#METABOLOMICS WORKBENCH juliehaines_20221216_071050 DATATRACK_ID:3653 STUDY_ID:ST002413 ANALYSIS_ID:AN003934 VERSION 1 CREATED_ON 12-19-2022 #PROJECT PR:PROJECT_TITLE IFN-inducible phospholipid levels govern endosomal antiviral immunity PR:PROJECT_SUMMARY The interferon-induced transmembrane proteins (IFITMs) are implicated in several PR:PROJECT_SUMMARY biological processes including antiviral defense, but their modes of action PR:PROJECT_SUMMARY remain debated. Here, taking advantage of pseudotyped viral entry assays and PR:PROJECT_SUMMARY replicating viruses, we uncover the requirement of host co-factors for endosomal PR:PROJECT_SUMMARY antiviral inhibition through high-throughput proteomics and lipidomics in PR:PROJECT_SUMMARY cellular models of IFITM restriction. Unlike plasma membrane (PM)-localized PR:PROJECT_SUMMARY IFITM restriction that targets infectious SARS-CoV2 and other PM-fusing viral PR:PROJECT_SUMMARY envelopes, inhibition of endosomal viral entry depends on lysines within the PR:PROJECT_SUMMARY conserved IFITM intracellular loop. These residues recruit PR:PROJECT_SUMMARY phosphatydilinositol-3-phosphate (PIP3) that we show here to be required for PR:PROJECT_SUMMARY endosomal IFITM activity. We identify PIP3 as an IFN-inducible phospholipid that PR:PROJECT_SUMMARY acts as a rheostat for endosomal antiviral immunity as its expression levels PR:PROJECT_SUMMARY correlated with the potency of endosomal IFITM restriction and exogenous PIP3 PR:PROJECT_SUMMARY enhanced inhibition of endocytic viruses including the recent SARS-CoV2 Omicron PR:PROJECT_SUMMARY variant. Together, our results identify PIP3 levels as a critical regulator of PR:PROJECT_SUMMARY endosomal IFITM restriction linking it to the Pi3K/Akt/mTORC pathway and PR:PROJECT_SUMMARY elucidate cell-compartment specific antiviral mechanisms, informing the PR:PROJECT_SUMMARY development of broadly acting antiviral strategies. PR:INSTITUTE University of Colorado Denver PR:LABORATORY Angelo D'Alessandro PR:LAST_NAME Haines PR:FIRST_NAME Julie PR:ADDRESS 12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA PR:EMAIL julie.haines@cuanschutz.edu PR:PHONE 3037243339 PR:DOI http://dx.doi.org/10.21228/M8ZB01 #STUDY ST:STUDY_TITLE IFN-inducible phospholipid levels govern endosomal antiviral immunity ST:STUDY_SUMMARY Untargeted lipidomics of 3 cell lines at baseline with focus on phospholipids to ST:STUDY_SUMMARY understand the role of these lipids in endosomal antiviral immunity ST:INSTITUTE University of Colorado Denver ST:LABORATORY Angelo D'Alessandro ST:LAST_NAME Haines ST:FIRST_NAME Julie ST:ADDRESS 12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA ST:EMAIL julie.haines@cuanschutz.edu ST:PHONE 3037243339 ST:SUBMIT_DATE 2022-12-16 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - F2-27-07 Group:K562 Oe-IFITM3 RAW_FILE_NAME=F2-27-07 SUBJECT_SAMPLE_FACTORS - F2-27-08 Group:K562 Oe-IFITM3 RAW_FILE_NAME=F2-27-08 SUBJECT_SAMPLE_FACTORS - F2-27-09 Group:K562 Oe-IFITM3 RAW_FILE_NAME=F2-27-09 SUBJECT_SAMPLE_FACTORS - F2-27-13 Group:mPB-CD34+ RAW_FILE_NAME=F2-27-13 SUBJECT_SAMPLE_FACTORS - F2-27-14 Group:mPB-CD34+ RAW_FILE_NAME=F2-27-14 SUBJECT_SAMPLE_FACTORS - F2-27-15 Group:mPB-CD34+ RAW_FILE_NAME=F2-27-15 SUBJECT_SAMPLE_FACTORS - F2-27-01 Group:THP1 Oe-IFITM3 