Supplementary Information For

2 Supplementary Information for

3 A CRISPR/Cas9 Screen Identiﬁes Mitochondrial Translation as an Essential Process in Latent

4 KSHV Infection of Human Endothelial Cells

5 Daniel L Holmes, Daniel T Vogt, and Michael Lagunoff

6 Michael Lagunoff. 7 E-mail: [email protected]

8 This PDF ﬁle includes:

9 Figs. S1 to S7 10 Tables S1 to S5 11 SI References

Daniel L Holmes, Daniel T Vogt, and Michael Lagunoff 1 of 16 Fig. S1. Dead Cell Screen Histogram: Genes plotted using the median log-fold change data from the dead cell screen. There is a bimodal distribution where a subset of genes in the library fall into a second peak where the median for those genes represents a 2.75 or greater log2 fold change in KSHV infected cells when compared to uninfected controls.

2 of 16 Daniel L Holmes, Daniel T Vogt, and Michael Lagunoff Fig. S2. Reduction in MT-COXII expression with MRPS34 knock-out: (A) Western blot for MT-COXII expression in TIME cells transduced with MRPS34 targeting sgRNAs. (B) Quantiﬁcation of bands in Fig. S2A, MT-COXII expression relative to non-template control normalized to GAPDH expression.

Daniel L Holmes, Daniel T Vogt, and Michael Lagunoff 3 of 16 Fig. S3. Antibiotic treatment leads to supression of cell proliferation in PELs: Chloramphenicol (A) and Tigecyline (B) treatment suppress proliferation to a greater extent in PELs than in BJAB and RAJI cells which lack KSHV.

4 of 16 Daniel L Holmes, Daniel T Vogt, and Michael Lagunoff Fig. S4. TIME Cells are Insensitive to Rotenone Treatment: Mock and KSHV infected TIME cells were treated with increasing concentrations of rotenone for 48 hours. Even at highest concentration used, 100 nM, which is double the highest concentration that led to cell death in PEL cells, neither mock nor KSHV infected TIME cells showed a decrease in cell confluence when compared to DMSO treated controls. Plots are of technical triplicates of a single representative experiment and SEMs. Percent confluence was normalized based on the confluence of DMSO treated cells for Mock and KSHV infected cells separately.

Daniel L Holmes, Daniel T Vogt, and Michael Lagunoff 5 of 16 Fig. S5. Pyruvate is Unable to Rescue Rotenone Sensitivity in KSHV Infected B-cells: Cell viability was measured 48 hours after treatment with rotenone by trypan blue staining. Neither 1mM (A) nor 10mM (B) concentrations of pyruvate were able to rescue cell death. A single experiment was done for each of the 1mM and 10mM experiments.

6 of 16 Daniel L Holmes, Daniel T Vogt, and Michael Lagunoff Fig. S6. Elimination of Mitochondrial Genomes Leads to Reduced Log Phase Doubling Time During KSHV Infection: (A) Induction of mutant Uracil-N-Glycosylase (mtUNG) with doxycycline leads to suppression of gene products encoded on the mitochondrial genome after two weeks, while the control wild-type (wtUNG) does not cause a substantial change in expression. (B) The log-phase doubling time of the TIME cells calculated from the slopes of the Incucyte growth curves in Fig. 4B. Plots are the mean of three experiments with SEM.

Daniel L Holmes, Daniel T Vogt, and Michael Lagunoff 7 of 16 Fig. S7. tRNAs from the Mitochondrial Genome are Increased During KSHV Infection: The RNA-Seq data from Sychev et al., 2017 was analyzed to look at changes in average gene expression across each chromosome. Average gene expression was elevated for transcripts encoded by the mitochondrial genome (A). When looking at the individual genes responsible for the increased average expression, the genes encoding tRNAs are elevated (B).

