Palmitoleate Protects against Zika virus infection-induced Endoplasmic Reticulum Stress and Apoptosis in Neurons

ABSTRACT Zika virus (ZIKV) infection during pregnancy is associated with the development of fetal complications such as microcephaly. We have recently demonstrated that palmitoleate protects against ZIKV-induced apoptosis in placental trophoblasts. In the present study, we hypothesize that palmitoleate prevents ZIKV infection-induced endoplasmic reticulum (ER) stress and apoptosis in neurons. Neurons were infected with 0.1-1 multiplicity of infection of recombinant MR766 or PRVABC59 strains of ZIKV for an hour followed by treatment of palmitoleate (100 µM-200 µM) for different post-infection time points. Apoptosis was measured by nuclear morphological changes, caspase 3/7 activity, and immunoblot analysis of pro-apoptotic mediators. Activation of ER stress markers and viral envelope levels were detected using qRT-PCR and immunoblot analysis. Infectious virus particles were measured by using plaque assay. ZIKV infection to neuronal cells showed increased levels of pro-apoptotic markers like cleaved-PARP, cleaved caspase-3, Bim, and Puma, whereas decreased levels of anti-apoptotic markers such as Mcl-1, Bcl-1, and Bcl-xL. Further, we observed activation of three arms of ER stress namely: inositol requiring enzyme 1 alpha (IRE1), protein kinase-like ER kinase (PERK), and activating transcription factor (ATF6) pathways with ZIKV infection. Treatment of palmitoleate dramatically decreased ZIKV infection-induced increase in percent apoptotic nuclei and caspase 3/7 activity. Further, treatment of palmitoleate decreased cleaved PARP and PUMA protein expressions. Treatment of palmitoleate reduced ZIKV-induced ER stress activation as evidenced by decreased levels of phosphorylated forms of IRE1 and eukaryotic initiation factor 2 alpha; decreased expressions of cleaved ATF6, spliced X-box associated protein 1 and C/EBP homologous protein compared to ZIKV infection alone. Further, treatment of palmitoleate attenuated ZIKV envelope levels and infectious titer in SH-SY5Y and primary fetal cortical neurons isolated from humanized STAT2 knockin mice. These data suggest that palmitoleate supplementation protects against ZIKV-induced neuronal ER stress, apoptosis and decreases Zika viral load thereby mitigates neuronal damage. Competing Interest Statement The authors have declared no competing interest. Footnotes The project described was supported by the Child Health Research Institute, University of Nebraska Medical Center, Nexus of Virology, Immunology and Bioengineering (NVIBE), University of Nebraska-Lincoln, the United States Department of Agriculture (USDA)-National Institute of Food and Agriculture (NIFA) Standard Grant (2023-67017-40223), University of Nebraska-Lincoln, Nebraska Agricultural Experimental Station with funding from the Hatch Act (Accession #7005822) and U54 GM115458, which funds the Great Plains IDeA-CTR Network (all to SKN). The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the USDA, National Institute of Health. List of abbreviations - ZIKV - Zika virus - rMR - recombinant clone of African strain, MR766 - PR - PRVABC59 Asian strain - MOI - multiplicity of infection - PARP - poly (ADP-ribose) polymerase - MCL-1 - Myeloid cell leukemia 1 - BCL-2 - B-cell lymphoma 2 - Bcl-xL - B-cell lymphoma-extra large - ATF4 - Activating transcription factor 4 - ATF6 - Activating transcription factor 6 - BIM - BCL2-interacting mediator of cell death - CHOP - C/EBP homologous protein - DAPI - 4’-6-diamidino-2-phenylindole dihydrochloride - ER - Endopasmic reticulum - PO - Palmitoleate - eIF2α - Eukaryotic initiation factor α - IRE1α - Inositol-requiring transmembrane kinase/endoribonuclease 1α - MAPK - Mitogen activated protein kinase - PERK - protein kinase RNA-like endoplasmic reticulum kinase - XBP1 - X-box binding protein 1 - Bip - immunoglobulin heavy chain binding protein - PUMA - p53 upregulated modulator of apoptosis - JNK - c-Jun N-terminal kinase - Bax - Bcl-2-associated X protein - Bak - Bcl-2 antagonist killer 1 - hSTAT2KI - humanized Signal Transducer and Activator of Transcription 2 knockin - PFU - plaque-forming unit - LD - lipid droplets - SEM - standard error of the mean