bioRxiv preprint doi: https://doi.org/10.1101/2021.06.29.450442; this version posted July 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Epigenetic regulation of frog innate immunity: Discovery of 2 frog microRNAs associated with antiviral responses and 3 ranavirus infection using a Xenopus laevis skin epithelial-like 4 cell line 5 6 Lauren A. Todd1, Maxwell P. Bui-Marinos1, and Barbara A. Katzenback1,* 7 8 1Department of Biology, Faculty of Science, University of Waterloo, 200 University Avenue 9 West, Waterloo, Ontario, Canada, N2L 3G1. 10 11 Email addresses: 12 [email protected] (L.A.T) 13 [email protected] (M.P.B-M) 14 [email protected] (B.A.K) 15 16 *Corresponding author 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.29.450442; this version posted July 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 17 Abstract 18 19 Epigenetic regulators such as microRNAs are emerging as conserved regulators of innate 20 antiviral immunity in vertebrates, yet their roles in amphibian antiviral responses remain 21 uncharacterized. We profiled changes in microRNA expressions in the Xenopus laevis skin 22 epithelial-like cell line Xela DS2 in response to poly(I:C) – an analogue of double-stranded viral 23 RNA and inducer of type I interferons – or frog virus 3 (FV3), an immunoevasive virus 24 associated with amphibian mortality events. We sequenced small RNA libraries generated from 25 untreated, poly(I:C)-treated, and FV3-infected cells. We detected 136 known X. laevis 26 microRNAs and discovered 133 novel X. laevis microRNAs. Sixty-five microRNAs were 27 differentially expressed in response to poly(I:C), many of which were predicted to target 28 regulators of antiviral pathways such as cGAS-STING, RIG-I/MDA-5, TLR signaling, and type I 29 interferon signaling, as well as products of these pathways (NF-ĸB-induced and interferon- 30 stimulated genes). In contrast, only 49 microRNAs were altered by FV3 infection, fewer of 31 which were predicted to interact with antiviral pathways. Interestingly, poly(I:C) treatment or 32 FV3 infection downregulated transcripts encoding factors of the host microRNA biogenesis 33 pathway. Our study is the first to suggest that host microRNAs regulate innate antiviral 34 immunity in frogs, and sheds light on microRNA-mediated mechanisms of immunoevasion by 35 FV3. 36 37 Keywords: Frog virus 3, microRNAs, antiviral immunity, poly(I:C), skin epithelial cells, 38 Xenopus laevis 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.29.450442; this version posted July 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 39 Introduction 40 Epigenetic mechanisms are emerging as key regulators of immune signaling in 41 vertebrates, and microRNAs (miRNAs) are a prime example of such a mechanism (Boosani and 42 Agrawal 2016). miRNAs are a class of small non-coding RNAs that function in post- 43 transcriptional regulation of gene expression. miRNA genes are transcribed in the nucleus by 44 RNA polymerase II and are processed into hairpin precursors (pre-miRNAs) by the RNase III 45 enzyme Drosha (Lee et al. 2002). Pre-miRNAs are exported to the cytoplasm where they are 46 further processed into mature miRNA duplexes by the RNase III enzyme Dicer (Lee et al. 2002). 47 One strand of the miRNA duplex is degraded, while the other guides the Argonaute-containing 48 RNA-induced silencing complex (RISC) to mRNAs which are complementary to the miRNA 49 guide (Lee et al. 2002). Direct interactions between the miRNA and mRNA [typically the 3’ 50 untranslated region (UTR)] induce degradation or inhibit translation of the mRNA (Lee et al. 51 2002). 52 Vertebrate miRNAs have been found to regulate the expression of genes involved in 53 antiviral signaling pathways that produce interferons (IFNs) and inflammatory cytokines in 54 response to pathogen infection (Boosani and Agrawal 2016). Host miRNAs have been shown to 55 regulate components of key pathogen sensing pathways such as the cytosolic RIG-I/MDA5 56 sensing pathway (Li et al. 2020), endosomal TLR3 pathway (Tili et al. 2007; Hou et al. 2009; 57 Imaizumi et al. 2010), endosomal/cell surface TLR4 pathway (Wendlandt et al. 2012), and 58 endosomal TLR7/8/9 pathways (Hou et al. 2009; Tang et al. 2009), as well as the JAK-STAT 59 signaling pathway that regulates the production of IFN-stimulated genes (ISGs) (Tang et al. 60 2009; Jarret et al. 2016). In some cases, these interactions promote efficient host immune 61 responses. However, viral infection can induce