#METABOLOMICS WORKBENCH cuiliang50_20220511_223811 DATATRACK_ID:3245 STUDY_ID:ST002164 ANALYSIS_ID:AN003546 PROJECT_ID:PR001377 VERSION 1 CREATED_ON May 13, 2022, 12:10 pm #PROJECT PR:PROJECT_TITLE TMEM41B and VMP1 modulate cellular lipid and energy metabolism for facilitating PR:PROJECT_TITLE Dengue virus infection PR:PROJECT_TYPE untargeted metabolomics PR:PROJECT_SUMMARY Lipid metabolism is an intricate yet crucial cellular process co-opted by PR:PROJECT_SUMMARY multiple viruses for replication and biogenesis. Transmembrane Protein 41B PR:PROJECT_SUMMARY (TMEM41B) and Vacuole Membrane Protein 1 (VMP1) are two recently identified PR:PROJECT_SUMMARY ER-resident lipid scramblases that play a role in autophagosome formation and PR:PROJECT_SUMMARY cellular lipid metabolism. Importantly, TMEM41B is also a newly validated host PR:PROJECT_SUMMARY dependency factor required for productive infection of several medically PR:PROJECT_SUMMARY important enveloped RNA viruses, such as flaviviruses and human coronaviruses. PR:PROJECT_SUMMARY However, the exact underlying mechanism of TMEM414B in modulating viral PR:PROJECT_SUMMARY infections remains an open question. Here, we uncovered that TMEM41B and VMP1 PR:PROJECT_SUMMARY deficiencies severely impaired replication of flavivirus and human coronavirus PR:PROJECT_SUMMARY via multiple parallel cellular mechanisms. In accordance with previous reports, PR:PROJECT_SUMMARY we validated that both TMEM41B and VMP1 are indispensable for all four serotypes PR:PROJECT_SUMMARY of dengue virus (DENV) and human coronavirus OC43 (HCoV-OC43) to infect human PR:PROJECT_SUMMARY cells, but not chikungunya virus, an alphavirus. Impaired dengue virus PR:PROJECT_SUMMARY replication in TMEM41B and VMP1 deficient cells could induce a robust activation PR:PROJECT_SUMMARY of innate immune RNA sensing as evidenced by hyperactivation of RIG-I and MDA5. PR:PROJECT_SUMMARY However, this phenomenon was a consequence but not the root cause of the PR:PROJECT_SUMMARY diminished viral replication. Notably, the impact of TMEM41B deficiency on DENV PR:PROJECT_SUMMARY replication could be reversed by complementing the cells using exogenous PR:PROJECT_SUMMARY unsaturated fatty acids, indicating a metabolic role for TMEM41B in flavivirus PR:PROJECT_SUMMARY infection. Furthermore, we found that derailed cellular energy metabolism could PR:PROJECT_SUMMARY be a contributing factor to block DENV infection as TMEM41B and VMP1 deficient PR:PROJECT_SUMMARY cells harbored higher levels of compromised mitochondria that exhibited aberrant PR:PROJECT_SUMMARY functions in facilitating beta-oxidation. Using lipidome and metabolome PR:PROJECT_SUMMARY profiling of TMEM41B and VMP1 deficient cells, we further revealed that each of PR:PROJECT_SUMMARY these genetic deficiencies result in distinctive cellular metabolic PR:PROJECT_SUMMARY dysregulations, underlining their necessity for a balanced metabolic landscape, PR:PROJECT_SUMMARY and strengthening the metabolic role of these ER membrane proteins in PR:PROJECT_SUMMARY facilitating virus infection. Our results highlighted that TMEM41B and VMP1 are PR:PROJECT_SUMMARY required for homeostasis of cellular metabolism, and this metabolic role PR:PROJECT_SUMMARY contributes to their essentiality in facilitating DENV infection PR:INSTITUTE Singapore-MIT Alliance for Research and Technology (SMART Centre) PR:LAST_NAME Cui PR:FIRST_NAME Liang PR:ADDRESS 1 CREATE Way, #03-12 Enterprise Wing, Singapore, Singapore, 