RESUMO
The vaccine strain against smallpox, vaccinia virus (VACV), is highly immunogenic yet causes relatively benign disease. These attributes are believed to be caused by gene loss in VACV. Using a targeted small interfering RNA (siRNA) screen, we identified a viral inhibitor found in cowpox virus (CPXV) and other orthopoxviruses that bound to the host SKP1-Cullin1-F-box (SCF) machinery and the essential necroptosis kinase receptor interacting protein kinase 3 (RIPK3). This "viral inducer of RIPK3 degradation" (vIRD) triggered ubiquitination and proteasome-mediated degradation of RIPK3 and inhibited necroptosis. In contrast to orthopoxviruses, the distantly related leporipoxvirus myxoma virus (MYXV), which infects RIPK3-deficient hosts, lacks a functional vIRD. Introduction of vIRD into VACV, which encodes a truncated and defective vIRD, enhanced viral replication in mice. Deletion of vIRD reduced CPXV-induced inflammation, viral replication, and mortality, which were reversed in RIPK3- and MLKL-deficient mice. Hence, vIRD-RIPK3 drives pathogen-host evolution and regulates virus-induced inflammation and pathogenesis.
Assuntos
Vírus da Varíola Bovina/fisiologia , Varíola Bovina/imunologia , RNA Interferente Pequeno/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Vaccinia virus/metabolismo , Proteínas Virais/metabolismo , Animais , Evolução Molecular , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Inflamação , Camundongos , Camundongos Knockout , Necroptose/genética , Orthopoxvirus , Filogenia , Proteínas Quinases/genética , Proteólise , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Análise de Sequência de RNA , Proteínas Virais/genética , Replicação ViralRESUMO
Optogenetics has emerged as an exciting tool for manipulating neural activity, which in turn, can modulate behavior in live organisms. However, detecting the response to the optical stimulation requires electrophysiology with physical contact or fluorescent imaging at target locations, which is often limited by photobleaching and phototoxicity. In this paper, we show that phase imaging can report the intracellular transport induced by optogenetic stimulation. We developed a multimodal instrument that can both stimulate cells with subcellular spatial resolution and detect optical pathlength (OPL) changes with nanometer scale sensitivity. We found that OPL fluctuations following stimulation are consistent with active organelle transport. Furthermore, the results indicate a broadening in the transport velocity distribution, which is significantly higher in stimulated cells compared to optogenetically inactive cells. It is likely that this label-free, contactless measurement of optogenetic response will provide an enabling approach to neuroscience.