Identification of a Region in the Common Amino-terminal Domain of Hendra Virus P, V, and W Proteins Responsible for Phase Transition and Amyloid Formation

Abstract

Henipaviruses are BSL-4 zoonotic pathogens responsible in humans for severe encephalitis. Their V protein is a key player in the evasion of the host innate immune response. We previously showed that the Henipavirus V proteins consist of a long intrinsically disordered N-terminal domain (NTD) and a β-enriched C-terminal domain (CTD). These terminals are critical for V binding to DDB1, which is a cellular protein that is a component of the ubiquitin ligase E3 complex, as well as binding to MDA5 and LGP2, which are two host sensors of viral RNA. Here, we serendipitously discovered that the Hendra virus V protein undergoes a liquid-to-hydrogel phase transition and identified the V region responsible for this phenomenon. This region, referred to as PNT3 and encompassing residues 200–310, was further investigated using a combination of biophysical and structural approaches. Congo red binding assays, together with negative-staining transmisison electron microscopy (TEM) studies, show that PNT3 forms amyloid-like fibrils. Fibrillation abilities are dramatically reduced in a rationally designed PNT3 variant in which a stretch of three contiguous tyrosines, falling within an amyloidogenic motif, were replaced by three alanines. Worthy to note, Congo red staining experiments provided hints that these amyloid-like fibrils form not only in vitro but also in cellula after transfection or infection. The present results set the stage for further investigations aimed at assessing the functional role of phase separation and fibrillation by the Henipavirus V proteins.

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

“…This apparent conflicting behavior might arise from the much higher aggregation propensity of the HeV W protein that may hinder protease cleavage. This observation mirrors our previous studies where the HeV V protein was found to jellify under conditions where NiV V did not [82,83].…”

“…In line with the ability of the HeV V PNT3 region, common to both the V and W proteins, to undergo a liquid-to-gel transition and to form amyloid-like fibers [82,83], we herein show that both the HeV and NiV W proteins form phase separated condensates that bind the amyloid-specific CR and ThT dyes.…”

“…Under the assumption that fibrillation also occurs in cellula, it is tempting to hypothesize that the phase separation and fibrillation abilities of the Henipavirus V and W proteins might be related to interference with the host innate immune response. In addition, it should be kept in mind that the minimal region of the HeV V protein conferring the ability to phase separate (i.e., PNT3) [82,83] is also part of the P protein that is a constituent of the viral factories of Mononegavirales members [102,103,109]. Therefore, it is conceivable that the Henipavirus P protein may phase separate and fibrillate as well, with this property being possibly functionally coupled to the formation of viral factories.…”

“…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].…”

Molecular determinants of fibrillation in a viral amyloidogenic domain from combined biochemical and biophysical studies

“…The PNT3 3A variant shows a significant increase in the relative solubility compared to PNT3 HeV wt (Fig. 4A and 4B), a result in agreement with previous findings that pointed out a much lower propensity to form amyloid-like fibrils for PNT3 3A [25]. The three variants where only one Tyr was replaced, however, show no significant difference in the PEG1/2 values compared to HeV PNT3 wt (Fig.…”