8 of 16 Daniel L Holmes, Daniel T Vogt, and Michael Lagunoff Table S1. List of Genes Signiﬁcantly Depleted or Enriched

Gene EnsemblID sgRNAs Depletion sgRNAs Median Enrichment Symbol Rank Depleted Log FC Rank NDUFB7 ENSG00000099795 5 1 4 -2.0775 7353 DCHS1 ENSG00000166341 5 2 4 -4.4392 6641 LSM11 ENSG00000155858 5 3 5 -3.0307 11467 SNX14 ENSG00000135317 5 4 3 -4.1888 7411 VCAM1 ENSG00000162692 5 5 4 -2.3906 7721 FLII ENSG00000177731 5 6 2 -0.1825 7802 DNAJC8 ENSG00000126698 5 7 4 -2.4065 10095 MED10 ENSG00000133398 5 8 5 -1.743 11466 TBC1D9B ENSG00000197226 5 9 4 -2.4606 6199 SMEK2 ENSG00000275052 5 10 5 -2.1333 11465 MAPKAPK5 ENSG00000089022 5 11 3 -0.5049 8246 RABL5 ENSG00000108774 5 12 3 -2.5109 7503 EPOR ENSG00000187266 4 13 4 -4.3151 11294 TMEM39A ENSG00000176142 5 14 2 -0.2036 7100 AKIP1 ENSG00000166452 4 15 2 -0.8423 8453 ICT1 ENSG00000167862 5 16 3 -1.4643 9160 AP4B1 ENSG00000134262 5 17 5 -1.6573 11458 PLXND1 ENSG00000004399 4 18 4 -2.014 11464 UBQLN1 ENSG00000135018 5 19 5 -3.3447 11363 CHCHD5 ENSG00000125611 4 20 2 -0.9262 7183 ARHGAP22 ENSG00000128805 5 21 2 -0.2947 1471 CCDC71L ENSG00000253276 5 22 4 -3.4777 10919 GLG1 ENSG00000090863 4 23 3 -1.14 8268 NSF ENSG00000073969 5 24 3 -0.9028 2034 COQ7 ENSG00000167186 5 25 4 -3.8584 10554 POLR1C ENSG00000171453 5 26 5 -2.8705 11408 HEXB ENSG00000049860 5 27 5 -3.1462 11309 DCUN1D5 ENSG00000137692 4 28 3 -2.1877 9700 FAM171B ENSG00000144369 5 29 5 -1.7835 11447 CRLS1 ENSG00000088766 5 30 5 -2.0521 11443 BRD2 ENSG00000204256 5 31 4 -3.0697 11051 SYDE2 ENSG00000097096 5 32 5 -1.7893 11463 CAPS ENSG00000105519 5 33 4 -3.2305 10652 C4orf29 ENSG00000164074 5 34 1 0.24346 5554 CCNL2 ENSG00000221978 5 35 5 -2.6784 11462 SPC24 ENSG00000161888 5 36 4 -1.8157 9811 PARP9 ENSG00000138496 5 37 3 -1.18 7898 CREBRF ENSG00000164463 5 38 5 -2.1626 11377 ZNF462 ENSG00000148143 5 39 4 -2.0698 10780 FKTN ENSG00000106692 5 40 4 -1.9937 10725 ZNF813 ENSG00000198346 5 41 2 -0.2427 5146 TXNRD2 ENSG00000184470 5 42 5 -1.5816 11461 CMKLR1 ENSG00000174600 4 43 3 -2.0477 10927 KCNJ12 ENSG00000184185 5 44 1 -0.1202 2805 ATL1 ENSG00000198513 5 45 5 -2.1666 11459 CACNB1 ENSG00000067191 5 46 4 -1.1397 9935 TOR1AIP1 ENSG00000143337 5 47 4 -1.4423 10892 PTRHD1 ENSG00000184924 5 48 4 -1.8351 9762 SLC6A15 ENSG00000072041 5 49 4 -1.4768 7292 RMI1 ENSG00000178966 5 50 4 -2.4976 11194 PTCD3 ENSG00000132300 5 51 5 -1.6687 11460 DYNC1I2 ENSG00000077380 5 52 4 -2.217 8269 ARHGEF18 ENSG00000104880 5 53 4 -2.0029 8836 PTPN14 ENSG00000152104 5 54 4 -2.4214 8487 HECTD1 ENSG00000092148 5 55 4 -3.5386 10961 Continued on the next page

