viruses Article MicroRNAs for Virus Pathogenicity and Host Responses, Identified in SARS-CoV-2 Genomes, May Play Roles in Viral-Host Co-Evolution in Putative Zoonotic Host Species Sigrun Lange 1,* , Elif Damla Arisan 2 , Guy H. Grant 3 and Pinar Uysal-Onganer 4,* 1 Tissue Architecture and Regeneration Research Group, School of Life Sciences, University of Westminster, London W1W 6UW, UK 2 Institute of Biotechnology, Gebze Technical University, Gebze, 41400 Kocaeli, Turkey; [email protected] 3 School of Life Sciences, University of Bedfordshire, Park Square, Luton LU1 3JU, UK; [email protected] 4 Cancer Research Group, School of Life Sciences, University of Westminster, London W1W 6UW, UK * Correspondence: [email protected] (S.L.); [email protected] (P.U.-O.) Abstract: Our recent study identified seven key microRNAs (miR-8066, 5197, 3611, 3934-3p, 1307-3p, 3691-3p, 1468-5p) similar between SARS-CoV-2 and the human genome, pointing at miR-related mechanisms in viral entry and the regulatory effects on host immunity. To identify the putative roles of these miRs in zoonosis, we assessed their conservation, compared with humans, in some key wild and domestic animal carriers of zoonotic viruses, including bat, pangolin, pig, cow, rat, and chicken. Out of the seven miRs under study, miR-3611 was the most strongly conserved across all species; miR-5197 was the most conserved in pangolin, pig, cow, bat, and rat; miR-1307 was most strongly conserved in pangolin, pig, cow, bat, and human; miR-3691-3p in pangolin, cow, and human; miR-3934-3p in pig and cow, followed by pangolin and bat; miR-1468 was most conserved Citation: Lange, S.; Arisan, E.D.; in pangolin, pig, and bat; while miR-8066 was most conserved in pangolin and pig. In humans, Grant, G.H.; Uysal-Onganer, P. miR-3611 and miR-1307 were most conserved, while miR-8066, miR-5197, miR-3334-3p and miR-1468 MicroRNAs for Virus Pathogenicity were least conserved, compared with pangolin, pig, cow, and bat. Furthermore, we identified that and Host Responses, Identified in changes in the miR-5197 nucleotides between pangolin and human can generate three new miRs, with SARS-CoV-2 Genomes, May Play differing tissue distribution in the brain, lung, intestines, lymph nodes, and muscle, and with different Roles in Viral-Host Co-Evolution in downstream regulatory effects on KEGG pathways. This may be of considerable importance as miR- Putative Zoonotic Host Species. Viruses 2021, 13, 117. https:// 5197 is localized in the spike protein transcript area of the SARS-CoV-2 genome. Our findings may doi.org/10.3390/v13010117 indicate roles for these miRs in viral–host co-evolution in zoonotic hosts, particularly highlighting pangolin, bat, cow, and pig as putative zoonotic carriers, while highlighting the miRs’ roles in KEGG Academic Editor: Andrew Davidson pathways linked to viral pathogenicity and host responses in humans. This in silico study paves the Received: 30 November 2020 way for investigations into the roles of miRs in zoonotic disease. Accepted: 13 January 2021 Published: 16 January 2021 Keywords: microRNA; SARS-CoV-2; COVID-19; zoonosis; co-evolution; viral pathogenesis Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. The COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus- 2 (SARS-CoV-2), a zoonotic virus, which belongs to the betacoronavirus family. While a num- ber of zoonotic hosts have been suggested that may possibly not show disease symptoms [1], SARS-CoV-2 causes significant pathogenicity in humans due to alterations in inflammation- Copyright: © 2021 by the authors. related pathways, including some resulting in exacerbated inflammatory responses, vascu- Licensee MDPI, Basel, Switzerland. lar responses, cutaneous manifestations [2], extensive lung pathology, cardiovascular and This article is an open access article cardiomyopathy [3–5], kidney damage [6,7], gastrointestinal involvement [8], as well as a distributed under the terms and wide range of neurological conditions including stroke, encephalopathy, encephalitis, central conditions of the Creative Commons nervous system (CNS) vasculitis and acute neuropathies [9–11]. The SARS-CoV-2 genome con- Attribution (CC BY) license (https:// tains 14 open reading frames (ORFs), preceded by transcriptional regulatory sequences (TRSs), creativecommons.org/licenses/by/ while the two main transcriptional units, ORF1a and ORF1ab, encode replicase polyprotein 1a 4.0/). Viruses 2021, 13, 117. https://doi.org/10.3390/v13010117 https://www.mdpi.com/journal/viruses Viruses 2021, 13, x FOR PEER REVIEW 2 of 18 acute neuropathies [9–11]. The SARS-CoV-2 genome contains 14 open reading frames (ORFs), preceded by transcriptional regulatory sequences (TRSs), while