (Inglis and been largely localized in the Golgi network (Wise et al., 2012).
Brown, 1981; Lamb and Choppin, 1981; Lamb and Lai, 1981,
1982). The M2 protein of influenza A viruses is approximately Segment 8: non-structural protein 1 (NS1) and
97 aa long, classified as a type III transmembrane protein. It pos- nuclear export protein/non-structural protein 2
sesses a short ecto-domain (24 aa residues), a transmembrane (NEP/NS2)
domain (19 aa), and a cytoplasmic domain (54 aa) with palmi- The NS1 and NEP proteins are the most studied and best
tate and phosphate modifications (Lamb et al., 1985; Zebedee characterized internal IAV proteins with an extensive literature
et al., 1985). It is found in infected cells and viral envelope as a (Fernandez-Sesma, 2007; Lin et al., 2007; Hale, 2008b, 2014;
homotetramer composed of two disulfide dimers linked together Paterson and Fodor, 2012; Marc, 2014; Krug, 2015). Only the
by non-covalent interactions (Lamb et al., 1985). Each monomer most salient features are discussed below. The NS1 is a multi-
possesses an amphiphilic transmembrane alpha-helical domain functional protein encoded in segment 8 (Inglis et al., 1979;
that conforms the protein transmembrane channel, with which Lamb and Choppin, 1979). NS1 exists as a homodimer and is
amantadine, the specific anti-influenza A drug, interacts (Sugrue abundantly expressed early in the infection. While recognized as
and Hay, 1991). M2 constitutes a proton channel and it is respon- a non-structural protein, a recent report suggests NS1 is present
sible for proton influx into the virion in response to low pH of the in substantial amounts as an internal component of purified virus
endosome (Pinto et al., 1992; Schroeder et al., 1994; Shimbo et particles (Hutchinson et al., 2014). Whether NS1 is actively
al., 1996), triggering dissociation of the vRNAs from the matrix packed into virus particles or merely incorporated as a passive
proteins and fusion of the endosomal and virus membranes, bystander remains to be determined. The primary function
releasing the vRNAs into the cytoplasm for further translocation of NS1 is to suppress the virus-induced host type I interferon
into the nucleus (Cady et al., 2009). In addition, it is found at the (IFN-α/β) response (García-Sastre et al., 1998). As a multifunc-
trans-Golgi network membrane, and there is evidence that sug- tional protein, NS1 is involved in a plethora of activities during
gest the involvement of M2 ion channel activity on stabilization the virus replication cycle, such as temporal regulation of virus
of the HA during its post-translational modification in the trans- RNA synthesis, control of virus mRNA splicing, prevention of
Golgi, regulating pH of the vesicles to ensure proper folding and the host’s mRNA translation, enhancement of virus mRNA trans-
to prevent earlier cleavage of HAs with a polybasic cleavage site lation, regulation of virus particle morphology, and involvement
such as H5 and H7 HAs (Sugrue and Hay, 1991). in strain-dependent pathogenesis. All of these functions of NS1
M2 equilibrates the pH across the viral membrane during rely on its ability to participate in a multitude of protein–protein
cell entry and across the trans-Golgi membrane of infected cells and protein–RNA interactions (Hale et al., 2008b).
during virus assembly (Pielak and Chou, 2011). Under condi- The NEP/NS2 protein (121 amino acids) is produced from a
tions of pH ≥ 7.5, the M2 proton channel is closed and inactive. virus mRNA transcript generated by splicing of segment 8 (Inglis
When the pH is ≤ 6.5, the M2 proton channel is open and active. et al., 1979; Lamb and Choppin, 1979). Amounts of spliced NEP/
M2’s ability to open and close is dependent on the action of a NS2 virus mRNA correspond to approximately 10% of unspliced
single transmembrane domain residue, tryptophan 41 (W41), NS1 virus mRNA (Lamb et al., 1980). During the virus replica-
which is highly conserved among all IAVs (reviewed in Manzoor tion cycle, NEP/NS2 interacts with M1 and vRNP and recruits
et al., 2017). M2 is essential for viral replication and the target for the nuclear export machinery to direct the export of newly syn-
the anti-influenza drugs amantadine and rimantadine (McCown thesized vRNP complexes (Martin and Helenius, 1991a; Yasuda
and Pekosz, 2005). Both amantadine and rimantadine bind and et al., 1993; O’Neill et al., 1998; Shaw and Palese, 2013a). NEP/
block the inner pore of M2. It must be noted, however, that fully NS2 interacts with members of the nuclear export machinery,
fit and competent resistant strains against these two drugs can including Crm1 and nucleoporins (O’Neill et al., 1998; Neumann
emerge readily in the avian reservoir (likely due to their misuse in et al., 2000). NEP/NS2 appears to help in virus budding (Gorai
poultry). A commonly observed mutation is the substitution of et al., 2012) and in regulating the switch between virus transcrip-
serine at position 31 for asparagine (S31N) within the inner pore tion and replication (Robb et al., 2009).
that ultimately prevents drug interaction while preserving proton
pump activity. Virus life cycle
Alternative splicing of segment 7 mRNA gives origin to an As the virus moves towards the target cell, the NA removes muco-
alternative M protein called M42 (Wise et al., 2012). Four mRNA polysaccharides present in the extracellular space to allow the HA
transcripts are involved on M proteins synthesis: mRNA 1 and 2 to find and bind the terminal SA receptors present on glycolipids