U251 cells were transduced with lentiviruses encoding AcGFP alone or AcGFP plus myc-tagged PEX11B for 48 h and then transfected with poly(I:C) (+) or an empty plasmid vector (?) for 12 h

U251 cells were transduced with lentiviruses encoding AcGFP alone or AcGFP plus myc-tagged PEX11B for 48 h and then transfected with poly(I:C) (+) or an empty plasmid vector (?) for 12 h. and nerve function, our studies provide important insights into the roles of peroxisomes in regulating ZIKV infection and potentially neuropathogenesis. and genes [10]. Secreted type I and III IFNs bind to receptors on the cell surface that signal through the JAK/STAT pathway to induce the transcription of IFN-stimulated genes (ISGs), resulting in an antiviral state [11,12]. However, many viruses, including flaviviruses, are known to deploy an array of counter-measures to suppress IFN induction and downstream antiviral signaling [13,14]. In addition to mitochondria, peroxisomes, which are membrane-bound organelles that have well characterized functions in lipid metabolism and regulation of reactive oxygen species [15,16], have recently been shown to play critical roles in antiviral defense. Specifically, activation of MAVS on peroxisomal as well as mitochondrial membranes PHA-767491 hydrochloride appears to be important for IFN induction and signaling [17,18,19]. Evidence indicating that viruses disrupt peroxisome biogenesis began to emerge shortly after, further supporting the importance of peroxisomes in antiviral defense. First, we showed that in cells infected with West Nile (WNV) or Dengue (DENV) viruses, a critical peroxisome biogenesis factor, PEX19, is selectively degraded [20]. This process, which involves the capsid proteins of WNV and DENV, results in reduced levels of peroxisomes and a dampened type III IFN response [20]. Subsequently, it was reported that the NS3-4A protease of hepatitis C virus cleaves MAVS localized on peroxisomes and mitochondria [18,21], whereas the nsp1 protein of porcine diarrhea virus reduces type III IFN induction, in part by reducing peroxisome pools via an unknown mechanism [22]. Finally, human immunodeficiency virus-1 (HIV-1) infection was shown to downregulate peroxisomes by upregulating cellular microRNAs that inhibit the expression of peroxisome biogenesis factors such as PEX2, PEX7, PHA-767491 hydrochloride PEX11 and PEX13 [23]. More recently, it was reported that the infection of Vero cells with ZIKV results in a 12% decrease in peroxisome density as well as a 50% loss of the peroxisomal membrane protein PMP70 [24]. It was hypothesized that during ZIKV infection, peroxisomes are consumed and, accordingly, that these organelles are actually required for ZIKV replication. However, this notion contrasts with mounting evidence supporting an antiviral role for peroxisomes [17,18,19,20,21,22]. Here, we investigated the interplay between ZIKV infection and peroxisomes in primary human fetal astrocytes (HFAs), the most abundant cell type in the brain and potentially a cellular reservoir for ZIKV [25]. Iinfection of HFAs resulted in a dramatic reduction in peroxisomes, regardless of the type of ZIKV strain employed. PEX11B, a biogenesis factor that induces PHA-767491 hydrochloride peroxisome proliferation, was found to be a restriction factor for ZIKV. Elevated expression of PEX11B was associated with increased levels of MAVS and enhanced IFN induction and downstream signaling. As peroxisomes are critical for brain development and function [26,27], it is tempting to speculate that the loss of these organelles in HFAs may play a role in the neurological deficits associated with in utero ZIKV infection. 2. Materials and Methods 2.1. Cells and Virus Infection A549, HEK293T, Vero and U251 cells were purchased from the American Type PHA-767491 hydrochloride Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbeccos modified Eagles medium (DMEM; Gibco; Waltham, MA, USA) supplemented with 100 U/mL penicillin and streptomycin, 1 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic RAC2 acid (HEPES)(Gibco; Waltham, MA, USA), 2 mM glutamine (Gibco; Waltham, MA, USA), 10% heat-inactivated fetal bovine serum (FBS; Gibco; Waltham, MA, USA) at 37 C in 5% CO2. Primary human fetal astrocytes (HFAs) were prepared as previously described [28] from 15C19 week aborted fetuses with written consent approved under the protocol 1420 by the University of Alberta Human Research Ethics Board (Biomedical). HFAs were grown in Minimum Essential Media (MEM) (1 g/L Glucose, 15 mM HEPES, Gibco; Waltham, MA, USA) supplemented with 10% FBS, L-glutamine, MEM non-essential amino acids, sodium pyruvate, and 1 g/mL glucose at 37 C in.