Daniel L Holmes, Daniel T Vogt, and Michael Lagunoff 9 of 16 Table S1 – continued from previous page Gene EnsemblID sgRNAs Depletion sgRNAs Median Enrichment Symbol Rank Depleted Log FC Rank EFCAB13 ENSG00000178852 5 56 4 -0.8558 10898 DCUN1D2 ENSG00000150401 5 57 3 -1.3216 3054 THEMIS2 ENSG00000130775 5 58 5 -1.4697 11457 ZNF382 ENSG00000161298 5 59 4 -1.2922 6458 STK17A ENSG00000164543 5 60 3 -2.3901 5891 ZNF483 ENSG00000173258 5 61 2 -0.1812 677 OSTF1 ENSG00000134996 5 62 2 -0.1688 8415 CRTC3 ENSG00000140577 5 63 3 -2.7604 10248 RBPMS ENSG00000157110 5 64 4 -2.1141 10161 BCKDHB ENSG00000083123 4 65 3 -0.9262 10066 SEC24B ENSG00000138802 5 66 4 -2.6164 8868 RARS ENSG00000113643 5 67 3 -0.9168 4159 ADD3 ENSG00000148700 5 68 4 -2.0512 6973 GBP4 ENSG00000162654 5 69 5 -2.0544 11306 ATXN7L3 ENSG00000087152 5 70 2 -0.1095 6060 WNT9A ENSG00000143816 4 71 1 1.0067 407 C7orf43 ENSG00000146826 5 72 4 -2.5332 11164 TFEB ENSG00000112561 4 73 3 -2.5307 639 GLCCI1 ENSG00000106415 5 74 4 -2.207 10680 EXOSC5 ENSG00000077348 5 75 3 -0.6525 4089 ZNF438 ENSG00000183621 5 76 4 -1.7947 1665 IL15 ENSG00000164136 5 77 5 -1.6989 11456 TDRD3 ENSG00000083544 4 78 4 -2.1723 11417 ITSN2 ENSG00000198399 5 79 3 -3.0093 6606 BLMH ENSG00000108578 5 80 2 -0.3238 6160 ABCB1 ENSG00000085563 5 81 5 -1.2863 11455 POU4F1 ENSG00000152192 5 82 4 -1.6358 9290 MMS22L ENSG00000146263 5 83 4 -2.2806 9756 MXD3 ENSG00000213347 5 84 4 -2.2366 5545 SP110 ENSG00000135899 5 85 4 -1.7516 11288 MRPL40 ENSG00000185608 5 86 4 -2.0208 5771 POLE4 ENSG00000115350 4 87 3 -2.4167 6224 ATP6V1F ENSG00000128524 5 88 2 -0.6075 4759 IL17RA ENSG00000177663 5 89 4 -1.6191 10470 HSPBP1 ENSG00000133265 5 90 3 -2.7214 6343 NPFF ENSG00000139574 5 91 4 -1.1309 6837 DTWD2 ENSG00000169570 5 92 5 -1.7013 11454 AARS2 ENSG00000124608 5 93 2 -0.1289 5288 POLE ENSG00000177084 5 94 4 -2.7027 10328 FIGNL1 ENSG00000132436 4 95 4 -1.2182 11453 ITFG2 ENSG00000111203 5 96 4 -1.7614 5933 HIP1R ENSG00000130787 5 97 1 0.15838 4369 ZNF837 ENSG00000152475 5 98 4 -2.5069 10653 TEX2 ENSG00000136478 5 99 2 -0.046 6881 CROT ENSG00000005469 4 100 4 -2.2664 11452 ATP11C ENSG00000101974 4 101 4 -1.478 11451 RRNAD1 ENSG00000143303 5 102 2 -0.3475 8166 SRFBP1 ENSG00000151304 5 103 2 -0.6282 7325 EIF3L ENSG00000100129 5 104 5 -1.2771 11450 MAGI1 ENSG00000151276 5 105 3 -0.6407 9704 SDCCAG8 ENSG00000054282 4 106 3 -2.1237 9712 CUL7 ENSG00000044090 5 107 3 -1.2412 3513 SDHD ENSG00000204370 5 108 3 -1.1765 3773 SLC7A1 ENSG00000139514 4 109 4 -1.6054 11449 ZNF566 ENSG00000186017 5 110 1 -0.0387 8521 ZNF343 ENSG00000088876 5 111 3 -2.0185 10049 Continued on the next page

