NMS基因敲除HEK293细胞

This study developed a system consisting of two rounds of screening cellular proteins involved in the nuclear egress of herpes simplex virus 1 (HSV-1). Using this system, we first screened cellular proteins that interacted with the HSV-1 nuclear egress complex (NEC) consisting of UL34 and UL31 in HSV-1-infected cells, which are critical for the nuclear egress of HSV-1, by tandem affinity purification coupled with mass spectrometry-based proteomics technology. Next, we performed CRISPR/Cas9-based screening of live HSV-1-infected reporter cells under fluorescence microscopy using single guide RNAs targeting the cellular proteins identified in the first proteomic screening to detect the mislocalization of the lamin-associated protein emerin, which is a phenotype for defects in HSV-1 nuclear egress. This study focused on a cellular orphan transporter SLC35E1, one of the cellular proteins identified by the screening system. Knockout of SLC35E1 reduced HSV-1 replication and induced membranous invaginations containing perinuclear enveloped virions (PEVs) adjacent to the nuclear membrane (NM), aberrant accumulation of PEVs in the perinuclear space between the inner and outer NMs and the invagination structures, and mislocalization of the NEC. These effects were similar to those of previously reported mutation(s) in HSV-1 proteins and depletion of cellular proteins that are important for HSV-1 de-envelopment, one of the steps required for HSV-1 nuclear egress. Our newly established screening system enabled us to identify a novel cellular protein required for efficient HSV-1 de-envelopment. The identification of cellular protein(s) that interact with viral effector proteins and function in important viral procedures is necessary for enhancing our understanding of the mechanics of various viral processes. In this study, we established a new system consisting of interactome screening for the herpes simplex virus 1 (HSV-1) nuclear egress complex (NEC), followed by loss-of-function screening to target the identified putative NEC-interacting cellular proteins to detect a defect in HSV-1 nuclear egress. This newly established system identified SLC35E1, an orphan transporter, as a novel cellular protein required for efficient HSV-1 de-envelopment, providing an insight into the mechanisms involved in this viral procedure.

Background:Neurodegenerative tauopathies may progress based on seeding by pathological tau assemblies, whereby an aggregate is released from one cell, gains entry to an adjacent or connected cell, and serves as a specific template for its own replication in the cytoplasm. seeding reactions typically take days, yet seeding into the complex cytoplasmic milieu can happen within hours. A cellular machinery might regulate this process, but potential players are unknown. Methods:We used proximity labeling to identify factors that control seed amplification. We fused split-APEX2 to the C-terminus of tau repeat domain (RD) to reconstitute peroxidase activity upon seeded intracellular tau aggregation. We identified valosin containing protein (VCP/p97) 5h after seeding. Mutations in VCP underlie two neurodegenerative diseases, multisystem proteinopathy and vacuolar tauopathy, but its mechanistic role is unclear. We utilized tau biosensors, a cellular model for tau aggregation, to study the effects of VCP on tau seeding. Results:VCP knockdown reduced tau seeding. However, distinct chemical inhibitors of VCP and the proteasome had opposing effects on aggregation, but only when given <8h of seed exposure. ML-240 increased seeding efficiency ~40x, whereas NMS-873 decreased seeding efficiency by 50%, and MG132 increased seeding ~10x. We screened VCP co-factors in HEK293 biosensor cells by genetic knockout or knockdown. Reduction of ATXN3, NSFL1C, UBE4B, NGLY1, and OTUB1 decreased tau seeding, as did NPLOC4, which also uniquely increased soluble tau levels. Reduction of FAF2 and UBXN6 increased tau seeding. Conclusions:VCP uses distinct cofactors to determine seed replication efficiency, consistent with a dedicated cytoplasmic processing complex that directs seeds towards dissolution vs. amplification.