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Human Cytomegalovirus pUL37x1 is Important to Remodeling of Host Lipid Metabolism

ABSTRACT: Human cytomegalovirus (HCMV) replication requires host metabolism. Infection alters the activity in multiple metabolic pathways, including increasing fatty acid elongation and lipid synthesis. The virus-host interactions regulating the metabolic changes associated with replication are essential to infection. While multiple host factors, including kinases and transcription factors, have been identified to be important to metabolic changes that occurr following HCMV infection, little is known about the viral factors required to alter metabolism. The HCMV UL37x1 protein (pUL37x1) localizes to the mitochondria and ER and controls Ca2+ flux from the ER to the cytosol which may influence metabolism. In this study, we tested the hypothesis that pUL37x1 is important to the metabolic remodeling that is necessary for HCMV replication by using a combination of metabolomics, lipidomics, and metabolic tracers to measure fatty acid elongation. We observed that fibroblast cells infected with wild-type (WT) HCMV had similar levels of metabolites as those infected with a mutant virus lacking the UL37x1 gene, subUL37x1. However, we found that subUL37x1-infected cells had reduced levels of two host proteins that were previously demonstrated to be important for lipid metabolism during HCMV infection—fatty acid elongase 7 (ELOVL7) and ER-stress related kinase PERK—relative to WT-infected cells. Moreover, we observed that HCMV infection results in an increase in phospholipids with very long-chain fatty acid tails (PL-VLCFAs) that contain 26 or more carbons in one of their two tails. The levels of many PL-VLCFAs were lower in subUL37x1-infected cells compared to WT-infected cells. We also show that uninfected cells expressing a high-level of pUL37x1 have a greater amount of lipids relative to control cells. Overall, we conclude that although pUL37x1 is not necessary for network-wide metabolic changes occurring during infection, it is important to the remodeling of lipid metabolic pathways during HCMV infection.

INSTRUMENT(S): Liquid Chromatography MS - negative - reverse phase, Liquid Chromatography MS - positive - reverse phase

SUBMITTER: John Purdy

PROVIDER: MTBLS1073 | MetaboLights | 2020-06-26

REPOSITORIES: MetaboLights

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Project description:Human cytomegalovirus (HCMV) replication requires host metabolism. Infection alters the activity in multiple metabolic pathways, including increasing fatty acid elongation and lipid synthesis. The virus-host interactions regulating the metabolic changes associated with replication are essential to infection. While multiple host factors, including kinases and transcription factors, have been identified to be important to metabolic changes that occurr following HCMV infection, little is known about the viral factors required to alter metabolism. The HCMV UL37x1 protein (pUL37x1) localizes to the mitochondria and ER and controls Ca2+ flux from the ER to the cytosol which may influence metabolism. In this study, we tested the hypothesis that pUL37x1 is important to the metabolic remodeling that is necessary for HCMV replication by using a combination of metabolomics, lipidomics, and metabolic tracers to measure fatty acid elongation. We observed that fibroblast cells infected with wild-type (WT) HCMV had similar levels of metabolites as those infected with a mutant virus lacking the UL37x1 gene, subUL37x1. However, we found that subUL37x1-infected cells had reduced levels of two host proteins that were previously demonstrated to be important for lipid metabolism during HCMV infection—fatty acid elongase 7 (ELOVL7) and ER-stress related kinase PERK—relative to WT-infected cells. Moreover, we observed that HCMV infection results in an increase in phospholipids with very long-chain fatty acid tails (PL-VLCFAs) that contain 26 or more carbons in one of their two tails. The levels of many PL-VLCFAs were lower in subUL37x1-infected cells compared to WT-infected cells. We also show that uninfected cells expressing a high-level of pUL37x1 have a greater amount of lipids relative to control cells. Overall, we conclude that although pUL37x1 is not necessary for network-wide metabolic changes occurring during infection, it is important to the remodeling of lipid metabolic pathways during HCMV infection.

