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Zika virus remodelled ER membranes contain an array of proviral factors involved in redox pathways

ABSTRACT: Zika virus (ZIKV) has emerged as a global health issue, yet neither antiviral therapy nor vaccine are available. ZIKV is an enveloped RNA virus, replicating in the cytoplasm in close association with ER membranes with the help of host cell factors that are poorly characterized. Here, we isolated ER membranes from ZIKV-infected cells and determined their proteome. 46 host cell factors were enriched in ZIKV remodeled membranes, several of these proteins having a role in redox pathways. 4 proteins were characterized in detail. Thioredoxin Reductase 1 (TXNRD1) contributing to folding of disulfide bond containing proteins and modulating ZIKV secretion; aldo-keto reductase family 1 member C3 (AKR1C3), regulating capsid protein abundance and thus, ZIKV assembly; biliverdin reductase B (BLVRB) involved in ZIKV induced lipid peroxidation and promoting stability of non-structural proteins having transmembrane domains; Adenosylhomocysteinase (AHCY) promoting m6A methylation of ZIKV RNA and thus immune evasion. These results highlight the involvement of redox enzymes in ZIKV life cycle and their recruitment to virally remodeled ER membranes.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

SUBMITTER: Quirin Emslander

LAB HEAD: Andreas Pichlmair

PROVIDER: PXD043372 | Pride | 2023-11-14

REPOSITORIES: Pride

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			Action	DRS
	20211015_QEp5_AnPi_A549_CNX_HA_Mock_10_2.raw	Raw
	20211015_QEp5_AnPi_A549_CNX_HA_Mock_11_2.raw	Raw
	20211015_QEp5_AnPi_A549_CNX_HA_Mock_12_2.raw	Raw
	20211015_QEp5_AnPi_A549_CNX_HA_Mock_13_2.raw	Raw
	20211015_QEp5_AnPi_A549_CNX_HA_Mock_14_2.raw	Raw

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Project description:Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infection causes an immune-mediated disease. We have recently shown that SARS-CoV-induced epithelial Calu-3 cytokines could exacerbate and dampen host inflammatory and T cell responses, respectively, through modulating the functions of macrophages and dendritic cells, thereby suggesting that not only are lung epithelial cells the primary cells of SARS-CoV infection, but they also involve in initiating and orchestrating the host innate and adaptive immunity. Comprehensive evaluation of the complex epithelial signaling to SARS-CoV is, thus, crucial for paving the way to better understand SARS pathogenesis and develop the innovative therapeutics against SARS. Here, based on the microarray-based functional genomics, we reported that 2B4 cells, a clonal derivative of Calu-3 cells, elicited a temporal and spatial activation of nuclear factor (NF)kappaB, activator protein (AP)-1 (ATF2/c-Jun), and interferon regulatory factor (IRF)-3/-7 at 12-, 24-, and 48-hrs post infection (p.i.), respectively, resulting in the activation of many antiviral genes, including interferon (IFN)-Î², -Î»s, SARS-related inflammatory mediators, and various IFN-stimulated genes (ISGs). While elevated responses of IFN-Î² and IFN-Î»s were not detected until 48-hrs p.i., as a consequence of a delayed IRF-3/-7 activation, we showed, for the first time, that both types of IFNs exerted previously under-described non-redundant, complementary, and/or synergistic effects on the epithelial defense against SARS-CoV. Collectively, our results highlight the molecular mechanisms of the sequential activation of virus- and IFN-dependent signaling of lung epithelial cells against SARS-CoV and identify novel cellular targets for future studies, aiming at advancing strategies against SARS. DNA microarrays were performed in the Molecular Genomics (MG) Core Facilities at UTMB. The detailed information about the procedures of microarray analysis has been posted on the MG Core Facilities website (genomics@scms.utmb.edu). To characterize the dynamic, spatial, and temporal changes of the gene expression induced by SARS-CoV, confluent 2B4 cells grown in T-75 flasks were infected with SARS-CoV (MOI=0.1) or remained uninfected (as control) for 12, 24, and 48hrs. Because 2B4 cells were also permissive to the productive infection of Dhori virus (DHOV), a member of the Orthomyxoviridae family within the Thogotovirus genus, resulting in robust responses of IFNs and other pro-inflammatory mediators, we also established parallel cultures of DHOV-infected 2B4 cells (MOI=0.1) for the comparative analysis of global gene expression elicited by SARS-CoV- versus DHOV-infected 2B4 cells. To meet the minimal number required for application of statistical algorithms, we performed the study in triplicate at each time point for mock-, SARS-CoV-, and DHOV-infected cultures, yielding a total of 27 arrays. Briefly, supernatants were harvested from differentially treated cultures at 12-, 24-, and 48-hrs p.i. for subsequent analyses of viral yields and cytokine profiling, whereas the cells were subjected to total RNA extraction by using an RNAqueous-4PCR kit and following the protocol recommended by the manufacturer (Ambion, Austin, TX). Purified RNA samples were sent to our core facility for conversion to cDNA, biotin-labeled, and hybridized to 27 Affymetrix Human Genome U133 Plus 2.0 âGene Chipsâ each of which contained 54,675 probe set identifiers representing more than ~47,400 transcripts that identify ~38,500 well-characterized genes, and various internal controls (Affymetrix, Santa Clara, CA). Mock-infected cells were compared to cells infected with SARS-CoV or DHOV at each time point.

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Zika virus remodelled ER membranes contain an array of proviral factors involved in redox pathways

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