Fig 1.
VZV ORF20 contains a RHIM that inhibits TNF-induced necroptosis in HT-29 adenocarcinoma cells.
(A) Schematic diagram of RHIM-containing proteins. RHIM location indicated by yellow box. (B) Amino acid sequence alignment of the new RHIM identified in VZV ORF20 (Dumas strain) with other known human cellular RHIMs from RIPK3, RIPK1, TRIF and ZBP1 and the viral RHIMs from MCMV (M45), HSV-1 (ICP-6) and HSV-2 (ICP10), also indicating the percentage of conservation and consensus sequence, with the RHIM core tetrad boxed. (C) Cytopathic effect and immunofluorescence staining for VZV antigens (IE62, pORF29 and the gE:gI glycoprotein complex, green) in VZV-infected and mock HT-29s. Following immunostaining cells were counterstained with DAPI (blue). Scale bars indicate 20 μm (D) Viability of mock and VZV infected HT-29s (72 h post-infection) following treatments with TNF (T; 30 ng/ml), BV-6 (S; 1 μM), z-VAD-fmk (V; 25 μM) and necrostatin-1 (Nec1; 30 μM) alone or in combination as indicated. Data was normalised to DMSO only control. Error bars show standard error of the mean, from 4 independent replicates, statistical significance was determined using a two-way ANOVA.
Fig 2.
VZV infection inhibits phosphorylation of MLKL during TNF-induced necroptosis.
(A) Immunofluorescence staining for phosphorylated MLKL (red) and VZV IE62 antigen (green) in mock and VZV infected HT-29 adenocarcinoma cells untreated (DMSO control) or treated with TNF (T; 30 ng/ml), BV-6 (S; 1 μM) and z-VAD-fmk (V; 25 μM) for 7–8 h to induce necroptosis. Following immunostaining cells were counterstained with DAPI (blue). (B) The percentage of cells that were pMLKL positive was determined by randomly imaging 10–20 non-overlapping regions of each slide and manually counting cells from 3 independent experiments. Error bars show standard error of the mean, statistical significance was determined using a one-way ANOVA. Scale bar indicates 20 μm.
Fig 3.
The VZV ORF20 RHIM is not involved in TNF-induced necroptosis.
(A) Viability of mock, VZV and VZV RHIM mutated (VZV-RHIMmut) virus infected HT-29s (72 h post-infection) following treatments with TNF (T; 30 ng/ml), BV-6 (S; 1 μM), z-VAD-fmk (V; 25 μM) and necrostatin-1 (Nec1; 30 μM) alone or in combination as indicated. Data was normalised to DMSO only control. Error bars show standard error of the mean, from 4 independent replicates and statistical significance was determined using a two-way ANOVA.
Fig 4.
ZBP1 restricts VZV-RHIMmut and VZV-RHIMKO spread in culture.
Empty vector or ZBP1 expressing HT-29s were inoculated with parental HT-29 cells either mock or infected with parent VZV (pOKA), VZV-RHIMmut or VZV-RHIMKO. z-VAD–fmk (25 μM) or necrosulfonamide (NSA; 1 μM) was added at the time of inoculation where indicated. (A) After 72 h cells were fixed and immunostained for VZV IE63 (red) to assess virus spread. Images are representative from at least 2 independent replicates. Scale bar indicates 500 μm. (B) The area of 18–20 plaques per virus (as indicated) was calculated using Zen 3.1 Blue edition (Zeiss), and statistical significance calculated using a one-way ANOVA. Line indicates mean and error bars represent standard error of the mean.
Fig 5.
ZBP1 does not restrict the spread of VZV or VZV-RHIMmut in RIPK3-deficient, apoptosis-capable cells.
Empty vector or ZBP1 expressing ARPE-19s were inoculated with parental ARPE-19 cells infected with parent VZV (pOKA) or VZV-RHIMmut. z-VAD-fmk (25 μM) was added at the time of inoculation where indicated. After 72 h cells were fixed and immunostained for VZV IE63 (red) to assess virus spread. Images are representative from at least 2 independent replicates. Scale bars represent 400 μm.
Fig 6.
ORF20 interacts with human RHIM proteins associated with cell death signalling.
293T cells were transfected with ORF20 constructs and ZBP1-GFP or GFP alone. Cells were harvested and immunoprecipitation (IP) performed on the soluble and insoluble fractions using GFP as bait. Western blot (IB) was performed on the immunoprecipitated and input cell lysates. (A) Immunoprecipitation of ZBP1-GFP in the soluble fraction of cell lysates. (B) Immunoprecipitation of ZBP1-GFP in the insoluble fraction of cell lysates. (C) Immunoprecipitation of RIPK3-GFP in the soluble fraction of cell lysates. Each blot is representative of at least 2 independent biological replicates. Arrows indicate protein size markers.
Fig 7.
The ORF20 RHIM forms homomeric amyloid structures.
(A) Transmission electron microscopy image of structures formed by Ub-ORF201-114. Scale bar represents 200 nm. (B) Transmission electron microscopy image of structures formed by mCherry-ORF201-114. Scale bar represents 200 nm. (C) ThT fluorescence over time of Ub-ORF20 constructs after dilution from 8 M urea into assembly buffer. Buffer sample contains equimolar ThT but no protein. Curves are derived from three independent replicates. Error bars indicate standard deviation. (D) Absorbance spectra of solutions containing Congo red and Ub-ORF201-114 and Ub-ORF201-114mut after dialysis against assembly buffer. Buffer refers to a Congo red sample in assembly buffer with no protein. (E) Transmission electron microscopy image of structures formed by Ub-ORF201-114mut. Scale bar represents 200 nm. (F) Transmission electron microscopy image of structures formed by mCherry-ORF201-114mut. Scale bar represents 200 nm.
Fig 8.
The VZV ORF20 RHIM forms heteromeric complexes with RHIMs from RIPK3 and ZBP1.
(A) Schematic representation of fluorescence detection from dilute solutions containing two different fluorophores in a nanolitre confocal volume. Samples are excited simultaneously with two overlapping lasers and emission from the YPet and mCherry fluorophores is recorded separately. Coincident bursts in the two channels indicate formation of heteromeric complexes containing two different proteins (B-I). Representative fluorescent time traces collected from ORF201-114-mCherry, ORF201-114mut-mCherry, YPet-RIPK3387-518 or YPet-ZBP1170-355 fusion proteins, alone or mixed in pairs under conditions that allow co-assembly. The proteins present in each mixture are indicated for each part of the figure. Inserts show detail of 1 s dual fluorescence recording, indicated by star on the full time trace.
Fig 9.
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-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.
Fig 10.
Proposed mechanism for VZV ORF20 RHIM during VZV infection.
Upon VZV infection of a host cell the viral DNA is transported to the nucleus with some potentially leaked into the cytoplasm. Viral DNA is then transcribed into RNA and exported from the nucleus. ZBP1 can sense either VZV RNA or DNA and then via RHIM:RHIM interactions activate RIPK3 to drive apoptosis. However, the ORF20 RHIM, either from incoming viral capsids, or following de novo synthesis can interact with ZBP1 to prevent it driving apoptosis and thus prolong the survival of the infected cell to allow for the complete viral replication cycle to occur.