IISER MOHALI

Abstract: 

The Hendra (HeV) and Nipah (NiV) viruses are zoonotic BSL-4 paramyxoviruses responsible for severe respiratory and neurological disease. In both viruses, the gene encoding the Phosphoprotein (P protein) also encodes the V and W proteins. The P, V and W proteins share a common, intrinsically disordered N-terminal domain (NTD) and have unique and distinct C-terminal domains (CTD). V and W are two virulence factors that are responsible for evasion of the host innate immune system. They act either counteracting or inhibiting Interferon (IFN) signaling. Using a combination of biophysical approaches, we showed that the NTD is intrinsically disordered not only in isolation but also in the context of the V protein, i.e. when appended upstream to the CTD of V that was found to adopt a zinc-finger conformation and to play a major role in V binding to DDB1. We serendipitously discovered that the HeV V protein undergoes a liquid-to-hydrogel phase transition and identified the V region responsible for this phenomenon. This region, referred to as PNT3, was also found to be able to form amyloid-like fibrils. Noteworthy, Congo red staining experiments provided hints that these amyloid-like fibrils form not only in vitro but also in cellula. Using mutational approaches coupled to various biophysical approaches and electron microscopy, we next deciphered the molecular grammar of PNT3 fibrillation. Subsequently, we showed that the ability to form fibrils is a property also shared by the intrinsically disordered W proteins. We then found that the cysteine oxidation state acts as a molecular switch controlling the formation of either amorphous aggregates or flexible fibrils, while residues 1 to 29 are essential for fibrillation. We also demonstrated that HeV W can self-assemble in cellula. HeV W forms distinct types of nuclear condensates that exhibit different dependencies on the cysteine redox-state. While deletion of residues 1-29 prevents formation of nuclear filaments, cysteine-to-serine substitution mainly impairs the formation of non-filamentous condensates. Finally, we showed that impaired ability to form redox-sensitive, non-filamentous condensates is associated with a reduced W ability to inhibit the NF-κB pathway, while it conversely enhances W ability to repress the interferon response. Collectively, these studies shed light on the molecular mechanisms underlying the ability of V/W to counteract the host innate immune response and contribute to a better understanding of Henipavirus pathogenesis.