the two main transcriptional units, ORF1a and ORF1ab, encode replicase polyprotein 1a (PP1a) and polyprotein 1ab (PP1ab), respectively (Figure 1A). The largest polyprotein PP1ab embeds non-structural proteins (NSP1-16), which form the complex replicase machinery. This includes enzyme activities that are rare or absent in other families of positive-stranded (+) RNA viruses. The viral genomes encode four structural proteins, called spike (S), enve- lope (E), membrane (M), and nucleocapsid (N), and nine putative open reading frames (ORFs) for accessory factors. Non-structural proteins (NSP1-16) control an array of func- tions for survival for mature viruses. These vital functions include RNA-dependent polymerase (RDRp; NSP12), mRNA capping (NSPs 14 and 16), and RNA proofreading (NSP14) [12–15]. microRNAs (miRs) are short non-coding RNAs that play multifaceted roles in gene regulation, and also act as important regulators of the cellular antiviral response. Con- sequently, viruses have been found to utilize the host’s nuclear RNA to evade the im- mune response and exploit cellular machinery to their advantage by redirecting miRs to promote their replication [16]. In the last decade, miRs have not only been investigated for their diagnostic utility, but have already been applied therapeutically in different disease entities, for example, infection with hepatitis C virus (HCV) or oncological dis- eases [17,18]. Furthermore, miR-mediated vaccine studies showed that a number of miRs have the potential to decrease viral replication with an extensive immune response [19]. As a proof of this understanding, viral infections alter host miRs and can cause a dra- Viruses 2021, 13, 117 matic change in host responses [19]. The aspect of the miR-mediated regulation of viral2 of 17 infection is though still an emerging topic, with relatively few studies so far, and there- fore warrants further exploration particularly also in relation to zoonotic diseases. Recently, we reported seven key miRs in the SARS-CoV-2 genomes that relate to host–pathogen(PP1a) and polyprotein interaction 1ab (PP1ab),and viral respectively pathogenicity, (Figure alongside1A). The largest a number polyprotein of human PP1ab comorbidities,embeds non-structural and their proteins expression (NSP1-16), was verified which form in miRs the complex that are replicase expressed machinery. in lung Thisbi- includes enzyme activities that are rare or absent in other families of positive-stranded (+) opsies of SARS-CoV-2 patients and in in vitro cell models in the PRJNA615032 Bioproject RNA viruses. The viral genomes encode four structural proteins, called spike (S), envelope trancriptome data [20]. The seven identified miRs are spread on the SARS-CoV-2 ge- (E), membrane (M), and nucleocapsid (N), and nine putative open reading frames (ORFs) for nome, with three miRs in ORF1a and four in ORF1b, whereof two are on the spike (S) accessory factors. Non-structural proteins (NSP1-16) control an array of functions for survival protein, and two on the nuclear (N) protein (Figure 1B). for mature viruses. These vital functions include RNA-dependent polymerase (RDRp; NSP12), mRNA capping (NSPs 14 and 16), and RNA proofreading (NSP14) [12–15]. FigureFigure 1. 1. SARS-CoV-2SARS-CoV-2 polycistronic genome.genome. ((AA)) TheThe genome genome of of SARS-CoV-2 SARS-CoV-2 organized organized in individualin individual ORFs. ORFs. (B) miR-(B) miR-1468,1468, 3691, 3691, 3611, 3611, 5197, 5197, 3934, 3934, 8066, 8066, and 1307and locations1307 locations on the on SARS-CoV-2 the SARS-CoV-2 genome genome (S spike; (S Espike; envelope; E envelope; M membrane; M mem- N brane;nucleocapsid; N nucleocapsid; NSP non-structural NSP non-structural proteins; proteins; ORF Open ORF reading Open frame).reading frame). OurmicroRNAs previous (miRs)study highlighted are short non-coding the roles RNAsof miRs that in playrelation multifaceted to SARS-CoV-2 roles ininfec- gene tionregulation, and the andmultifaceted also act as symptoms important associated regulators with of the COVID-19 cellular antiviral[20]. While response. the spread Conse- of quently, viruses have been found to utilize the host’s nuclear RNA to evade the immune response and exploit cellular machinery to their advantage by redirecting miRs to promote their replication [16]. In the last decade, miRs have not only been investigated for their diagnostic utility, but have already been applied therapeutically in different disease entities, for example, infection with hepatitis C virus (HCV) or oncological diseases [17,18]. Fur- thermore, miR-mediated vaccine studies showed that a number of miRs have the potential to decrease viral replication with an extensive immune response