RAW_FILE_NAME=F2-27-01 SUBJECT_SAMPLE_FACTORS - F2-27-02 Group:THP1 Oe-IFITM3 RAW_FILE_NAME=F2-27-02 SUBJECT_SAMPLE_FACTORS - F2-27-03 Group:THP1 Oe-IFITM3 RAW_FILE_NAME=F2-27-03 #COLLECTION CO:COLLECTION_SUMMARY The human embryonic kidney 293T (HEK293T) and K562 cells were maintained in CO:COLLECTION_SUMMARY Iscove's modified Dulbecco’s medium (IMDM; Sigma), human THP1 cells were CO:COLLECTION_SUMMARY maintained in Roswell Park Memorial Institute medium (RPMI; Lonza). All media CO:COLLECTION_SUMMARY were supplemented with 10% fetal bovine serum (FBS; Gibco), penicillin (100 CO:COLLECTION_SUMMARY IU/ml), streptomycin (100 µg/ml) and 2% glutamine. A549 and Vero E6 cells were CO:COLLECTION_SUMMARY cultured in Dulbecco's Modified Eagle Medium (DMEM, Sigma) supplemented with 10% CO:COLLECTION_SUMMARY fetal bovine serum (FBS; Gibco), 1% Non-essential amino acid (NEAA, Gibco), 1% CO:COLLECTION_SUMMARY Sodium Pyruvate (Gibco). Calu3 were cultured in Dulbecco's Modified Eagle Medium CO:COLLECTION_SUMMARY (DMEM, Sigma) supplemented with 20% fetal bovine serum (FBS; Gibco), 1% CO:COLLECTION_SUMMARY Non-essential amino acid (NEAA, Gibco), 1% Sodium Pyruvate (Gibco) and 2% CO:COLLECTION_SUMMARY GlutaMAX (Gibco). All cells were maintained in a 5% CO2 humidified atmosphere at CO:COLLECTION_SUMMARY 37°C. A549-hACE2 cell lines were generated by lentiviral transduction. CO:COLLECTION_SUMMARY Lentiviral vectors were produced following a standard procedure based on calcium CO:COLLECTION_SUMMARY phosphate co-transfection with 3rd generation helper and transfer plasmids. CO:COLLECTION_SUMMARY Primary cells Human CD34+ HSPC were isolated through positive magnetic bead CO:COLLECTION_SUMMARY selection according to manufacturer’s instructions (Milteney) from umbilical CO:COLLECTION_SUMMARY cord blood or from mononuclear cells collected upon informed consent from CO:COLLECTION_SUMMARY healthy volunteers according to the Institutional Ethical Committee approved CO:COLLECTION_SUMMARY protocol (TIGET01). Otherwise, CB, bone marrow (BM) or G-CSF mPB-CD34+ cells CO:COLLECTION_SUMMARY were directly purchased from Lonza or Hemacare. All cells were maintained in a CO:COLLECTION_SUMMARY 5% CO2 humidified atmosphere at 37°C. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY Transduction All cells were transduced at the indicated multiplicity of TR:TREATMENT_SUMMARY infection (MOI) as calculated by titration of vector stocks on 293T cells and TR:TREATMENT_SUMMARY expressed as transducing units (TU)/293T cell. Cell lines were transduced with TR:TREATMENT_SUMMARY LV for 16 hours, washed and kept in culture until reading transduction TR:TREATMENT_SUMMARY efficiency by flow cytometry. Human CB-derived HSPC were cultured in serum-free TR:TREATMENT_SUMMARY StemSpan medium (StemCell Technologies) supplemented with penicillin (100 TR:TREATMENT_SUMMARY IU/ml), streptomycin (100 µg/ml), 100 ng/ml recombinant human stem cell factor TR:TREATMENT_SUMMARY (rhSCF), 20 ng/ml recombinant human thrombopoietin (rhTPO), 100 ng/ml TR:TREATMENT_SUMMARY recombinant human Flt3 ligand (rhFlt3), and 20 ng/ml recombinant human IL6 TR:TREATMENT_SUMMARY (rhIL6) (all from Peprotech) 16 to 24 hours prior to transduction. HSPC were TR:TREATMENT_SUMMARY then transduced at a concentration of 1×106 cells