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Herpesviruses are ubiquitous human pathogens that cause a wide range of health complications. Currently, there is an incomplete understanding of cellular factors that contribute to herpesvirus infection. Here, we report an antiviral necroptosis-based genetic screen to identify novel host cell factors required for infection with the β-herpesvirus murine cytomegalovirus (MCMV). Our genome-wide CRISPR-based screen harnessed the capacity of herpesvirus mutants that trigger antiviral necroptotic cell death upon early viral gene expression. Vascular endothelial growth factor (VEGF) and semaphorin-binding receptor Neuropilin-1 (Nrp-1) emerge as crucial determinants of MCMV infection. We find that elimination of Nrp-1 impairs early viral gene expression and reduces infection rates in endothelial cells, fibroblasts, and macrophages. Furthermore, preincubation of virus with soluble Nrp-1 dramatically inhibits infection by reducing virus attachment. Thus, Nrp-1 is a key determinant of the initial phase of MCMV infection., Competing Interests: The authors declare no competing interest.

Necroptosis is an inflammatory form of programmed cell death executed through plasma membrane rupture by the pseudokinase mixed lineage kinase domain-like (MLKL). We previously showed that MLKL activation requires metabolites of the inositol phosphate (IP) pathway. Here we reveal that I(1,3,4,6)P 4 , I(1,3,4,5,6)P 5 , and IP 6 promote membrane permeabilization by MLKL through directly binding the N-terminal executioner domain (NED) and dissociating its auto-inhibitory region. We show that IP 6 and inositol pentakisphosphate 2-kinase (IPPK) are required for necroptosis as IPPK deletion ablated IP 6 production and inhibited necroptosis. The NED auto-inhibitory region is more extensive than originally described and single amino acid substitutions along this region induce spontaneous necroptosis by MLKL. Activating IPs bind three sites with affinity of 100–600 μM to destabilize contacts between the auto-inhibitory region and NED, thereby promoting MLKL activation. We therefore uncover MLKL's activating switch in NED triggered by a select repertoire of IP metabolites. • MLKL N-terminal executioner domain (NED) is auto-inhibited by the linker-brace • Inositol pentakisphosphate 2-kinase (IPPK) synthesizes IP 6 to regulate necroptosis • Selective inositol phosphate (IP) repertoire binds three distinct sites in NED • Hierarchical direct activation of MLKL is induced by three IPs (IP 6 >IP 4 ≥IP 5) McNamara et al. demonstrate that MLKL is extensively auto-inhibited and that several inositol phosphates and their enzymes, but not the putative lipid ligand phosphatidylinositol-4,5-bisphosphate, are required for MLKL activation in necroptosis. These inositol phosphates cooperate with RIPK3 phosphorylation potently activating MLKL to rupture the plasma membrane in necroptosis. [ABSTRACT FROM AUTHOR]