Henipavirus W Proteins Interact with 14-3-3 To Modulate Host Gene Expression

Abstract

Nipah virus (NiV) and Hendra virus (HeV), members of the Henipavirus genus in the Paramyxoviridae family, are recently emerged, highly lethal zoonotic pathogens. The NiV and HeV nonsegmented, negative-sense RNA genomes encode nine proteins, including the W protein. Expressed from the P gene through mRNA editing, W shares a common N-terminus with P and V but has a unique C-terminus. Expressed alone, W modulates innate immune responses by several mechanisms, and elimination of W from NiV alters the course of infection in experimentally infected ferrets. However, the specific host interactions that allow W to modulate innate immunity are incompletely understood. This study demonstrates that the NiV and HeV W proteins interact with all seven isoforms of the 14-3-3 family, regulatory molecules that preferentially bind phosphorylated target proteins to regulate a wide range of cellular functions. The interaction is dependent on the penultimate amino acid residue in the W sequence, a conserved, phosphorylated serine. The cocrystal structure of the W C-terminal binding motif with 14-3-3 provides only the second structure of a complex containing a mode III interactor, which is defined as a 14-3-3 interaction with a phosphoserine/phosphothreonine at the C-termini of the target protein. Transcriptomic analysis of inducible cell lines infected with an RNA virus and expressing either wild-type W or W lacking 14-3-3 binding, identifies new functions for W. These include the regulation of cellular metabolic processes, extracellular matrix organization, and apoptosis. IMPORTANCE Nipah virus (NiV) and Hendra virus (HeV), members of the Henipavirus genus, are recently emerged, highly lethal zoonotic pathogens that cause yearly outbreaks. NiV and HeV each encode a W protein that has roles in regulating host signaling pathways, including antagonism of the innate immune response. However, the mechanisms used by W to regulate these host responses are not clear. Here, characterization of the interaction of NiV and HeV W with 14-3-3 identifies modulation of 14-3-3-regulated host signaling pathways not previously associated with W, suggesting new avenues of research. The cocrystal structure of the NiV W:14-3-3 complex, as only the second structure of a 14-3-3 mode III interactor, provides further insight into this less-well-understood 14-3-3 binding motif.

Evolutionary history of cotranscriptional editing in the paramyxoviral phosphoprotein gene

“…For example, NiV W can sequester unphosphorylated STAT proteins in the nucleus, via its N-terminal STAT1 binding site and C-terminal NLS, potentially inhibiting IFN signalling. NiV and HeV W were also recently discovered to modulate host gene expression by interacting with the 14-3-3 family of regulatory proteins, an interaction that depends upon phosphorylation of the penultimate serine residue in WCT ( Edwards et al (2020) ; Fig. 2 ).…”

“…Under our model, the W protein has evolved a novel function associated with its unique C-terminal region on three independent occasions ( Figs. 2 and 4 ): once for NDV ( Yang et al 2019 ; Karsunke et al 2019 ), once for the henipaviral clade comprised of HeV and NiV ( Shaw et al 2005 ; Lo et al 2009 ; Edwards et al 2020 ), and once for the respiroviral clade composed of BPIV-3, HPIV-3, and CPIV-3 ( Pelet et al 1991 ; Durbin et al 1999 ). There are varying levels of experimental evidence supporting the existence of a W protein function in these three clades (see Section 4.3).…”

Recognition of high-risk HPV E6 oncoproteins by 14-3-3 proteins studied by interactomics and crystallography

“…To the best of our knowledge, the only two reported structures with pS/pTXX-COOH consensus peptides are those of 14-3-3γ with the influenza virus protein NS1 (4O46.pdb) and our recently reported 14-3-3σ complex with the synthetic papillomavirus 16E6 phosphopeptide (6TWZ.pdb 24 ), however, both of them have rather low resolution (2.8-2.9 Å) (Supplementary Table 1). Together with the henipavirus protein W binding to 14-3-3σ via its C-terminal motif III peptide RRMpSN-COOH 66 , the adeno-associated virus protein Rep68 binding to 14-3-3 proteins using its Cterminal motif III peptide RGHpSL-COOH 67 and the hepatitis B virus protein X binding to 14-3-3ζ via an internal motif I peptide RPLpSGP 68 , these examples illustrate that viral proteins commonly use their elements, efficiently mimicking the host 14-3-3-binding peptides, to hijack the cellular functions controlled by 14-3-3 proteins. One of the possible mechanisms is the 14-3-3-mediated stabilization of viral proteins to evade dephosphorylation and degradation, which may prolong the half-life and increase chances for successful replication and further infections 21,68 .…”

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

“…Briefly, an initial pattern-recognition receptor (PRR) pathway recognises and responds to unique viral components (pathogen-associated molecular patterns; PAMPs). In Paramyxoviridae infections, PRRs recognising components of the single-stranded RNA (ssRNA) genome or replication intermediates, such as double-stranded RNA (dsRNA), trigger signalling cascades leading to the activation of latent transcription factors IRF-3 and NF-κB, which translocate to the nucleus, inducing the expression of inflammatory cytokines and IFN genes [ 52 , 53 ].…”