ppat.1008473.g009.tif (2.57 MB)

The VZV ORF20 RHIM forms heteromeric amyloid fibrils with ZBP1, which are dependent on the core tetrad for stable higher-order assembly.

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posted on 10.07.2020, 17:39 by Megan Steain, Max O. D. G. Baker, Chi L. L. Pham, Nirukshan Shanmugam, Yann Gambin, Emma Sierecki, Brian P. McSharry, Selmir Avdic, Barry Slobedman, Margaret Sunde, Allison Abendroth

(A) Transmission electron micrograph of protein assemblies formed from combinations of Ub-ORF201-114, Ub-ORF201-114mut, and YPet-ZBP1170-355 fusion proteins. Scale bar represents 200 nm. (B) Confocal microscope images of heterofibrils formed from a mixture of ORF201-114-mCherry and YPet-ZBP1170-355 in ThT-containing assembly buffer. Scale bar represents 20 μm. (C) The IQIG core tetrad from ORF20 is required to trap ZBP1 in insoluble, detergent-stable aggregates. SDS-AGE analysis of monomeric or assembled forms of Ub-ORF201-114, Ub-ORF201-114mut, and YPetZBP1170-355, or combinations thereof. Monomeric proteins were maintained in 8 M urea before electrophoresis. Assembled samples were incubated with either water or 2% SDS at room temperature for 10 min before electrophoresis. Identity and treatment of protein indicated above each sample.