Species-Specific Inhibition of Necroptosis by HCMV UL36

Abstract

Viral infection activates cellular antiviral defenses including programmed cell death (PCD). Many viruses, particularly those of the Herpesviridae family, encode cell death inhibitors that antagonize different forms of PCD. While some viral inhibitors are broadly active in cells of different species, others have species-specific functions, probably reflecting the co-evolution of the herpesviruses with their respective hosts. Human cytomegalovirus (HCMV) protein UL36 is a dual cell death pathway inhibitor. It blocks death receptor-dependent apoptosis by inhibiting caspase-8 activation, and necroptosis by binding to the mixed lineage kinase domain-like (MLKL) protein and inducing its degradation. While UL36 has been shown to inhibit apoptosis in human and murine cells, the specificity of its necroptosis-inhibiting function has not been investigated. Here we show that UL36 interacts with both human and murine MLKL, but has a higher affinity for human MLKL. When expressed by a recombinant mouse cytomegalovirus (MCMV), UL36 caused a modest reduction of murine MLKL levels but did not inhibit necroptosis in murine cells. These data suggest that UL36 inhibits necroptosis, but not apoptosis, in a species-specific manner, similar to ICP6 of herpes simplex virus type 1 and MC159 of molluscum contagiosum virus. Species-specific necroptosis inhibition might contribute to the narrow host range of these viruses.

Keywords: HCMV; ICP6; M45; MCMV; MLKL; RIPK1; RIPK3; UL36; apoptosis; herpesvirus; necroptosis.

Figure 1

Inhibition of apoptosis and necroptosis by HSV-1, HCMV, and MCMV. (a) Schematic representation of death receptor-dependent (extrinsic) apoptosis and necroptosis signaling pathways. The viral inhibitors (marked with virus symbols) and their targets are shown. (b) Known functions of the viral cell death inhibitors in human and murine cells. Figure generated with BioRender.com (accessed on 21 October 2021).

Figure 2

HCMV UL36 co-precipitates and co-localizes with human and murine MLKL. (a) HEK-293A cells were co-transfected with plasmids expressing HA-tagged UL36 and either Flag-tagged mMLKL or Flag-tagged hMLKL. HA-UL36 was immunoprecipitated, and co-precipitating Flag-tagged MLKLs were detected by Western blot. Flag-tagged IFI16 was used as a negative control. (b) HEK-293A cells were transfected with plasmids as described above. Flag-tagged proteins were immunoprecipitated, and co-precipitating HA-UL36 was detected by Western blot. (c) HEK-293A cells were co-transfected with plasmids expressing HA-tagged M36 or UL36 and Flag-tagged mMLKL or hMLKL. HA-tagged proteins were immunoprecipitated, and co-precipitating Flag-tagged MLKLs were detected by Western blot. (d) NIH-3T3 cells were transfected with plasmids encoding HA-UL36 (Red), and Flag-tagged hMLKL or mMLKL (Green). At 24 h post-transfection, the proteins were detected by immunofluorescence using tag-specific antibodies. Nuclei were stained with Hoechst 33342 (Blue). Scale bar, 10 µm.

Figure 3

HCMV UL36 interacts with the N-terminal domain of MLKL. (a) Schematic depiction of hMLKL, consisting of an N-terminal four-helix bundle and a C-terminal pseudokinase domain, and truncation mutants. Hatched regions were deleted. (b) HEK-293A cells were co-transfected with plasmids expressing HA-tagged UL36 and Flag-tagged hMLKL truncation mutants. HA-UL36 was immunoprecipitated, and co-precipitating Flag-MLKLs were detected by Western blot. Flag-tagged IFI16 served as a negative control. (c) HEK-293A cells were transfected with plasmids as in (b). Flag-MLKLs were immunoprecipitated, and co-precipitating HA-UL36 was detected by Western blot. (d) Schematic depiction of mMLKL truncation mutants. (e) HEK-293A cells were co-transfected with plasmids expressing HA-UL36 and Flag-mMLKL truncation mutants. HA-UL36 was immunoprecipitated, and co-precipitating Flag-MLKLs were detected by Western blot. Flag-IFI16 served as a negative control. (f) HEK-293A cells were transfected with plasmids as in (e). Flag-MLKLs were immunoprecipitated, and co-precipitating HA-UL36 was detected by Western blot. + indicates transfection with pcDNA HA-UL36.

Figure 4

HCMV UL36 has stronger affinity to human than to murine MLKL. HEK-293A cells were co-transfected with 2.6 µg of plasmid expressing HA-tagged UL36, 2.6 µg of plasmids expressing myc-tagged mMLKL and increasing concentration (from 0 µg to 2.6 µg, black triangles) of plasmid expressing Flag-tagged hMLKL. HA-tagged UL36 was immunoprecipitated, and co-precipitating myc-tagged mMLKL and Flag-tagged hMLKL were detected by Western blot. Empty vector (EV) plasmid was used to normalize the total amount of transfected DNA. + indicates transfection with HA-UL36 and myc-mMLKL plasmids, respectively.

Figure 5

UL36 expression reduces mMLKL levels in MCMV-infected cells. (a) SVEC4-10 cells were infected with MCMV WT or MCMV-UL36. mMLKL, UL36, and M45 (infection control) levels were determined at different hours post-infection (hpi) by Western blot. (b) SVEC4-10 cells were infected with MCMV-UL36. Six hours before harvest, cells were treated with a lysosomal acidification inhibitor (NH₄Cl) or a proteasome inhibitor (MG-132) or left untreated. mMLKL, UL36, and M45 levels were determined by immunoblot at 24 and 48 hpi. mMLKL levels relative to those of a housekeeping gene product were determined by densitometry and normalized to mock infected cells. + indicates treatment and – lack of treatment with the respective inhibitor.

Figure 6

UL36 inhibits necroptosis in human but not in murine cells. (a) Schematic of MCMV mutants, generated with BioRender (accessed on September 1, 2021). (b) SVEC4-10 cells were infected with MCMVs (MOI 5) and treated 6 hpi with TNFα (T, 30 ng/mL), the SMAC mimetic BV6 (B, 1 µM), the caspase inhibitor zVAD-fmk (V, 75 µM), and the RIPK3 inhibitor GSK’872 (3 µM). Cell viability was determined by measuring ATP levels at 24 hpi. Values were normalized by group to cells treated with both inhibitors (T+B+V+G). (c) SVEC4-10 cells were treated with a caspase inhibitor (zVAD-fmk, 50 µM), a RIPK3 inhibitor (GSK’872, 5 µM), or vector (DMSO, 0.1%), and infected 1 h post-treatment with MCMV mutants. Cell viability was determined by measuring ATP levels at 24 hpi. For MCMV-UL36-M45mutRHIM two independent clones were used. Values were normalized by group to V+G treated cells (d) HT-29 cells were MCMVs infected and treated 6 hpi as in B. Cell viability was determined by measuring ATP levels at 24 hpi. Values were normalized by group to T+B+V+G-treated cells. For MCMV-UL36-M45mutRHIM two independent clones were used. Arrowheads indicate the samples to be compared.