Insights into ZIKV-Mediated Innate Immune Responses in Human Dermal Fibroblasts and Epidermal Keratinocytes

Abstract

Zika virus (ZIKV) has emerged as a global pathogen causing significant public health concern. ZIKV infections in humans principally occur via mosquito bites. Thus, host skin cells are permissive to ZIKV infection and are the first line of defense against the virus. Here, we examined the role and mechanisms of antiviral skin immunity against ZIKV infection. ZIKV infection (African lineage MR766) in human dermal fibroblasts, human epidermal keratinocytes, and HaCaT keratinocytes resulted in distinct expression changes in RIG-I-like receptors, such as RIG-I and MDA5. Inhibition of RIG-I using small interfering RNA resulted in increased viral gene expression and reduced induction of IFNs and IFN-stimulated genes. Furthermore, ZIKV NS1 directly interacted with RIG-I or MDA5 and down-regulated RIG-I-like receptor-mediated antiviral signaling pathways. Asian lineage ZIKV (PRVABC59) infection also showed a distinct pattern of antiviral immunity in human skin cells, compared with other ZIKV strains. Additionally, ZIKV infections in human neural progenitor cells induced the robust activation of RIG-I-like receptor-mediated signaling, followed by highly enhanced IFN-stimulated gene expression. Our findings provide important insights into ZIKV tropism and subsequent antiviral signaling pathways that regulate ZIKV replication in human dermal fibroblasts and human epidermal keratinocytes.

Coxsackievirus B3 Infection of Human Neural Progenitor Cells Results in Distinct Expression Patterns of Innate Immune Genes

“…The preparation of hNPCs was as described previously [12,13]. Briefly, human embryonic stem cells (hESCs) were grown under standard culture conditions with a feeder layer and transferred to a matrigel-coated plate with mouse embryonic fibroblasts (MEFs) conditioned medium.…”

“…Total RNA was isolated using Trizol reagent (Invitrogen), as previously reported [13,16]. RNA quality was assessed by Agilent 2100 bioanalyzer using RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, The Netherlands), and RNA quantification was performed using ND-2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).…”

“…QuantSeq results were validated by RT-PCR on specific target genes. First-strand cDNA was synthesized from 0.5 µg of total RNA using ImProm-II Reverse Transcription System (Promega) according to manufacturer's instructions with the previously described primer sequences [13,24]. QuantStudio 6 Flex Real-time PCR system (Thermo Fisher Scientific) was utilized for cDNA amplification with Power SYBR ® Green Master Mix (Applied Biosystems) under the following conditions: 95 • C for 10 min, followed by 40 cycles of 95 • C for 30 s and 60 • C for 1 min.…”

Zika virus replicates in skeletal muscle contributing to peripheral viral amplification prior to reach neural tissue

“…Although ZIKV tropism in peripheral tissues was not well characterized yet, clinical studies have shown the viral persistence in several body fluids (21). First’s reports evaluating cell permissiveness demonstrated that ZIKV replicates at placental trophoblasts, endothelial cells, human skin fibroblast and neonatal keratinocytes (21, 35, 36). ZIKV also replicates and induce morphological alterations in human skin explants (35), that are consistent with skin lesions presented in many ZIKV-infected patients (37).…”

Attenuation of cGAS/STING Activity During Mitosis

“…We chose to investigate activity of the cGAS/STING pathway in HaCaTs, a spontaneously immortalized human keratinocyte cell line [57]. This cell line is a good model for primary keratinocytes that serve a barrier function and as host cells for a number of viral infections including papillomaviruses, herpesviruses, mosquito-transmitted alphaviruses and flaviviruses [58][59][60][61][62][63][64], and numerous bacterial and fungal pathogens [65,66]. As not all cell types express every PRR, we sought to ensure the cGAS/STING pathway was functional in HaCaTs and that this pathway was the primary mode of IRF3 phosphorylation in response to foreign DNA.…”