10 of 16 Daniel L Holmes, Daniel T Vogt, and Michael Lagunoff Table S1 – continued from previous page Gene EnsemblID sgRNAs Depletion sgRNAs Median Enrichment Symbol Rank Depleted Log FC Rank SULT1E1 ENSG00000109193 5 112 4 -2.7214 11121 DHRS13 ENSG00000167536 5 113 2 -0.2312 8639 PPP1R37 ENSG00000104866 5 114 3 -0.8495 6052 MEIS1 ENSG00000143995 5 115 5 -1.9804 11448 CBX8 ENSG00000141570 5 116 2 -0.4158 7586 TMEM45A ENSG00000181458 5 117 4 -1.8195 10859 FUK ENSG00000157353 5 118 4 -2.7796 11200 JAK2 ENSG00000096968 5 119 5 -1.9911 11446 HOOK2 ENSG00000095066 5 120 2 -0.5103 8656 MED9 ENSG00000141026 4 121 3 -1.3286 6271 SLC48A1 ENSG00000211584 4 122 4 -1.6701 11129 LYAR ENSG00000145220 5 123 5 -1.5619 11445 SKIL ENSG00000136603 5 124 3 -0.7862 8322 FOXJ3 ENSG00000198815 5 125 2 -0.0829 5283 SH3RF2 ENSG00000156463 5 126 3 -2.3305 8471 PLCB1 ENSG00000182621 5 127 4 -2.764 9692 MEAF6 ENSG00000163875 5 128 5 -1.4906 11444 TAZ ENSG00000102125 5 129 3 -0.89 10808 NCAPH ENSG00000121152 5 130 2 -0.3705 3285 IL1RL1 ENSG00000115602 5 131 5 -1.2253 11442 NECAP2 ENSG00000157191 5 132 3 -3.201 9896 CKAP4 ENSG00000136026 5 133 4 -1.4295 11231 ZNF736 ENSG00000234444 5 135 5 -1.5939 11440 C5orf22 ENSG00000082213 5 136 3 -2.2377 3027 NPRL2 ENSG00000114388 5 137 5 -2.2841 11439 CIAO1 ENSG00000144021 5 138 2 -0.1755 2679 ZNF358 ENSG00000198816 5 139 3 -0.8152 798 PRRC2B ENSG00000130723 5 141 2 -0.1937 2869 ATP6V1C2 ENSG00000143882 5 140 1 -0.0346 4690 UCHL1 ENSG00000154277 5 143 4 -1.9911 10492 KIF11 ENSG00000138160 5 145 2 0.46874 2397 GLI2 ENSG00000074047 5 144 4 -1.6002 9361 AHR ENSG00000106546 5 146 4 -1.9769 9550 FUT1 ENSG00000174951 5 147 5 -1.6629 11438 CPE ENSG00000109472 5 148 3 -0.9763 4116 KCTD10 ENSG00000110906 5 10892 0 3.4405 1 HSPA4 ENSG00000170606 5 11409 0 3.2069 2 Table S1. Table S1: Hit list from Live Cell Whole Genome CRISPR/Cas9 Screen. The "Gene Symbol" and "EnsemblID" columns refer to the common gene symbol and EnsemblID,respectively. The "sgRNA" column refers to the number of sgRNAs detected in the sequencing data for a particular gene. "Depletion Rank" is the ranking of genes depleted in KSHV infected cells relative to mock infection, by MAGeCK score. "sgRNAs Depleted" is the number of sgRNAs whose log fold change is less than zero during KSHV infection. "Median Log FC" is the median log fold change for all sgRNAs detected for a particular gene. "Enrichment Rank" is the ranking of genes enriched in KSHV infected cells relative to mock infection, by MAGeCK score.