per milliliter with TR:TREATMENT_SUMMARY VSV-gp-pseudotyped SINLV for 16 hours at the indicated multiplicity of infection TR:TREATMENT_SUMMARY (MOI) in the same medium. G-CSF mobilized peripheral blood CD34+ cells were TR:TREATMENT_SUMMARY maintained in culture in CellGro medium (Cell Genix) containing a cocktail of TR:TREATMENT_SUMMARY cytokines: 60 ng/ml IL-3, 100 ng/ml TPO, 300 ng/ml SCF, and 300 ng/ml FLT-3L TR:TREATMENT_SUMMARY (all from Cell Peprotech). Transductions were performed with the indicated dose TR:TREATMENT_SUMMARY of vectors for 16 hours in the same cytokine-containing medium. For single hit TR:TREATMENT_SUMMARY reporter LV transductions cells were washed and maintained in serum-free medium TR:TREATMENT_SUMMARY supplemented with cytokines as above until the reading of the percentage of TR:TREATMENT_SUMMARY positive cells by FACS. To perform OE, KD and KO experiments cells were TR:TREATMENT_SUMMARY transduced with the respective LV and then challenged with a second transduction TR:TREATMENT_SUMMARY with or without drugs. Except for primary cells, KD and KO cells were selected TR:TREATMENT_SUMMARY with Puromycin before the second hit of transduction. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Lipids were extracted from frozen cell pellets using LC-MS grade cold methanol SP:SAMPLEPREP_SUMMARY at a concentration of 2e6 cells/mL. Samples were briefly vortexed to mix then SP:SAMPLEPREP_SUMMARY incubated for 30 min at -20C. Supernatants were clarified by centrifugation (10 SP:SAMPLEPREP_SUMMARY min, 18,213 g, 4C) then 25 uL aliquots were transferred to autosampler vials. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY 17 minute positive CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (150 x 2.1mm,1.8um) CH:COLUMN_TEMPERATURE 45 CH:FLOW_GRADIENT 0–10 min 30–100% B and 0.325 mL/min; 10–12 min 100% B and 0.325 mL/min; CH:FLOW_GRADIENT 12–12.5 min 100–30% B and 0.4 mL/min; 12.5–14.9 min 30% B and 0.4 mL/min; CH:FLOW_GRADIENT 14.9–15 min 30% B and 0.325 mL/min. CH:FLOW_RATE varies, see solvent gradient CH:SOLVENT_A 25% acetonitrile/75% water; 5 mM ammonium acetate CH:SOLVENT_B 90% isopropanol/10% acetonitrile; 5 mM ammonium acetate CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:MS_COMMENTS the mass spectrometer operated in top 10 data dependent MS2 mode with NCE of 30 MS:MS_COMMENTS eV (HCD) and dynamic exclusion of 10 s. MS1 scans were acquired at a resolution MS:MS_COMMENTS of 70,000 across the m/z range of 125-1500. MS2 scans were acquired at a MS:MS_COMMENTS resolution of 17,500. Sheath and auxiliary gases (both N2) were set at 45 and 15 MS:MS_COMMENTS (unitless), respectively. Instrument stability and quality control were assessed MS:MS_COMMENTS via injection of technical replicates every 6 runs. Untargeted lipid results MS:MS_COMMENTS were obtained using LipidSearch (Thermo) with precursor ion tolerance set to 5 MS:MS_COMMENTS ppm and product ion tolerance at 8 ppm. MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples F2-27-07 F2-27-08 F2-27-09 F2-27-13 F2-27-14 F2-27-15 F2-27-01 F2-27-02 F2-27-03 Factors Group:K562 Oe-IFITM3 Group:K562 Oe-IFITM3 Group:K562 Oe-IFITM3 Group:mPB-CD34+ Group:mPB-CD34+ Group:mPB-CD34+ Group:THP1 Oe-IFITM3 Group:THP1 Oe-IFITM3 Group:THP1 Oe-IFITM3 LPC(20:0) 