Figure 1.
Roles of NEP in influenza virus replication.
(A) The influenza virion is an enveloped particle with an outer surface dominated by the receptor-binding protein haemagglutanin (HA) and the sialic acid-cleaving neuraminidase (NA), as well as small amounts of the M2 ion channel. The viral matrix protein (M1) is situated beneath the lipid envelope. The segmented virus genome is packaged in the form of viral ribonucleoproteins (vRNPs) that comprise the three viral polymerase subunits PA, PB1, and PB2 and multiple copies of the nucleoprotein (NP) bound to the viral genomic RNA. NEP was first thought to be nonstructural in function, however it is now recognised that NEP is resident within influenza virions where it may interact with M1. (B) NEP stimulates the synthesis of the viral cRNP replication intermediate that is proposed to result in the increased production of vRNPs late in infection for packaging into progeny virions. In order to achieve this, NEP may act in concert with recently described small viral RNAs (svRNAs). (C) NEP acts as an adaptor protein to mediate the export of vRNPs from the nucleus for packaging into progeny virions at the cell periphery. NEP mediates nuclear export by interacting with the cellular nuclear export protein Crm1 and the viral M1 protein, which is in turn bound to vRNPs. (D) NEP has been shown to recruit the F1Fo ATPase that is involved in the budding of progeny virions.
Figure 2.
Organisation and structure of influenza virus A NEP.
(A) A schematic representation of NEP including amino acid conservation at each position marked graphically above. Conservation at each amino acid position was calculated using Bioedit (Ibis Biosciences) from alignments of 16065 full-length influenza A NEP sequences obtained from http://www.fludb.org (accessed March 14, 2012). Alignments of all sequences were conducted using MAFFT [76]. The N-terminal nuclear export signal (NES) enables binding of NEP to the cellular β-importin Crm1. A highly conserved serine-rich motif at positions 23–25 (purple) can be phosphorylated during virus infection. The C-terminal α-helices interact along their lengths to form an almost perfectly anti-parallel hairpin, the structure of which has been solved (PDB:1pd3; [26]). (B) Hydrophobic residues on the NEP C-terminal domain are depicted in orange. The opposing hydrophobic and hydrophilic faces result in the NEP C-terminal domain forming an amphipathic molecule. In full-length NEP the N-terminal domain is proposed to pack against the hydrophobic face, effectively burying it. The putative M1-binding tryptophan residue (W78, orange) projects prominently from within a ring of glutamate residues (red) on the hydrophilic face of the NEP C-terminal domain.
Figure 3.
The daisy chain model for NEP-mediated nuclear export of influenza virus vRNPs.
The β-importin Crm1 mediates export of the vRNP complex by binding to the N-terminal domain of NEP, as well as to its cofactor, the small GTPase Ran. The C-terminus of NEP binds to the nuclear localisation signal (NLS) on the N-terminal domain of the viral matrix protein M1. The C-terminus of M1 in turn binds strongly to the vRNP through interaction with NP (grey).
Figure 4.
Mutations in NEP are responsible for overcoming host-range restriction.
Adaptive mutations in NEP can result in an increase in viral RNA accumulation, thereby allowing avian influenza viruses to overcome host restriction in mammalian cells. Avian influenza viruses show restricted replication in mammalian hosts due to inefficient viral RNA synthesis. This has been proposed to result from decreased cRNP stability. Adaptive mutations in NEP can overcome this restriction by increasing viral RNA accumulation.