changes in host miRNA expression that detriment 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.29.450442; this version posted July 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 62 the host. For example, Coxsackievirus B3-induced miR-30a represses TRIM25 and RIG-I 63 function, resulting in enhanced viral replication (Li et al. 2020), vesicular stomatitis virus- 64 induced miR-146a represses type I IFN production and promotes viral replication by repressing 65 TRAF6, IRAK2, and IRAK1 (Hou et al. 2009), and hepatitis C virus upregulates miR-208b and 66 miR-499a-5p which enhances viral replication by dampening type I IFN signaling through 67 repression of the type I IFN receptor IFNAR1 (Jarret et al. 2016). miRNA-mediated regulation of 68 host immune responses is therefore complex, and while a growing number of functional studies 69 have been conducted, our understanding of the broad functions host miRNAs play in antiviral 70 responses remains limited. 71 Ranaviruses, type species frog virus 3 (FV3), are large double-stranded DNA viruses 72 causing emerging infectious diseases that threaten amphibian populations. Despite the 73 devastating amphibian morbidities and mortalities associated with ranaviral infection, our 74 understanding of frog antiviral defenses remains in its infancy, and the roles of frog miRNAs in 75 regulating these antiviral responses are largely unexplored. As frog skin is an important barrier to 76 pathogen entry (Varga et al. 2019), we have recently generated a Xenopus laevis dorsal skin 77 epithelial-like cell line (Xela DS2) (Bui-Marinos et al. 2020) that is permissive to FV3 (Bui- 78 Marinos et al. submitted) to facilitate our understanding of antiviral responses in frog skin 79 epithelial cells. All viruses, including FV3 (Doherty et al. 2016), produce viral double-stranded 80 RNA (dsRNA) at some point in their replication. Along with others, we have studied frog cell 81 antiviral responses through treatment with poly(I:C) (Sang et al. 2016; Wendel et al. 2018; Bui- 82 Marinos et al. 2020), a synthetic viral dsRNA analog and known inducer of type I IFNs. While 83 poly(I:C) is not a perfect mimic of viral dsRNA, as the sequence and length of viral dsRNA is 84 known to impact the induction of host antiviral responses (Poynter and DeWitte-Orr 2015), it 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.29.450442; this version posted July 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 85 permits comparisons of type I IFN responses across cell types and species. By modeling 86 “typical” antiviral responses in frogs using poly(I:C), we can further our understanding of how 87 miRNAs function to regulate effective antiviral responses. By comparing “typical” antiviral 88 miRNA responses in frog skin epithelial cells [modeled by poly(I:C)] to antiviral miRNA 89 responses to immunoevasive viruses such as FV3, we aim to elucidate how viral pathogens may 90 subvert epigenetic regulatory responses as direct or indirect mechanisms of immunoevasion. 91 In this study, we sought to perform initial investigations into the role of host miRNAs in 92 innate antiviral immune responses in frog skin epithelial cells. The goals of this study were to (1) 93 profile changes in miRNA expression in Xela DS2 skin epithelial-like cells during antiviral 94 responses to poly(I:C) and FV3, (2) determine the antiviral targets of differentially expressed 95 miRNAs, (3) compare normal antiviral miRNA responses modeled by poly(I:C) – a known 96 inducer of robust antiviral responses – to miRNA responses induced by FV3, an immunoevasive 97 virus, and (4) expand the number of currently annotated frog miRNAs through the discovery of 98 novel X. laevis miRNAs. 99 100 Materials and methods 101 Cell line maintenance 102 Xela DS2, a skin epithelial-like cell line previously generated from X. laevis dorsal skin 103 (Bui-Marinos et al. 2020), was maintained in seven parts Leibovitz’s L-15 (AL-15) medium 104 (Wisent, Mont-Saint-Hilaire, Canada) diluted with 3 parts sterile cell culture water to adjust for 105 amphibian osmolarity, and is herein referred to as amphibian L-15 (AL-15) medium. AL-15 106 medium was supplemented with 15% fetal bovine serum (FBS; lot #234K18; VWR, Radnor, 107 United States) and used to culture Xela DS2 cells at 26 °C in 75 cm2 plug-seal tissue-culture 5 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.29.450442; this version posted July 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder.