Molecular Determinants of Fibrillation in a Viral Amyloidogenic Domain from Combined Biochemical and Biophysical Studies

Abstract

The Nipah and Hendra viruses (NiV and HeV) are biosafety level 4 human pathogens classified within the Henipavirus genus of the Paramyxoviridae family. In both NiV and HeV, the gene encoding the Phosphoprotein (P protein), an essential polymerase cofactor, also encodes the V and W proteins. These three proteins, which share an intrinsically disordered N-terminal domain (NTD) and have unique C-terminal domains (CTD), are all known to counteract the host innate immune response, with V and W acting by either counteracting or inhibiting Interferon (IFN) signaling. Recently, the ability of a short region within the shared NTD (i.e., PNT3) to form amyloid-like structures was reported. Here, we evaluated the relevance of each of three contiguous tyrosine residues located in a previously identified amyloidogenic motif (EYYY) within HeV PNT3 to the fibrillation process. Our results indicate that removal of a single tyrosine in this motif significantly decreases the ability to form fibrils independently of position, mainly affecting the elongation phase. In addition, we show that the C-terminal half of PNT3 has an inhibitory effect on fibril formation that may act as a molecular shield and could thus be a key domain in the regulation of PNT3 fibrillation. Finally, the kinetics of fibril formation for the two PNT3 variants with the highest and the lowest fibrillation propensity were studied by Taylor Dispersion Analysis (TDA). The results herein presented shed light onto the molecular mechanisms involved in fibril formation.

Key Factors Controlling Fibril Formation of Proteins

“…The SARS-CoV-2 nucleocapsid protein (N protein) accelerates αS fibrillation through electrostatic interactions, inducing cell death [192]. It has been shown that SARS-CoV-2 proteins can also form aggregates in isolation [193,194], and similar results have been obtained for other virus species, such as the Hendra and Nipah viruses [195]. Based on the observation that human amyloids can interact with viruses, interfering with their replication, protein aggregation has been proposed as a strategy to discover new antiviral agents [196].…”

Host–Pathogen Interactions Influencing Zoonotic Spillover Potential and Transmission in Humans

“…The maturation and nucleation of the condensates formed via liquid–liquid phase separation can give rise to fibrils with amyloidogenic properties [ 103 , 104 ]. Despite the extensive literature regarding phase separation [ 100 ], the study of virus-derived amyloid-like fibrils and their role in pathogenesis is still in its infancy [ 102 , 105 , 106 , 107 ]. Attention to this field has notably increased in the last couple of years with the demonstration of amyloidogenesis by the Spike, nucleoprotein and envelope proteins of SARS-CoV-2 and their possible involvement in pathogenesis [ 108 , 109 , 110 , 111 ].…”

Biological Liquid–Liquid Phase Separation, Biomolecular Condensates, and Membraneless Organelles: Now You See Me, Now You Don’t

“…Juliet F. Nilsson, Hakima Baroudi, Frank Gondelaud, Giulia Pesce, Christophe Bignon, Denis Ptchelkine, Joseph Chamieh, Hervé Cottet, Andrey V. Kajava, and Sonia Longhi also studied the formation of amyloid fibrils, but analyzed the aggregation behavior of an interesting viral protein, the phosphoprotein (P protein) from the Nipah and Hendra viruses (NiV and HeV), which is an essential polymerase cofactor [21]. A peculiar feature of this system is that the gene encoding the P protein in both NiV and HeV also encodes the V and W proteins.…”

“…This analysis revealed that the ability to form fibrils is dramatically reduced by the removal of a single tyrosine independently of its position. Furthermore, the authors showed that PNT3 fibrillation can be regulated by the C-terminal half of this protein, showing an inhibitory effect on fibril formation [21]. In their research article, Mohtadin Hashemi, Siddhartha Banerjee, and Yuri L. Lyubchenko investigated the effects of membrane and free cholesterol on the early stages of the aggregation process of amyloid β (Aβ) [20].…”

“…This analysis revealed that the ability to form fibrils is dramatically reduced by the removal of a single tyrosine independently of its position. Furthermore, the authors showed that PNT3 fibrillation can be regulated by the C-terminal half of this protein, showing an inhibitory effect on fibril formation [21].…”