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

Abstract

Henipaviruses are severe human pathogens responsible for severe encephalitis. Their V protein is a key player in the evasion of the host innate immune response. We have previously reported a biophysical characterization of the Henipavirus V proteins and shown that they interact with DDB1, a cellular protein that is a component of the ubiquitin ligase E3 complex. Here, we serendipitously discovered that the Hendra virus V protein undergoes a liquidhydrogel phase transition. By combining experimental and bioinformatics approaches, we have identified the V region responsible for this phenomenon. This region (referred to as PNT3), which falls within the long intrinsically disordered region of V, was further investigated using a combination of biophysical and structural approaches. ThioflavinT and Congo red binding assays, together with negative-staining electron microscopy studies, show that this region forms amyloid-like, β-enriched structures. Such structures are also formed in mammal cells transfected to express PNT3. Those cells also exhibit a reduced viability in the presence of a stress agent. Interestingly, mammal cells expressing a rationally designed, nonamyloidogenic PNT3 variant (PNT3 3A ), appear to be much less sensitive to the stress agent, thus enabling the establishment of a link between fibril formation and cell toxicity. The present findings therefore pinpoint a so far never reported possible mechanism of virusinduced cell toxicity.

Relevance of Electrostatic Charges in Compactness, Aggregation, and Phase Separation of Intrinsically Disordered Proteins

“…Although this protein is classified as a strong polyampholyte (FCR = 0.364, |NCPR| = 0.164), NaCl does not affect its ability to form aggregates (at least up to 300 mM). In support for the hypothesis of the involvement of π–π interactions, the most amyloidogenic region contains three contiguous tyrosine residues whose replacement with alanine residues abrogates fibrillation [ 147 ]. Finally, we can cite the case of γ44-gliadin, a wheat storage protein with an intrinsically disordered domain that undergoes LLPS in a salt-dependent manner in spite of its very weakly charged nature (FCR = 0.04 and NCPR = 0) [ 148 ].…”

“…Examples also exist where salt, namely NaCl, does not exert any significant impact on LLPS, suggesting that the formation of coacervates relies on hydrophobic interactions. An illustrative example is provided by PNT3, a viral protein region that undergoes LLPS with concomitant formation of amyloid-like fibrils [ 147 ]. Although this protein is classified as a strong polyampholyte (FCR = 0.364, |NCPR| = 0.164), NaCl does not affect its ability to form aggregates (at least up to 300 mM).…”

Liquid–Liquid Phase Separation by Intrinsically Disordered Protein Regions of Viruses: Roles in Viral Life Cycle and Control of Virus–Host Interactions

“…In the same vein, the V protein from Hendra virus (HeV, a paramyxovirus) was shown to undergo a liquid-to-gel phase transition in vitro. The minimal V region responsible for this property, referred to as PNT3, has been identified within the intrinsically disordered N-terminal domain of V [207]. PNT3 was found to form amyloid-like fibrils both in the liquid and hydrogel state.…”

Experimental Evidence of Intrinsic Disorder and Amyloid Formation by the Henipavirus W Proteins

“…In line with these properties, we previously reported that HeV V jellifies in vitro. The minimal HeV V region responsible for this ability (referred to as PNT3, aa 200-310 of HeV V) was identified within its intrinsically disordered NTD [82,83]. Binding assays to an amyloid-specific dye and negative-staining transmission electron microscopy (TEM) studies, showed that PNT3 forms amyloid-like fibrils [82,83].…”