Daniel L Holmes, Daniel T Vogt, and Michael Lagunoff 11 of 16 Table S2. Primary Antibodies

Target Protein Company Product # Species Western Blot Dilution IFA Dilution ATP5A UQCRC2 SDHB Abcam ab110411 Mouse 1:1000 N/A MT-CO2 NDUFB8 MT-CO2 Abcam ab110258 Mouse 1:1000 1:500 GAPDH Proteintech 60004-1-1g Mouse 1:5000 N/A COXIV Cell Signaling 4850 Rabbit N/A 1:200 COXIV Cell Signaling 11967 Mouse N/A 1:200 LANA Gift from A. Polson and D. Ganem Rabbit 1:2000 1:1000 ORF59 Advanced Biotechnologies 13-211-100 Mouse N/A 1:3000 FLAG Tag Sigma-Aldrich F3165 Mouse 1:1000 1:500 V5 Tag Proteintech 14440-1-ap Rabbit 1:1000 1:200 MRPS34 Novus NBP2-45432 Mouse 1:1000 N/A

12 of 16 Daniel L Holmes, Daniel T Vogt, and Michael Lagunoff Table S3. Secondary Antibodies

Antibody Name Target Species Company Product # Species Western Blot Dilution IFA Dilution IRDye 680RD Mouse LI-COR 926-68070 Goat 1:10,000 N/A IRDye 680RD Rabbit LI-COR 926-68071 Goat 1:10,000 N/A IRDye 800CW Mouse LI-COR 926-32210 Goat 1:10,000 N/A IRDye 800CW Rabbit LI-COR 926-32211 Goat 1:10,000 N/A Alexa Fluor 488 F(ab’)2 fragment Mouse Invitrogen A-11017 Goat N/A 1:1000 Alexa Fluor 488 F(ab’)2 fragment Rabbit Invitrogen A-11070 Goat N/A 1:1000 Alexa Fluor 594 F(ab’)2 fragment Mouse Invitrogen A-11020 Goat N/A 1:1000 Alexa Fluor 594 F(ab’)2 fragment Rabbit Invitrogen A-11072 Goat N/A 1:1000

Daniel L Holmes, Daniel T Vogt, and Michael Lagunoff 13 of 16 Table S4. Plasmids

Name Type Product # Source Depositor Application Publication Kind gift of Daniel pRRL LentiCRISPR Plasmid CRISPR/Cas9 Gene Knock-out Stetson (University of Washington) Human Activity-Optimized Whole Genome Pooled Library 1000000067 Addgene David Sabatini, Eric Lander (1) CRISPR Knockout Library CRISPR/Cas9 Screening Mitochondria pMA3287 Plasmid 46883 Addgene Mikhail Alexeyev (2) genome curing Mitochondria pMA3288 Plasmid 46885 Addgene Mikhail Alexeyev (2) genome curing Tet-on effector pLenti CMV rtTA3 Hygro (w785-1) Plasmid 26730 Addgene Eric Campeau expression Gateway™ pLX302 Plasmid 25896 Addgene David Root (3) Destination Vector For making Gateway™ pDONR221 Plasmid 12536017 ThermoFisher Entry Plasmids p3xFLAGCMV 10 Plasmid This paper Expression 3XFLAG10-vCyc Plasmid This paper Expression 3XFLAG10-KapC Plasmid This paper Expression 3XFLAG10-KapB Plasmid This paper Expression pcDNA3.1 Plasmid This paper Expression Kind gift of pcDNA3 3XFLAG LANA Plasmid Expression Tim Rose (University of Washington) pLX302_3xFLAG_vFLIP_HS Plasmid This paper Expression pLX302_vFLIP_HS_V5 Plasmid This paper Expression Lentiviral psPAX2 Plasmid 12260 Addgene Didier Trono packaging plasmid VSV-G Env pMD2.G Plasmid 12259 Addgene Didier Trono Expressing Plasmid 14 of 16 Daniel L Holmes, Daniel T Vogt, and Michael Lagunoff Table S5. Oligonucleotide Sequences

