The Henipavirus V protein is a prevalently unfolded protein with a zinc-finger domain involved in binding to DDB1

Abstract

Henipaviruses are severe human pathogens within the Paramyxoviridae family. Beyond the P protein, the Henipavirus P gene also encodes the V protein which shares with P its N-terminal, intrinsically disordered region (PNT) and possesses a unique C-terminal domain predicted to be folded and to bind zinc (ZnFD). Henipavirus V proteins antagonize IFN signaling through PNT-mediated binding to STAT1, and several paramyxoviral V proteins promote STAT1 degradation through binding to DDB1. Structural and molecular information on Henipavirus V proteins is lacking, and their ability to interact with DDB1 has not been documented yet. We cloned the V genes from Nipah and Hendra viruses and purified the V proteins from E. coli and DDB1 from insect cells. Using analytical size-exclusion chromatography, CD and SAXS we characterized the V proteins and their domains. Using pull-down and MST we assessed their binding abilities towards DDB1. We show that PNT remains disordered also in the context of the V protein, while the ZnFD adopts a predominant β conformation. We also show that the V proteins interact with DDB1 predominantly via their ZnFD. This is the first experimental characterization of the Henipavirus V proteins and the first experimental evidence of their interaction with DDB1. The DDB1-ZnFD interaction constitutes a promising target for antiviral strategies. These studies provide a conceptual asset to design new antiviral strategies expected to reduce or abrogate the ability of these viruses to escape the innate immune response. They also contribute to illuminating the conformational behaviour of proteins encompassing large intrinsically disordered domains.

Phase transition and amyloid formation by a viral protein as an additional molecular mechanism of virus-induced cell toxicity

“…This activity relies on the ability of their V protein to bind to DNA damage-binding protein 1 (DDB1), a component of the ubiquitin ligase E3 complex, and then to recruit STAT proteins onto this complex [22]. In line with this, we have recently reported that Henipavirus V proteins interact with DDB1, and have unveiled a critical contribution of their C-terminal domain [23]. Incidentally, we also confirmed that the latter is folded and has a high β content both in isolation and when appended to PNT.…”

“…Out of the two already available HeV domains (i.e. ZnFD and PNT) [8,23], only PNT was found to be able to form a hydrogel under conditions similar to those used for HeV V (Figure 1C). We subsequently divided the HeV PNT domain in four overlapping fragments (referred to as PNT1-PNT4) of 110 residues each ( Figure 1A).…”

“…The constructs encoding Henipavirus V proteins, as well as their Zinc-finger and PNT domains, have already been described [8,23].…”

“…The purification protocol of the Henipavirus V proteins and of the HeV V ZnFD have been already reported [23], as was that of HeV PNT [8]. The PNT1-PNT4, PNT3 3A and PNT3-GFP proteins were purified as follows.…”

Ensemble description of the intrinsically disordered N-terminal domain of the Nipah virus P/V protein from combined NMR and SAXS

“…The crystal structures of NiV PMD (PDB code 4N5B) 8 and of the NiV N°-P NTD complex (PDB code 4CO6) 4 are shown. The homology-derived models of NiV XD 9 and of the ZnFD of V are also shown 10 . All structures were drawn using Pymol 2.0.1 (https ://pymol .org/2/) 11 .…”