6093.6723 6184.9856 5892.3019 6352.0005 6331.4686 6311.5397 22579.9471 17247.6843 81194.0171 LPS(18:0) 155742.4350 223765.3801 265345.4998 217784.1323 217745.4471 274428.7855 1441840.3310 827187.3942 2922432.3730 LPS(18:1) 69613.5450 124366.9169 58559.9653 100278.3878 87758.7690 141366.6124 168370.4812 171547.7631 332907.6390 PC(11:0_20:4) 10999.0719 11006.2749 11047.2176 144187.0892 22255.0220 90108.9899 377912.8125 122534.2153 416331.6971 PC(12:0e_18:0) 23684.1606 21949.8225 14330.3376 25765.8524 13963.1326 41522.5235 1677626.9500 929877.5975 1797400.2200 PC(12:1e_18:2) 37564.3376 51217.5926 56229.2526 236555.1549 61907.7530 204293.8826 2124480.9070 2514586.9150 5852603.7810 PC(25:0) 1194709.7550 1330675.5760 1280978.6700 1011362.9920 1287797.5880 1230591.7040 1077737.6850 1278946.8470 1020838.7150 PC(29:0) 39845.7590 29471.0990 52049.7837 1014336.6790 273088.0006 688187.1047 2541192.9580 1117424.8180 1684793.9290 PC(29:1e) 14866.9596 13881.4778 26095.6570 28515.7531 15331.8360 28455.1675 1818678.5780 541422.7204 924057.6035 PC(31:2) 53076.0868 86519.5117 62447.9867 60562.0167 63327.2544 67631.7847 81128.3916 55319.4785 58716.9832 PC(31:7e) 9061.5961 7153.6976 7837.7955 10768.2398 12030.0372 17639.4008 14290.8995 10638.5469 21856.0627 PC(33:3) 9444.1672 8389.4751 9873.9615 79737.2352 10286.5133 47310.1107 151846.5956 243502.5786 159963.9476 PC(34:1) 322701.0346 306852.4235 450617.7223 347061.4098 485845.9322 299956.2390 488056.8578 302858.2613 374190.1795 PC(34:4) 109895.3463 43442.9039 158760.4226 77016.2186 12830.7010 63810.4825 612987.3928 650574.8126 525858.1318 PC(34:4e) 49377.8636 128416.2992 87669.4063 30984.0491 20466.2421 93052.0626 6479183.7010 5722941.9580 14199085.4700 PC(34:5) 41273.1187 14509.8172 62265.2368 609163.8950 174219.9132 543778.7471 1026976.5720 1974033.5930 4774049.6900 PC(35:4) 451014.2422 771757.9540 558859.5996 7191375.3870 1764818.1430 7459482.1050 13213038.4900 14092629.5500 22934789.2600 PC(35:5) 9650731.9240 8711397.0640 8299064.3510 8677983.8610 8407241.8860 8900455.8790 9137391.8050 8474996.4890 8725640.1450 PC(35:6) 269475.6607 130931.7773 426849.6527 2192363.2460 522053.5521 1745957.3680 2825683.8310 4310971.8970 4322667.8010 PC(36:1) 103537.4036 74697.4160 95009.3995 117275.6958 113588.2556 74094.0967 217917.3933 82056.0771 142008.8517 PC(36:5) 15505.0704 77054.3311 13848.7013 15088.2121 14598.7519 52906.7722 61787.1568 106423.7837 288387.2777 PC(36:6) 116528.7048 58779.1327 158421.0400 825130.8263 185667.6910 1132731.1300 5929822.1730 6280176.4810 10543073.2300 PC(37:5) 207302.3741 171026.8035 329429.5497 465231.0039 1718373.5240 644219.5626 2026561.5460 3142888.6700 3907414.5410 PC(38:1) 189550.0973 195814.9533 173689.2663 208988.0278 201110.1921 212014.1888 316976.4443 227532.2466 261175.1650 PC(38:2) 15126.3324 41649.5355 31809.3851 29610.7543 59101.8634 15909.1007 103515.4871 17168.8958 96852.3443 PC(38:3e) 683602.7251 293421.6924 966707.7763 269376.3953 54966.7073 424450.5918 18210966.8900 15245384.5700 39590469.3900 PC(39:2e) 475145.6806 461640.9736 466849.3400 473218.8848 417713.1227 533320.8475 966106.3230 1026942.9160 1688058.4270 PC(39:3) 18634.8368 16152.7917 24057.8511 27019.7753 19998.1504 19474.3863 694658.4952 991454.6966 2435811.3320 PC(39:6) 9300.5198 7668.2866 15698.8472 81155.4222 