Abstract
Viruses manipulate cellular signalling by inducing the degradation of crucial signal transducers, usually via the ubiquitin–proteasome pathway. Here, we show that the murine cytomegalovirus (Murid herpesvirus 1) M45 protein induces the degradation of two cellular signalling proteins, the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) essential modulator (NEMO) and the receptor-interacting protein kinase 1 (RIPK1), via a different mechanism: it induces their sequestration as insoluble protein aggregates and subsequently facilitates their degradation by autophagy. Aggregation of target proteins requires a distinct sequence motif in M45, which we termed ‘induced protein aggregation motif’. In a second step, M45 recruits the retromer component vacuolar protein sorting 26B (VPS26B) and the microtubule-associated protein light chain 3 (LC3)-interacting adaptor protein TBC1D5 to facilitate degradation of aggregates by selective autophagy. The induced protein aggregation motif is conserved in M45-homologous proteins of several human herpesviruses, including herpes simplex virus, Epstein–Barr virus and Kaposi’s sarcoma-associated herpesvirus, but is only partially conserved in the human cytomegalovirus UL45 protein. We further show that the HSV-1 ICP6 protein induces RIPK1 aggregation and degradation in a similar fashion to M45. These data suggest that induced protein aggregation combined with selective autophagy of aggregates (aggrephagy) represents a conserved viral immune-evasion mechanism.
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Data availability
The datasets generated and analysed during the course of this study are available from the corresponding author upon request without restrictions. Uncropped western blot images of all figures in the manuscript (Figs. 1–5, Extended Data Figs. 3, 4, 6 and 7) and numerical data with statistical analysis (Figs. 2, 3 and 5) are provided as supplementary source data.
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Acknowledgements
We thank J. Connor, J. Bertin and E. Mocarski for RIPK3 kinase-dead mice, S. Jonjic, Y. Kawaguchi and R. Teasdale for reagents, F. Giraudo for technical assistance and T. Potgieter for critical readings of the manuscript. This study was supported by funding from Deutsche Forschungsgemeinschaft (BR 1730/3-2 to WB). The Heinrich Pette Institute is supported by the Free and Hanseatic City of Hamburg and the Federal Ministry of Health. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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E.M. designed, performed and analysed most experiments. R.S. performed a few biochemical experiments. E.K. and S.L. performed SILAC and AP–MS analyses. A.S. supervised the mass spectrometry analyses. E.C. performed and analysed FRAP experiments. M.R. generated and analysed TBC1D5-deficient cell clones. C.S. and R.R. performed correlative light and electron microscopy analyses. Y.-H.K. provided mice and biochemical reagents. V.J.L. provided biochemical reagents. E.O. performed in vivo experiments. W.B. designed and supervised the study, acquired funding and provided resources. E.M. and W.B. wrote and edited the manuscript.
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Extended data
Extended Data Fig. 1 M45 induced aggregates.
(a) Atg5-/- MEFs were transfected with an M45-mCherry plasmid. 24 h post transfection cells were fixed and analyzed by fluorescence microscopy. Nuclei were stained with Hoechst 33342. Scale bar, 10 µm. (b) NIH-3T3 cells transfected with plasmids expressing HA-tagged full-length M45 or M45-Ct3. HA-tagged proteins were detected by immunofluorescence (green), protein aggregates by using the ProteoStat dye (red). (c) Another two ultrathin sections of the same WT MEF cell shown in Fig. 1f (ix – xi). The arrow points to a different aggregate than the one in Fig. 1f. Magnification of the same aggregate (A) close to an autophagosome (*) in the two different sections (x – xii). Scale bars, 5 µm (ix, xi) and 200 nm (x, xii). (d) Maximum intensity projection of NIH-3T3 cell transfected with a plasmid encoding M45-mCherry and observed 6 h post transfection by live cell imaging for 30 h. Scale bar, 10 µm. These data are representative of three (a-b) or two (d) biologically independent experiments. This data (c) is representative of three independent infected cells per group where at least 10 different sections (50 nm) per cells were analyzed.
Extended Data Fig. 2 Proteins co-purifying with M45 detected by AP-MS.
NIH-3T3 cells were labelled by SILAC and infected with MCMV WT or MCMV-M45HA. Cell lysates were harvested 15 hpi and subjected to anti-HA affinity-purification. Purified were analyzed by mass spectrometry.
Extended Data Fig. 3 M45 interacts with VPS26B, independent of VPS26B’s interaction with VPS35, and TBC1D5.