Oligo Name Sequence (5’-3’) Application AAAGGACGAAACACC NTC_S GACATTGTTAGTAACGACTC Cloning NTC sgRNA into pRRL LentiCRISPR GTTTTAGAGCTAGAAATAGCAAG CTTGCTATTTCTAGCTCTAAAAC NTC_AS GAGTCGTTACTAACAATGTC GGTGTTTCGTCCTTT AAAGGACGAAACACC MRPS34_A_S GAAATGGAGACACAAGCACCG Cloning MRPS34 targeting sgRNA into pRRL LentiCRISPR GTTTTAGAGCTAGAAATAGCAAG CTTGCTATTTCTAGCTCTAAAAC MRPS34_A_AS CGGTGCTTGTGTCTCCATTTC GGTGTTTCGTCCTTT AAAGGACGAAACACC MRPS34_B_S GGAAGACTGAGAGCGAGGCG Cloning MRPS34 targeting sgRNA into pRRL LentiCRISPR GTTTTAGAGCTAGAAATAGCAAG CTTGCTATTTCTAGCTCTAAAAC MRPS34_B_AS CGCCTCGCTCTCAGTCTTCC GGTGTTTCGTCCTTT HPRT_F GAACGTCTTGCTCGAGATGTG RT-qPCR Reference for Gene Expression HPRT_R CCAGCAGGTCAGCAAAGAATT PROX1_F CCAAGGTTCTGAGCAGGATGT qPCR Reference for Genomic DNA PROX1_R CATACGAGTTCGCCCTCTTCA MT-CO2_F ACGCATCCTTTACATAACAGAC RT-qPCR for mitochondrial transcripts using random hexamer MT-CO2_R GCCAATTGATTTGATGGTAAGG primers and for mitochondrial genome quantiﬁcation MT-ND6_F GCTTTGTATGATTATGGGCGT RT-qPCR for mitochondrial transcripts using MT-ND6_R CACCAACAAACAATGTTCAACC random hexamer primers MT-ND4_F CCCTTCCTTGTACTATCCCT RT-qPCR for mitochondrial transcripts using MT-ND4_R TTTGTCGTAGGCAGATGGAG random hexamer primers AATGATACGGCGACCACCGAGATCTA sgRNA_barcode_F Generation of template for Illumina Sequencing CACCGACTCGGTGCCACTTTT CAAGCAGAAGACGGCATACGAGATC sgRNA_barcode_R_set1 Generation of template for Illumina Sequencing GCTGGATT TTTCTTGGGTAGTTTGCAGTTTT CAAGCAGAAGACGGCATACGAGATC sgRNA_barcode_R_set2 Generation of template for Illumina Sequencing TAACTCGG TTTCTTGGGTAGTTTGCAGTTTT CAAGCAGAAGACGGCATACGAGATC sgRNA_barcode_R_set3 Generation of template for Illumina Sequencing TAACAGTT TTTCTTGGGTAGTTTGCAGTTTT CAAGCAGAAGACGGCATACGAGATC sgRNA_barcode_R_set4 Generation of template for Illumina Sequencing ATACTCAA TTTCTTGGGTAGTTTGCAGTTTT CGGTGCCACTTTTTCAAGTTGATAACGGA Illumina_HiSeq_Read1 Read 1 HiSeq Primer CTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC TTTCAAGTTACGGTAAGCATATGATAGTCC Illumina_HiSeq_Index Indexing HiSeq Primer ATTTTAAAACATAATTTTAAAACTGCAAACTACCCAAGAAA

Daniel L Holmes, Daniel T Vogt, and Michael Lagunoff 15 of 16 13 References

14 1. T Wang, JJ Wei, DM Sabatini, ES Lander, Genetic Screens in Human Cells Using the CRISPR-Cas9 System. Science 343, 15 80–84 (2014). 16 2. IN Shokolenko, GL Wilson, MF Alexeyev, Persistent damage induces mitochondrial DNA degradation. DNA Repair 12, 17 488–499 (2013). 18 3. X Yang, et al., A public genome-scale lentiviral expression library of human ORFs. Nat. Methods 8, 659–661 (2011).

16 of 16 Daniel L Holmes, Daniel T Vogt, and Michael Lagunoff