18535.0546 42889.0675 189656.2738 190648.9515 210914.4028 PC(40:5) 110594.5323 161496.4618 108631.4007 553951.8890 213879.3286 552911.0305 3098330.3570 5116825.4270 12454203.0300 PC(40:8) 301300.4585 149047.8380 268927.8736 7508621.9380 1609978.6740 5269400.9370 1101202.6700 3523051.3450 4506320.1300 PC(41:2e) 1395227.8670 1437846.0610 1292397.4440 1225638.3370 1418293.3960 1433503.7200 2368625.3860 2123628.7590 4413612.2680 PC(41:3) 23220.0283 15948.4514 27204.7052 206654.7400 61022.6301 245090.5970 869901.0537 469470.2651 840493.2061 PC(41:5) 15580.6397 16048.3112 22353.3926 15901.5885 86065.4440 21132.4483 461332.6569 195935.4947 390564.8525 PC(42:10) 15150.0214 41108.1771 20475.7906 1152037.1510 72993.3783 785482.2457 653645.5759 363474.2474 443009.1096 PC(42:2e) 1400595.8300 1244288.4880 1391648.8640 1356617.6900 1415233.5570 1414862.3790 2762056.0760 2566451.5850 3945052.2510 PC(42:7) 26217.2543 24581.4908 21779.6179 617042.1113 80251.2363 430081.0877 86059.3492 71794.1382 137799.2681 PC(42:9) 43867.1809 34126.9720 90383.9624 1956164.0560 390192.9580 1481084.5510 211444.4011 836490.0435 667060.9678 PC(43:2e) 1950659.6340 1851182.5570 1990213.1170 1949139.0400 2109528.0180 1866418.1900 2449255.9690 1786954.8740 2170555.4300 PC(44:2e) 1036669.9520 1027712.5790 992816.4607 1026331.8290 1138148.7030 1114087.6520 1585527.7960 1067215.3090 1595017.2110 PC(44:3e) 251509.7008 235379.5738 235381.3108 220712.9819 246624.4116 238876.6860 2224580.9470 2148212.7700 7124841.6070 PC(44:7) 16090.6130 15356.2141 16419.5063 1605668.8210 161375.1192 1160984.2150 90838.8388 79589.7365 145154.8559 PE(16:0p_18:3) 13817.9360 14133.4471 13829.7317 22642.0744 16554.6041 15425.8009 272952.6529 142019.9846 593813.6004 PE(18:1_22:6) 500520.8729 302899.3901 811297.3662 7896868.0780 2376590.8350 7767688.1880 642603.8079 1413974.1320 677904.6341 PG(18:0_18:1) 171699.8344 70967.9578 276791.0900 2071064.9670 374304.4830 1601424.3850 9822919.2340 8832585.0710 31649552.4400 PI(16:0_20:3) 32257.4114 15466.3348 60807.3618 113433.5279 12713.6431 69235.0506 672313.6932 280528.3303 761884.1799 PS(16:0_18:1) 69762.6456 37961.4574 100041.1364 529925.8052 132016.5511 452020.0321 1857846.1760 1495795.7160 2036611.7380 PS(18:0_20:3) 101310.5571 71621.7679 189800.9219 625300.7409 252123.4023 475608.2332 846969.0419 748414.8501 396458.2024 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name pubchem_id inchi_key kegg_id other_id other_id_type ri ri_type moverz_quant LPC(20:0) 7.892 LPS(18:0) 6.064 LPS(18:1) 5.34 PC(11:0_20:4) 9.342 PC(12:0e_18:0) 9.817 PC(12:1e_18:2) 9.802 PC(25:0) 5.327 PC(29:0) 9.971 PC(29:1e) 9.969 PC(31:2) 7.549 PC(31:7e) 5.883 PC(33:3) 9.821 PC(34:1) 6.353 PC(34:4) 10.173 PC(34:4e) 9.664 PC(34:5) 9.694 PC(35:4) 9.955 PC(35:5) 5.152 PC(35:6) 9.828 PC(36:1) 7.329 PC(36:5) 8.686 PC(36:6) 9.381 PC(37:5) 9.832 PC(38:1) 8.045 PC(38:2) 11.528 PC(38:3e) 10.423 PC(39:2e) 11.166 PC(39:3) 10.246 PC(39:6) 10.491 PC(40:5) 9.851 PC(40:8) 9.705 PC(41:2e) 11.333 PC(41:3) 10.884 PC(41:5) 10.751 PC(42:10) 9.679 PC(42:2e) 11.398 PC(42:7) 10.249 PC(42:9) 9.748 PC(43:2e) 11.471 PC(44:2e) 11.524 PC(44:3e) 11.334 PC(44:7) 10.093 PE(16:0p_18:3) 9.046 PE(18:1_22:6) 10.073 PG(18:0_18:1) 9.393 PI(16:0_20:3) 9.232 PS(16:0_18:1) 9.822 PS(18:0_20:3) 10.09 METABOLITES_END #END