The Intrinsically Disordered W Protein Is Multifunctional during Henipavirus Infection, Disrupting Host Signalling Pathways and Nuclear Import

Abstract

Nipah and Hendra viruses are highly pathogenic, zoonotic henipaviruses that encode proteins that inhibit the host's innate immune response. The W protein is one of four products encoded from the P gene and binds a number of host proteins to regulate signalling pathways. The W protein is intrinsically disordered, a structural attribute that contributes to its diverse host protein interactions. Here, we review the role of W in innate immune suppression through inhibition of both pattern recognition receptor (PRR) pathways and interferon (IFN)-responsive signalling. PRR stimulation leading to activation of IRF-3 and IFN release is blocked by henipavirus W, and unphosphorylated STAT proteins are sequestered within the nucleus of host cells by W, thereby inhibiting the induction of IFN stimulated genes. We examine the critical role of nuclear transport in multiple functions of W and how specific binding of importin-alpha (Impα) isoforms, and the 14-3-3 group of regulatory proteins suggests further modulation of these processes. Overall, the disordered nature and multiple functions of W warrant further investigation to understand henipavirus pathogenesis and may reveal insights aiding the development of novel therapeutics.

Keywords: IRF-3; STAT; W protein; henipaviruses; intrinsically disordered.

Figure 1

Henipavirus genome organization and virion structure (not true to scale). (a) The henipavirus genome is a single stranded negative polarity RNA containing six viral genes. (b) The P gene encodes P, V, W, and C proteins possess IFN-antagonist activity. P, V, and W share a common sequence encoding their N-terminal domain (2404–3628 nucleotides (nt)), however at site AAAAAGGGC (3619–3628 nt) RNA editing can occur with a single G-nucleotide (1G) or double G (2G) insertion shifting the reading frame resulting in unique C-terminal domains for V and W proteins respectively; C-terminus of the P protein is longer (202 aa) when compared to W and V (43 and 49 aa, respectively). (c) Pleomorphic virion structure is controlled by M protein which lies under the virion envelope. Attachment protein G and fusion protein F are located on the envelope surface and protrude as spikes. The viral ribonucleoprotein (RNP) core consists of a single-stranded negative sense RNA, with N, L, and P proteins required for viral transcription.

Figure 1

Henipavirus genome organization and virion structure (not true to scale). (a) The henipavirus genome is a single stranded negative polarity RNA containing six viral genes. (b) The P gene encodes P, V, W, and C proteins possess IFN-antagonist activity. P, V, and W share a common sequence encoding their N-terminal domain (2404–3628 nucleotides (nt)), however at site AAAAAGGGC (3619–3628 nt) RNA editing can occur with a single G-nucleotide (1G) or double G (2G) insertion shifting the reading frame resulting in unique C-terminal domains for V and W proteins respectively; C-terminus of the P protein is longer (202 aa) when compared to W and V (43 and 49 aa, respectively). (c) Pleomorphic virion structure is controlled by M protein which lies under the virion envelope. Attachment protein G and fusion protein F are located on the envelope surface and protrude as spikes. The viral ribonucleoprotein (RNP) core consists of a single-stranded negative sense RNA, with N, L, and P proteins required for viral transcription.

Figure 2

Representation of W protein domains and amino acids sequence involved in host protein interactions. (a) The bioinformatics program IUPred2A [40] was used to predict disordered regions across the HeV W protein sequence (UniProt accession number P0C1C6). IUPred2A uses statistical potentials to generate energy estimations of amino acid residues (whether they are likely to form favourable interactions with each other). Residues with scores <0.5 are predicted to be disordered, whereas residues scored >0.5 are likely ordered. (b) The N-terminal domain of W protein (grey) is an intrinsically disordered region and contains region aa 114-140 (green) that is paramount for signal transducer and activator of transcription (STAT) binding (green) and inhibition. Soyuz1 and soyuz2 regions (dark grey) are highly conserved in henipaviruses and may play a role in interferon (IFN) antagonism. The C-terminal domain of residues 408 to 450 is required for interferon regulatory factor 3 (IRF-3) inhibition (blue) and includes the Impα2 (yellow) and Impα3-binding region (orange), vital for nuclear localisation, and the 14-3-3-binding region (pink). (c) Solved structures of W NLS (red) in complex with binding partners: Impα1 in yellow (PDB 6BW0), Impα3 in orange (PDB 6BVV), and 14-3-3 σ in pink (PDB 6W0L).

Figure 3

Type I IFN synthesis pathway and production of pro-inflammatory cytokines. Virus-responsive induction of the IFN-β promoter is directed by cytoplasmic helicases RIG-I and MDA5 [74]. Intracellular RNA pathogen-associated molecular patterns (PAMPs; e.g., dsRNA) are recognized by cellular pattern recognition receptors (PRRs; e.g., RIG-I, MDA5, TLR3), triggering antiviral signalling cascades. Activation of two signalling kinases TBK-1 and IKK-ε results in phosphorylation and activation of latent IRF-3 transcription factor that is subsequently translocated to the nucleus upon dimerization [67,68]. IRF-3 binds to p300 and CBP to form the DRAF1 transcription complex for IFN-β production. Cytoplasmic V protein is known to inhibit TBK-1 driven activation of IRF-3, whereas W protein successfully inhibits both TBK-1 and IKK-ε driven activation of IRF-3, with significant effects on TIR-domain-containing adapter-inducing interferon β (TRIF)-mediated activation [29].

Figure 4

IFN signalling pathway and production of IFN-stimulated gene (ISG) products. Virus-mediated induction of the type I IFN production activates IFN signalling pathways. Type I IFNs (IFNα/β) bind cognate IFNAR receptors that activate Janus kinase (JAK) proteins JAK1 and TYK2. When activated, JAKs phosphorylate STATs. The activated STAT-1 and STAT-2 proteins form heterodimers that associate with IRF-9, creating the ISGF3 complex. ISGF3 associates with coactivators to induce the expression of genes downstream of promoters with IFN-stimulated response elements (ISREs). This upregulates many host antiviral products (ISGp). Type II interferon (IFN-γ) binds IFNGR receptors to stimulate JAK activity and phosphorylation of STAT-1 homodimers (GAF complex). GAF binds promoters of gamma-activated sequences (GAS)-associated genes that upregulate host antiviral products (ISGp). W protein binds and sequesters unphosphorylated STAT in the nucleus as a high molecular weight complex, antagonizing the function of STAT in the JAK-STAT pathway.