138602, Singapore PR:EMAIL liangcui@smart.mit.edu PR:PHONE 65-84328978 #STUDY ST:STUDY_TITLE TMEM41B and VMP1 modulate cellular lipid and energy metabolism for facilitating ST:STUDY_TITLE Dengue virus infection ST:STUDY_TYPE untargeted analysis ST:STUDY_SUMMARY Lipid metabolism is an intricate yet crucial cellular process co-opted by ST:STUDY_SUMMARY multiple viruses for replication and biogenesis. Transmembrane Protein 41B ST:STUDY_SUMMARY (TMEM41B) and Vacuole Membrane Protein 1 (VMP1) are two recently identified ST:STUDY_SUMMARY ER-resident lipid scramblases that play a role in autophagosome formation and ST:STUDY_SUMMARY cellular lipid metabolism. Importantly, TMEM41B is also a newly validated host ST:STUDY_SUMMARY dependency factor required for productive infection of several medically ST:STUDY_SUMMARY important enveloped RNA viruses, such as flaviviruses and human coronaviruses. ST:STUDY_SUMMARY However, the exact underlying mechanism of TMEM414B in modulating viral ST:STUDY_SUMMARY infections remains an open question. Here, we uncovered that TMEM41B and VMP1 ST:STUDY_SUMMARY deficiencies severely impaired replication of flavivirus and human coronavirus ST:STUDY_SUMMARY via multiple parallel cellular mechanisms. In accordance with previous reports, ST:STUDY_SUMMARY we validated that both TMEM41B and VMP1 are indispensable for all four serotypes ST:STUDY_SUMMARY of dengue virus (DENV) and human coronavirus OC43 (HCoV-OC43) to infect human ST:STUDY_SUMMARY cells, but not chikungunya virus, an alphavirus. Impaired dengue virus ST:STUDY_SUMMARY replication in TMEM41B and VMP1 deficient cells could induce a robust activation ST:STUDY_SUMMARY of innate immune RNA sensing as evidenced by hyperactivation of RIG-I and MDA5. ST:STUDY_SUMMARY However, this phenomenon was a consequence but not the root cause of the ST:STUDY_SUMMARY diminished viral replication. Notably, the impact of TMEM41B deficiency on DENV ST:STUDY_SUMMARY replication could be reversed by complementing the cells using exogenous ST:STUDY_SUMMARY unsaturated fatty acids, indicating a metabolic role for TMEM41B in flavivirus ST:STUDY_SUMMARY infection. Furthermore, we found that derailed cellular energy metabolism could ST:STUDY_SUMMARY be a contributing factor to block DENV infection as TMEM41B and VMP1 deficient ST:STUDY_SUMMARY cells harbored higher levels of compromised mitochondria that exhibited aberrant ST:STUDY_SUMMARY functions in facilitating beta-oxidation. Using lipidome and metabolome ST:STUDY_SUMMARY profiling of TMEM41B and VMP1 deficient cells, we further revealed that each of ST:STUDY_SUMMARY these genetic deficiencies result in distinctive cellular metabolic ST:STUDY_SUMMARY dysregulations, underlining their necessity for a balanced metabolic landscape, ST:STUDY_SUMMARY and strengthening the metabolic role of these ER membrane proteins in ST:STUDY_SUMMARY facilitating virus infection. Our results highlighted that TMEM41B and VMP1 are ST:STUDY_SUMMARY required for homeostasis of cellular metabolism, and this metabolic role ST:STUDY_SUMMARY contributes to their essentiality in facilitating DENV infection. ST:INSTITUTE Singapore-MIT Alliance for Research and Technology (SMART Centre) ST:LAST_NAME Cui ST:FIRST_NAME Liang ST:ADDRESS 1 CREATE Way, #03-12 Enterprise Wing, Singapore, Singapore, 138602, Singapore ST:EMAIL liangcui@smart.mit.edu ST:PHONE 65-84328978 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - DH1 genotype:wild type RAW_FILE_NAME=DH1-2.d