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Protein Interaction Mapping Identifies RBBP6 as a Negative Regulator of Ebola Virus Replication.

Cell. 2018;

Batra J, Hultquist JF, Liu D, Shtanko O, Von Dollen J, Satkamp L, Jang GM, Luthra P, Schwarz TM, Small GI, Arnett E, Anantpadma M, Reyes A, Leung DW, Kaake R, Haas P, Schmidt CB, Schlesinger LS, LaCount DJ, Davey RA, Amarasinghe GK, Basler CF, Krogan NJ.

Products/Services Used	Details	Operation
Mutagenesis Services	1) Heinz Feldmann (NIH, Hamilton, MT) N/A Chemicals, Peptides, and Recombinant Proteins Lipofectamine2000 Invitrogen Cat#11668500 TransIT-X2 Mirus Cat#MIR6003 Lipofectamine LTX Thermo Fisher Scientiﬁc Cat#15338500 Lipofectamine RNAiMAX Thermo Fisher Scientiﬁc Cat#13778-150 PolyJet SignaGen Laboratories Cat#SL100688 Protease inhibitor cocktail Sigma Aldrich (Roche) Cat#11836170001 RBBP6 peptide (SLPATPVFVPVPPPPLYPPPPHT) Genscript N/A Perfecta SYBR Green Fastmix VWR Cat#101414-270 Doxycycline Clontech Cat#631311 PhosSTOP Phosphatase Inhibitor Sigma-Aldrich Cat#4906845001 Nonidet P 40 Substitute Fluka Analytical/Sigma-Aldrich Cat#74385 D-Desthiobiotin IBA Lifesciences Cat# 2-1000-001 Sequencing Grade Modiﬁed Trypsin Promega Cat#V5113 Critical Commercial Assays QIAGEN Plasmid Plus Maxi Kit QIAGEN Cat#12963 RNeasy Mini Kit QIAGEN Cat#74104 QIAquick Gel Extraction Kit QIAGEN Cat#28704 QIAquick PCR Puriﬁcation Kit QIAGEN Cat#28104 Dual-Glo Luciferase Assay System Promega Cat#E2940 QuikChange multi site-directed mutagenesis kit Agilent Technologies Cat#200514 SuperScript III First-Strand Synthesis System Thermo Fisher Scientiﬁc Cat#18080051 Hampton Research crystal screen kits Neon transfection system 10 ml kit Hampton research Cat#HR2-110 Thermo Fisher Scientiﬁc Cat#MPK1096 (Continued on next page) Cell 175, 1917–1930.... The coding sequences of EBOV, MARV, RESTV, and LLOV VP30 were synthesized in vitro (GenScript) and cloned into the pCAGGS vector (Addgene) by restriction digest for mammalian expression.	Get A Quote

摘要

Ebola virus (EBOV) infection often results in fatal illness in humans, yet little is known about how EBOV usurps host pathways during infection. To address this, we used affinity tag-purification mass spectrometry (AP-MS) to generate an EBOV-host protein-protein interaction (PPI) map. We uncovered 194 high-confidence EBOV-human PPIs, including one between the viral transcription regulator VP30 and the host ubiquitin ligase RBBP6. Domain mapping identified a 23 amino acid region within RBBP6 that binds to VP30. A crystal structure of the VP30-RBBP6 peptide complex revealed that RBBP6 mimics the viral nucleoprotein (NP) binding to the same interface of VP30. Knockdown of endogenous RBBP6 stimulated viral transcri... More