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

(A) Transmission electron micrograph of protein assemblies formed from combinations of Ub-ORF20_1-114, Ub-ORF20_1-114mut, and YPet-ZBP1_170-355 fusion proteins. Scale bar represents 200 nm. (B) Confocal microscope images of heterofibrils formed from a mixture of ORF20_1-114-mCherry and YPet-ZBP1_170-355 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-ORF20_1-114, Ub-ORF20_1-114mut, and YPetZBP1_170-355, 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.