SUBJECT_SAMPLE_FACTORS - DH2 genotype:wild type RAW_FILE_NAME=DH2-2.d SUBJECT_SAMPLE_FACTORS - DH3 genotype:wild type RAW_FILE_NAME=DH3-2.d SUBJECT_SAMPLE_FACTORS - DT1 genotype:TMEM41B knockout RAW_FILE_NAME=DT1-2.d SUBJECT_SAMPLE_FACTORS - DT2 genotype:TMEM41B knockout RAW_FILE_NAME=DT2-2.d SUBJECT_SAMPLE_FACTORS - DT3 genotype:TMEM41B knockout RAW_FILE_NAME=DT3-2.d SUBJECT_SAMPLE_FACTORS - DV1 genotype:VMP1 knockout RAW_FILE_NAME=DV1-2.d SUBJECT_SAMPLE_FACTORS - DV2 genotype:VMP1 knockout RAW_FILE_NAME=DV2-2.d SUBJECT_SAMPLE_FACTORS - DV3 genotype:VMP1 knockout RAW_FILE_NAME=DV3-2.d #COLLECTION CO:COLLECTION_SUMMARY 1x108 293FT, TMEM41B KO and VMP1 KO cells were washed with PBS thrice before CO:COLLECTION_SUMMARY adding 560 µl extraction solvent (methanol:water = 2:5), pre-cooled at −4°C. CO:COLLECTION_SUMMARY Cells were then scraped into the extraction solvent on ice and transferred into CO:COLLECTION_SUMMARY eppendorf tubes. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY NO TREATMENT #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 1x108 293FT, TMEM41B KO and VMP1 KO cells were washed with PBS thrice before SP:SAMPLEPREP_SUMMARY adding 560 µl extraction solvent (methanol:water = 2:5), pre-cooled at −4°C. SP:SAMPLEPREP_SUMMARY Cells were then scraped into the extraction solvent on ice and transferred into SP:SAMPLEPREP_SUMMARY eppendorf tubes. Then, 800 µL methyl tert-butyl ether (MTBE) was added and the SP:SAMPLEPREP_SUMMARY samples were sonicated for 10 min. After centrifugation for 15 min at 3,000 rpm SP:SAMPLEPREP_SUMMARY at 4°C to separate phases, the upper layer and lower layer were collected for SP:SAMPLEPREP_SUMMARY lipidomics and metabolomics analysis, respectively. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 ultrahigh pressure liquid chromatography system CH:COLUMN_NAME Agilent rapid resolution HT Zorbax SB-C18 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6550 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Mass data were collected between m/z 100 and 1000 at a rate of two scans per MS:MS_COMMENTS second. The ion spray voltage was set at 4,000 V, and the heated capillary MS:MS_COMMENTS temperature was maintained at 350°C. The drying gas and nebulizer nitrogen gas MS:MS_COMMENTS flow rates were 12.0 L/min and 50 psi, respectively. Two reference masses were MS:MS_COMMENTS continuously infused to the system to allow constant mass correction during the MS:MS_COMMENTS run: m/z 121.0509 (C5H4N4) and m/z 922.0098 (C18H18O6N3P3F24). Raw spectrometric MS:MS_COMMENTS data were analyzed by MassHunter Qualitative Analysis software (Agilent MS:MS_COMMENTS Technologies, US) and the molecular features characterized by retention time, MS:MS_COMMENTS chromatographic peak intensity and accurate mass, were obtained by using the MS:MS_COMMENTS Molecular Feature Extractor algorithm. The features were then analyzed by MS:MS_COMMENTS MassHunter Mass Profiler Professional software (Agilent Technologies, US). Only MS:MS_COMMENTS features with an intensity ≥ 20,000 counts (approximately three times the MS:MS_COMMENTS limit of detection of our LC-MS instrument), and found in at least 80% of the MS:MS_COMMENTS samples at the same sampling time point signal were kept for further processing. MS:MS_COMMENTS Next, a tolerance window of 0.15 min and 2 mDa was used for alignment of RT and MS:MS_COMMENTS m/z values. MS:MS_RESULTS_FILE ST002164_AN003546_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END