Project description:Lipid metabolism is an intricate yet crucial cellular process co-opted by multiple viruses for replication and biogenesis. Transmembrane Protein 41B (TMEM41B) and Vacuole Membrane Protein 1 (VMP1) are two recently identified ER-resident lipid scramblases that play a role in autophagosome formation and cellular lipid metabolism. Importantly, TMEM41B is also a newly validated host dependency factor required for productive infection of several medically important enveloped RNA viruses, such as flaviviruses and human coronaviruses. However, the exact underlying mechanism of TMEM414B in modulating viral infections remains an open question. Here, we uncovered that TMEM41B and VMP1 deficiencies severely impaired replication of flavivirus and human coronavirus via multiple parallel cellular mechanisms. In accordance with previous reports, we validated that both TMEM41B and VMP1 are indispensable for all four serotypes of dengue virus (DENV) and human coronavirus OC43 (HCoV-OC43) to infect human cells, but not chikungunya virus, an alphavirus. Impaired dengue virus replication in TMEM41B and VMP1 deficient cells could induce a robust activation of innate immune RNA sensing as evidenced by hyperactivation of RIG-I and MDA5. However, this phenomenon was a consequence but not the root cause of the diminished viral replication. Notably, the impact of TMEM41B deficiency on DENV replication could be reversed by complementing the cells using exogenous unsaturated fatty acids, indicating a metabolic role for TMEM41B in flavivirus infection. Furthermore, we found that derailed cellular energy metabolism could be a contributing factor to block DENV infection as TMEM41B and VMP1 deficient cells harbored higher levels of compromised mitochondria that exhibited aberrant functions in facilitating beta-oxidation. Using lipidome and metabolome profiling of TMEM41B and VMP1 deficient cells, we further revealed that each of these genetic deficiencies result in distinctive cellular metabolic dysregulations, underlining their necessity for a balanced metabolic landscape, and strengthening the metabolic role of these ER membrane proteins in facilitating virus infection. Our results highlighted that TMEM41B and VMP1 are required for homeostasis of cellular metabolism, and this metabolic role contributes to their essentiality in facilitating DENV infection.

Project description:To investigate the early host response triggered by three different strains of Trypanosoma cruzi at a local infection site, changes in host gene expression were monitored in a murine intradermal infection model using Affymetrix oligonucleotide arrays. Robust induction of IFN-stimulated genes (ISGs) was observed in excised skin 24 hours post-infection where the level of ISG induction was parasite strain-dependent with the least virulent strain triggering a muted IFN response. Infection of mice immunodepleted of IFNÎ³-producing cells or infection of IFNÎ³-deficient mice had minimal impact on the IFN response generated in T. cruzi infected mice. In contrast, infection of mice lacking the type I IFN receptor demonstrated that type I IFNs are largely responsible for the IFN response generated at the site of infection. These data highlight type I IFNs as important components of the innate immune response to T. cruzi the site of inoculation and their role in shaping the early transcriptional response to this pathogen. We used microarrays to detail the local host transcriptional response to intradermal T. cruzi infection in WT mice and mice depleted of NK cells, or deficient in IFN-gamma or type I IFN responses. Additionally we compared the local host-transcriptional response generated to infection with 3 different strains of Trypanosoma cruzi (Y, Brazil, and G). Experiment Overall Design: Mice were infected by intradermal injection of 10^6 T. cruzi trypomastigotes in 100uL of saline split between 2 adjacent sites on the shaved side flank. Control mice were injected with an equal volume of saline. 24 hours post-injection approximately 75mm^2 of skin immediately surrounding the injection site was excised and RNA was isolated from the tissue. Balb/c mice were used for most experiments and IFN-gamma KO mice were on the Balb/c background. WT 129 mice were also used as IFNAR-/- mice were on the 129 background. In total 33 arrays were performed. 7 WT (Balb/c) control, 3 Y strain infected, 3 Brazil strain infected, 3 G strain infected, 2 IFN-gamma KO control, 2 IFN-gamma KO infected, 1 NK cell depleted control, 1 NK cell depleted infected, 3 WT (129) control, 3 WT (129) infected, 3 IFNAR KO control, 3 IFNAR KO infected

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Human Cytomegalovirus pUL37x1 is Important to Remodeling of Host Lipid Metabolism

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