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1) an Arrayed Pipeline for Genetic Interaction Mapping of HIV Host-Dependency Factors 2) the HIV Ve-MAP Highlights Complexes

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1) an Arrayed Pipeline for Genetic Interaction Mapping of HIV Host-Dependency Factors 2) the HIV Ve-MAP Highlights Complexes A viral epistasis map (vE-MAP) reveals genetic interactions underlying HIV infection David E. Gordon1, 2, 5, Ariane Watson3, 5, Assen Roguev1,2, Gwendolyn M. Jang1, Joshua Kane1,2, Jiewei Xu1, Kathy Franks-Skiba1, Erica Stevenson1,2, Danielle Swaney1,2, Michael Shales1, Alexander Marson2,4, Gerard Cagney3, Nevan J. Krogan1,2* 1University of California San Francisco, Department of Cellular and Molecular Pharmacology, San Francisco, CA, USA, 2Gladstone Institutes for Virology and Immunology, San Francisco, CA, USA, 3University College Dublin, School of Medicine, Dublin, IRL, 4University of California San Francisco, Department of Microbiology and Immunology, San Francisco, CA, USA 5These authors contributed equally to this work. *Please send correspondence to [email protected] 1) An arrayed pipeline for genetic interaction 2) The HIV vE-MAP highlights complexes 4) The CNOT complex forms numerous mapping of HIV host-dependency factors and pathways impacting HIV infection negative genetic interactions in the vE-MAP 356 Knockdowns Pairwise knockdowns coupled with a luciferase reporter A B C Donor 1 Donor 2 Donor 3 Donor 4 D 200 1.2 1 2 3 AP 175 -M RNA 1.0 CNOT1 CNOT2 CNOT3 ELOC CUL2 ELOB virus facilitate high-throughput genetic interaction analysis 75 Transcription Granule RNA ESCRT HGS TSG101 CNOT1 ELOC nfectivity validated i 0.8 CNOT2 CUL2 HGS HIV E in primary he 1 CNOT3 ELOB TSG101 T-cells HIV 0.6 f t panel C of HIV host-dependency factors 50 ed o 2 z 0.4 Non-targeting CNOT1-1 CCNT1 CNOT1-1 + CCNT1 li CCNT1 CDC73 CTR9 PAF1 SUPT5H RTF1 DDX3X HGS TSG101 3 a CNOT1 CNOT2 rm 0.2 4 genes CNOT2 anti-CNOT1 f No 4 COPS3 COPS2 COPS4 COPS6 GPS1 5 6 25 o CNOT3 CNOT1 CNOT3 0.0 1 4 g 1 4 g 1 4 g 1 4 g 356 X 356 Automated High-throughput COPS3 crRNA 356 Knockdowns CR CR CR CR COPS2 CAND1 NEDD8 RBX1 Genetic Interaction Score anti-CCNT1 CCNT1 CCNT1 CX CCNT1 CX CCNT1 CX CX combinations microscopy luminometry RELA RELB COPS4 CAND1 Number CNOT1- CNOT1- CNOT1- COPS6 NEDD8 RELA 0 0.00 -0.83 -1.67 -2.50 CNOT1- 0 5 10 15 20 25 30 35 Non-targetin Non-targetin Non-targetin GPS1 RBX1 RELB Single KD HIV Phenotype Non-targetin Query Number of interactions with S-score < -1.5 anti-GAPDH Transfect Gene HIV Luciferase substrate (Log2 deviation from median) 5 CNOT1-1 + CCNT1 CNOT1-1 + CCNT1 CNOT1-1 + CCNT1 esiRNA knockdown infection with detergent CNOT1-1 + CCNT1 9 -3 -2 -1 0 1 2 3 7 8 KARS DARS EPRS SARS TNPO3 72 h 48 h 10 min CDC73 CTR9 PAF1 SUPT5H RTF1 KARS CDC73 ANAPC1 ANAPC2 Library DARS CTR9 ANAPC1 esiRNA PAF1 ANAPC2 EPRS Cells SUPT5H SARS expressing RTF1 TNPO3 (63,012 pairwise mCherry combinations) Cell count HIV reporter expression 5) CNOT1, CNOT10 and CNOT11 are required 6 S-score for HIV infection in primary CD4+ T-cells -2.50 -1.67 -0.83 Calculate genetic interactions impacting 0.00 0.83 1.67 2.50 HIV infection and expression 7 8 9 1.5 Donor 1 1.5 Donor 1 Donor 2 Genetic interaction scores (S-scores) are based upon Donor 2 nfectivity nfectivity CNOT1-1 CNOT1-2 i 1.0 nfectivity nfectivity Guide RNA Guide RNA i 1.0 HIV ed HIV deviation from the expected combinatorial phenotype. z CNOT10/11 BD MIF4G CN9BD NOT1 266.9 kDa CNOT1 Large Isoform li a ed z li CNOT1 Small Isoform MIF4G CN9BD NOT1 rm 0.5 a 0.5 Clustering of S-scores highlights gene complexes and No Four alternative rm start codons No pathways mediating the phenotype under study 3) Viral genetic interaction mapping can also in exon 8 0.0 0.0 0 1 g crRNA crRNA CXCR4 CNOT1 CNOT1 CNOT1-1 Observed