Abstract
Human cytomegalovirus (HCMV) is an important human pathogen and a paradigm of intrinsic, innate, and adaptive viral immune evasion. Here, we employed multiplexed tandem mass tag-based proteomics to characterize host proteins targeted for degradation late during HCMV infection. This approach revealed that mixed lineage kinase domain-like protein (MLKL), a key terminal mediator of cellular necroptosis, was rapidly and persistently degraded by the minimally passaged HCMV strain Merlin but not the extensively passaged strain AD169. The strain Merlin viral inhibitor of apoptosis pUL36 was necessary and sufficient both to degrade MLKL and to inhibit necroptosis. Furthermore, mutation of pUL36 Cys131 abrogated MLKL degradation and restored necroptosis. As the same residue is also required for pUL36-mediated inhibition of apoptosis by preventing proteolytic activation of procaspase-8, we define pUL36 as a multifunctional inhibitor of both apoptotic and necroptotic cell death.
Highlights
Human cytomegalovirus (HCMV) infects the majority of the world’s population, with a seroprevalence of 60 to 80% in Western Europe and the United States, and up to 100% in developing countries [1, 2]
MG132 is known to inhibit calpains and lysosomal cathepsins in addition to the proteasome [10] and was used to generate a comprehensive list of proteins targeted for degradation by HCMV rather than to identify proteins degraded by the proteasome
The present study provides a systematic, searchable database that examines host protein degradation at 48 h of HCMV strain Merlin infection, ∼50 to 66% of the way through the lytic replication cycle in HFFFs
Summary
Human cytomegalovirus (HCMV) infects the majority of the world’s population, with a seroprevalence of 60 to 80% in Western Europe and the United States, and up to 100% in developing countries [1, 2]. We have shown previously that >900 host proteins are down-regulated more than threefold over the course of HCMV infection, with 133 proteins degraded during the early phase, of which 89% are targeted to the proteasome These data led directly to the identification of candidate natural killer (NK) cell ligands and identified helicase-like transcription factor (HLTF) as an antiviral restriction factor [10, 11]. It is not yet known which proteins are degraded later during infection, or throughout a whole infection time course. Necroptosis can be activated by cytoplasmic sensing of murine cytomegalovirus (MCMV) DNA by Z-DNA-binding protein 1 (ZBP1), or ligation of Toll-like receptors 3 or 4 (TLR3/4), Significance
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