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Quantification of organelle membrane contact site protein abundances during infection with human viruses

ABSTRACT: Viruses rely on organelles to invade, replicate, and spread between host cells. Likewise, organelles underlie the host's ability to sense and respond to pathogen invasion. To subvert cellular functions and turn them to the benefit of virus production and spread, viruses remodel organelle structure and function during infection. We predicted that membrane contact sites (MCSs) underlie virus-driven organelle remodeling events. MCSs use protein interactions to bridge organelle membranes for the direct transfer of biomolecules, coordinating fundamental organelle dynamics. The extent of contact and potential functions of MCSs are dictated by the abundance of MCS-specific proteins at organelle interfaces. Here, we design a parallel reaction monitoring (PRM) targeted MS assay to quantify MCS protein abundances across time and space. Our assay encompasses nearly all MCS functions and all major cellular organelles (ER, mitochondria, peroxisome, endosomes, lysosomes, autophagosomes, lipid droplets, plasma membrane, Golgi), with 2-5 signature peptides per protein and internal controls. Utilizing MCS-PRM, we uncover temporal- and organelle-specific regulation of MCSs during the infectious cycles of critical human viruses: the beta-herpesvirus human cytomegalovirus (HCMV, also known as HHV-5), herpes simplex virus type 1 (HSV-1), the orthomyxovirus influenza A (Infl. A PR8), and the beta-coronavirus HCoV-OC43.

ORGANISM(S): Homo Sapiens Human Coronavirus Oc43 Influenza A Virus (a/puerto Rico/8/1934(h1n1)) Human Betaherpesvirus 5 Human Alphaherpesvirus 1

SUBMITTER: Katelyn Cook

PROVIDER: PXD023761 | panorama | Fri Jul 08 00:00:00 BST 2022

REPOSITORIES: PanoramaPublic

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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:Pairwise compatibility between virus and host proteins can dictate the outcome of infection. During transmission, both inter- and intraspecies variabilities in receptor protein sequences can impact cell susceptibility. Many viruses possess mutable viral entry proteins and the patterns of host compatibility can shift as the viral protein sequence changes. This combinatorial sequence space between virus and host is poorly understood, as traditional experimental approaches lack the throughput to simultaneously test all possible combinations of protein sequences. Here, we created a pseudotyped virus infection assay where a multiplexed target-cell library of host receptor variants can be assayed simultaneously using a DNA barcode sequencing readout. We applied this assay to test a panel of 30 ACE2 orthologs or human sequence mutants for infectability by the original SARS-CoV-2 spike protein or the Alpha, Beta, Gamma, Delta, and Omicron BA1 variant spikes. We compared these results to an analysis of the structural shifts that occurred for each variant spike’s interface with human ACE2. Mutated residues were directly involved in the largest shifts, although there were also widespread indirect effects altering interface structure. The N501Y substitution in spike conferred a large structural shift for interaction with ACE2, which was partially recreated by indirect distal substitutions in Delta, which does not harbor N501Y. The structural shifts from N501Y greatly influenced the set of animal orthologs the variant spike was capable of interacting with. Out of the thirteen non-human orthologs, ten exhibited unique patterns of variant-specific compatibility, demonstrating that spike sequence changes during human transmission can toggle ACE2 compatibility and potential susceptibility of other animal species, and cumulatively increase overall compatibilities as new variants emerge. These experiments provide a blueprint for similar large-scale assessments of protein compatibility during entry by diverse viruses. This dataset demonstrates the complex compatibility relationships that occur between variable interacting host and virus proteins.

Project description:Previous microarray data indicated a significant increase in interferon (IFN) stimulated gene expression in cells exposed to conditioned medium from primary human trophoblasts compared to non conditioned medium. Here we want to compare the effects of conditioned media, non conditioned media, and non conditioned media spiked with 100 units of IFN beta in wild type HT1080 cells as well as in HT1080 cells deficient in STAT1 (a key signaling molecular in the IFN pathway). Interferon stimulated genes (ISGs) target viruses at various stages of their infectious life cycles, including at the earliest stage of viral entry. Here we identify ArfGAP with dual pleckstrin homology (PH) domains 2 (ADAP2) as a gene upregulated by type I IFN treatment in a STAT1-dependent manner. ADAP2 functions as a GTPase-activating protein (GAP) for Arf6 and binds to phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) and PI(3,4)P2. We show that overexpression of ADAP2 suppresses dengue virus (DENV) and vesicular stomatitis virus (VSV) infection in an Arf6 GAP activity-dependent manner, while exerting no effect on coxsackievirus B (CVB) or Sendai virus (SeV) replication. We further show that ADAP2 expression induces macropinocytosis and that ADAP2 strongly associates with actin-enriched membrane ruffles and with Rab8a- and LAMP1-, but not EEA1- or Rab7-, positive vesicles. Utilizing two techniques—light-sensitive neutral red (NR)-containing DENV and fluorescence assays for virus internalization—we show that ADAP2 primarily restricts DENV infection at the stage of virion entry and/or intracellular trafficking and that incoming DENV and VSV particles associate with ADAP2 during their entry. Taken together, this study identifies ADAP2 as an ISG that exerts antiviral effects against RNA viruses by altering Arf6-mediated trafficking to disrupt viral entry. compare the cells exposed to CM and the cells exposed to NC media spiked with IFN to the control NC exposed cells, in wild type (2f-TGH) vs Stat -/- cells (HT1080U3A)

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Quantification of organelle membrane contact site protein abundances during infection with human viruses

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