combinatorial Genetic interaction query small molecules and virus mutants CNOT1-2 Non-targetin Non-targeting phenotype scoring (both isoforms) KD-a + KD-b (large isoform only) +2.0 Positive: Greater HIV HIV Luciferase expression +1.5 MLN-4924 Reporter Virus than expected a b c1 c2 c3 c4 d e CNOT1, 10 and 11 suppress innate immunity HIV Luciferase +1.0 a Host gene + drug Complex 1 6 reporter genetic interactions expression b 24 h 48 h IFIT1 +0.5 IFI27 c1 Transfect 4 48 h IFI44 IFI44L 2a α Donor 1 Donor 2 KD-a 0 Library 16 Donor 1 Neutral c2 IFI6 Complex 2 Capsid Mutant 14 Donor 2 esiRNA 2 c3 12 KD-b -0.5 CNOT10 KO vs. NT DDX60 Host gene + viral mutant 2a 2a c4 10 α α Non-targeting CNOT1-1 CNOT2 CNOT10 CNOT11 Non-targeting CNOT1-1 CNOT2 CNOT10 CNOT11 -1.0 genetic interactions 8 IFN IFN d -2 2 4 6 Complex 3 72 h 48 h 6 Negative: e CNOT1-1 KO vs. NT anti-IFIT1 -1.5 4 Less HIV -2 expression Query with HIV capsid mutant N74A replicates published Log P-value * Z-score 2 -2.0 0 10 20 30 0 HIV phenotype epistasis with IFN anti-GAPDH 2 1 0 1 than expected negative neutral positive Type I interferon g 2a signaling pathway α genetic interactions impacting HIV infection Regulation of IFN CNOT nuclease activity CNOT1 CNOT1 Cellular response to CNOT1- exogenous dsRNA Non-targetin 40 Regulation of tumor siTNPO3 Key: necrosis factor secretion TNPO3 30 = CPSF6 Protein Nucleus Nucleus Nucleus Nucleus We focused on human genes known to physically interact = Wild-type Capsid 20 Count CAPSID WT TNPO3 TNPO3 depletion TNPO3 + CPSF6 TNPO3 depletion + = Mutant Capsid N74A (2.33) depletion HIV capsid mutation The antiviral phenotype of CNOT10 knockout is mediated with the HIV virus, as well as published host-dependency 10 TNPO3 maintains CPSF6 Cytoplasmic CPSF6 binds CPSF6 depletion Capsid N74 mutation CPSF6 (4.2) nuclear localization HIV capsid and reduces alleviates HIV restriction reduces CPSF6 binding infection and restriction by IRF7 factors and genetic interactors of host-dependency factors 0 HIV ++++ + ++++ +++ -5 -4 -3 -2 -1 0.0 1 2 3 4 5 Infection S-score CNOT3 5 CNOT10 KO + 5 H. sapiens CCR4- The neddylation inhibitor MLN-4924 genetically interacts with ) IRF7-1 or IRF7-3 KO ) CNOT6/6L PR : 53 NOT Complex Mitochondrion organization 3 ed 4 ed 4 POL : 33 Cell proliferation 4 CNOT2 ERAD 5 CNOT1 REV : 25 the COP9 signalosome, neddylation machinery, and Cullin-2 3 3 C-terminus CNOT7/8 Apoptosis 9 NEF : 12 RT : 2 Cell signalling 10 NC : 12 Protein folding 10 2 2 11 infect (% 2 r infect (% 2 r Chromosome organization 60 50 CNOT10 CNOT1 TAT : 29 DNA damage response 11 CNOT10 KO + IFNa2a + MA : 21 Cell cycle 12 MLN-4924 MLN-4924 MLN-4924 ono 1 Library KOs ono 1 Library KOs DNA replication 12 50 Nedd8 D D Required for CNOT11 40 CAND1 (0.35) CNOT9 Poorly understood 12 siNEDD8 suppression of 13 CUL2 (0.39) Autophagy 40 0 0 interferon Cytoskeleton organization 15 COPS2 (1.05) UBA1 (0.40) CNOT1 30 Ube2M 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 stimulated genes N-terminus VIF : 25 tRNA aminoacylation 15 COPS3 (1.59) Nedd8 Translational regulation 16 30 IN : 30 COPS4 (1.67) RBX1 RBX1 Donor 1 (% infected) Donor 1 (% infected) Nuclear-Cytoplasmic transport 17 COPS6 (1.95) 20 NEDD8 (0.59) Cullin 2 Neddylation Cullin 2 Vesicle-mediated transport 19 Frequency Frequency 20 RBX1 (0.56) GP120 : 1 Transcriptional regulation 24 GPS1 (2.13) De-neddylation EloC Immune system process 26 CAND1 GAG : 14 10 EloB IRF7 HIV infection VPR : 40 Metabolic process 30 10 CA : 5 Ubiquitin-dependent protein degradation 40 COP9 RNA processing 42 Active Cullin 0 0 Inactive Cullin Signalosome Other : 50 -5 -4 -3 -2 -1 0.0 1 2 3 4 5 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 Ubiquitin Ligase VPU : 44 0 10 20 30 40 50 Count S-score Correlation coefficient.
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