(a) Schematic of WT and mutant VPS26B. (b) HEK-293A cells were co-transfected with VPS26B-myc and VPS35-Flag plasmids. Immunoprecipitations were done as indicated. (c) HEK-293A cells co-transfected with plasmids expressing M45- HA and myc-tagged VPS26B or VPS26A (negative control). M45-HA was immunoprecipitated. (d) NIH-3T3-Vps26B-myc cells infected with MCMV-M45HA, MCMV-M45mut2HA or HA- tagged M45 C-terminal truncation mutants (MOI 3). Immunoprecipitation was done with an anti-HA antibody. (e) MEFs were infected with MCMV-M45HA or MCMVΔM45 (MOI 3). (f) VPS35, VPS26A, VPS29, and viral protein levels were determined by immunoblot at different times post infection. HEK-293A cells co-transfected with plasmids expressing M45-HA and Flag-TBC1D5 or Flag-IFI16 (negative control) or Flag-NEMO (positive control). HA was immunoprecipitated. (g) HEK-293A cells co-transfected with plasmids expressing M45- HA full-length or C-terminus truncation mutants (HA tagged) and Flag-TBC1D5. Immunoprecipitation was done with an anti-HA antibody. (h) WT and Vps26b-/- MEFs were infected with MCMV-M45HA (MOI 3). Immunoprecipitation was done with an anti-HA antibody. Immunoblot labels are in kDa. These data are representative of two (b-e-h) or three (c-d-f-g) biologically independent experiments.
Extended Data Fig. 4 HSV-1 ICP6 induces aggregate formation.
(a) Ripk3-/- fibroblasts were transfected with plasmids expressing WT or mutant ICP6. 24 h post transfection protein aggregates were detected by using the ProteoStat dye (red) and HA-tagged ICP6 by immunofluorescence (green). Scale bar, 10 µm. (b) HFF were infected with WT or ICP6mut HSV-1 (MOI 1). 24 hpi cells were fixed and stained for LC3BII (green). Nuclei were stained with Hoechst 33342. Scale bar, 10 µm. (c) HFF infected as in b. LC3BII was detected by immunoblot at 8 and 24 hpi. (d) HFF were infected with HSV-1 ICP6HA or ICP6mutHA (MOI 1). 24 hpi cells were fixed and stained for gamma-tubulin (green) and HA (red). Nuclei were stained with Hoechst 33342. Scale bar, 10 µm. Immunoblot labels are in kDa. These data are representative of two (a-b-c) or three (d) biologically independent experiments.
Extended Data Fig. 5 M45 aggregates co-localize with LC3BII.
(a, b) NIH-3T3 infected with MCMV-M45HA or MCMV- M45mut2HA (MOI 3). 24 hpi cells were fixed and stained for HA (red) and either LC3BII (a) or Caveolin-1 (b) (green). Nuclei were stained with Hoechst 33342. Scale bar, 10 µm. These data (a-b) are representative of three biologically independent experiments.
Extended Data Fig. 6 M45-interacting regions in NEMO and RIPK1 do not share a common motif.
(a) HEK-293A cells were co-transfected with plasmids expressing M45-HA and Flag-tagged RIPK1. Full-length (FL) and deletion mutants lacking the N-terminus, the C-terminus, or the death domain (DD) were used. M45-HA was immuno- precipitated and co-precipitating proteins were detected by immunoblot. (b) HEK-293A cells were co-transfected with plasmids expressing M45-HA and Flag-tagged NEMO (FL or N- terminal truncation mutants). M45-HA was immuno- precipitated and co-precipitating proteins were detected by immunoblot. (c) Sequence alignment of M45-interacting regions in NEMO and RIPK1 with APOBEC3B. Immunoblot labels (a-b) are in kDa. These data are representative of two (a) or three (b) biologically independent experiments.
Extended Data Fig. 7 M45 aggregates do not co-localize with HSP70.
(a) NIH-3T3 were infected with MCMV-M45HA or MCMV- M45mut2HA (MOI 3). 24 hpi cells were fixed and immunostained for HA (red) and HSP70 (green). Nuclei were stained with Hoechst 33342. Scale bar, 10 µm. (b) Immunoblot analysis of the soluble and insoluble fractions of MCMV- M45HA and MCMV-M45mut2 infected Atg5-/- MEFs (MOI 5). These data (a-b) are representative of three biologically independent experiments.
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Muscolino, E., Schmitz, R., Loroch, S. et al. Herpesviruses induce aggregation and selective autophagy of host signalling proteins NEMO and RIPK1 as an immune-evasion mechanism. Nat Microbiol 5, 331–342 (2020). https://doi.org/10.1038/s41564-019-0624-1
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DOI: https://doi.org/10.1038/s41564-019-0624-1
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