Coronaviruses (CoVs) belong to the subfamily Orthocoronavirinae of the family Coronaviridae. CoVs are enveloped (+) RNA viruses with unusually long genomes. Severe acute respiratory syndrome CoV (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV), and the novel coronavirus  have been identif ied as causing global pandemics. Clinically tested vaccines are widely used to control rapidly spreading, acute, and often severe infections; however, effective drugs are still not available. The genomes of SARS-CoV-2 and SARS-CoV are approximately 80 % identical, while the genomes of SARS-CoV-2 and MERS-CoV are approximately 50 % identical. This indicates that there may be common mechanisms of coronavirus pathogenesis and, therefore, potential therapeutic targets for each virus may be the same. The enzymes and effector proteins that make up the replication-transcription complex (RTC) of coronaviruses are encoded by a large replicase gene. These enzymes and effector proteins represent promising targets for potential therapeutic drugs. The enzyme targets include papain- and 3C-like cysteine proteinases that process two large viral polyproteins, RNA-dependent RNA polymerase, RNA helicase, viral genome-modifying enzymes, and enzymes with 3’–5’ exoribonuclease or uridylate-specif ic endonuclease activity. Currently, there are many studies investigating the complex molecular mechanisms involved in the assembly andfunction of the RTC. This review will encompass current, modern studies on the properties and complexes of individualnon-structural subunits of the RTC, the structures of individual coronavirus RTC subunits, domain organizationand functions of subunits, protein-protein interactions, properties and architectures of subunit complexes, the effectof mutations, and the identif ication of mutations affecting the viability of the virus in cell culture.Key words: non-structural proteins CoVs; subunits of replicase CoVs; replication-transcription complex of CoVs; architectureof non-structural protein complexes CoVs. The 2019 coronavirus infection has spread globally, oftencausing severe respiratory, intestinal, and systemic illnesses.Coronaviruses (CoVs) belong to the Orthocoronavirinaesubfamily of the Coronaviridae family. The subfamily isfurther divided into α-, β-, ϒ-, and δ-coronaviruses. Severeacute respiratory syndrome CoV (SARS-CoV), Middle Eastrespiratory syndrome CoV (MERS-CoV), novel coronavirus, mouse hepatitis virus (MHV),and bovine coronavirus (BCoV) are all β-coronaviruses . Coronaviruses are enveloped viruses with an unusuallylong, single-stranded (+) RNA genome (26–32 kb).The SARS-CoV-2 genome is similar to the SARS-CoVgenome (sequence identity ~80 %), while the SARS-CoV-2and MERS-CoV genomes are less similar (sequence identity~50 %) . The structure and function of proteinsare preserved at levels as low as 30 % of amino acid sequenceidentity . This indicates that there may be commonmechanisms of pathogenesis among the CoVs and, therefore,the viruses may have the same potential therapeutic targets.The 5′ proximal region of each CoV genome includes a cap,a 5′ untranslated region (UTR) and a long replicase gene encoding16 non-structural proteins (comprising two-thirds ofthe genome). The 3′ regions encode structural proteins, includingS (spike), E (surface), M (membrane), and N (nucleocapsid),auxiliary proteins (the number of these varies amongCoVs), a 3′ UTR, and a poly(A) tract.The replication-transcription complex (RTC) of CoVs is acomplex consisting of viral and, probably, cellular proteins.The RTC produces the (+) RNA genome and a set of subgenomicCoVs RNA in infected cells. The CoV replicase genehas two overlapping open reading frames, ORF1a and ORF1b,which encode the viral components of the RTC. Expression ofthe gene leads to the formation of the pp1a polyprotein, whichis encoded by ORF1a. A ribosomal frameshift of –1 before theORF1a translation termination codon and ORF1b are requiredfor the formation of the pp1ab polyprotein, which is a continuationof pp1a. Polyproteins pp1a and pp1ab are processed bytwo viral cysteine proteinases, papain-like proteinase PLpro(PLP) and 3C-like proteinase 3СLpro (Mpro), which results inthe release of intermediate precursors and 16 mature highlyconserved non-structural proteins (nsps) capable of associatingwith each other and being subunits of the RTC. The pp1apolyprotein includes nsp1–nsp11, while pp1ab includes allpp1a nsps, as well as nsp12–nsp16 .The molecular mechanisms of the assembly and function ofthe RTC has not been studied. However, the structural andfunctional properties of conserved non-structural RTC subunitsand their complexes have been extensively researchedand are extremely important for the identification of key drugtargets against CoVs:nsp1 interacts with the 40S ribosome subunit and inhibitstranslation initiation of host proteins, including interferonresponse factors. Interaction of nsp1 with ribosomes alsoleads to the degradation of host RNA. Thus, nsp1 suppressescellular defence antiviral mechanisms ;nsp2 is not part of the RTC in cell culture. However, theabsence of nsp2 in cells infected with the MHVΔnsp2 orSARS-CoVΔnsp2 deletion mutants reduces the productionof the virus and viral RNA ;nsp3 and nsp5 are proteinases that process the pp1a andpp1ab polyproteins, resulting in the release of individual RTCcomponents. The PLP nsp3 domain(s) process the N-proximalregions of pp1a and pp1ab. The MHV nsp3 has two domains,PL1pro (PL1P) and PL2pro (PL2P). The PL1P domain cleavesthe nsp1/nsp2 and nsp2/nsp3 sites, while the PL2P domaincleaves the nsp3/nsp4 site . The SARS-CoV nsp3 has a single PL2Pdomain that cleaves all three nsp sites .The SARS-CoV PLP is an intracellular immune responseantagonist. PLP blocks the activation of transcription factorsIRF3 and NF-κB, which induces the expression of IFN(I)and antiviral genes. It does this by indirectly inhibiting IKKiand TBK1 kinases that activate IRF3 and stabilizing IκBα, aninhibitor of NF-κB . PLP also hydrolyzeselements of ubiquitin and the product of interferon-stimulatinggene 15 of the ubiquitin-like protein, thereby blocking thecellular mechanism of post-translational ubiquitination and,in turn, enhancing viral replication .However, nsp3 stabilizes the host E3 ubiquitin ligase RCHY1through the interaction of its SUD and PLP domains withRCHY1. This activates the RCHY1-mediated degradation ofp53, a cellular inhibitor of SARS-CoV replication . SARS-CoV nsp3 interacts with nsp5, nsp6,nsp12, nsp13, nsp14, and nsp16 in the yeast two-hybrid (Y2H)system and is thought to serve as a scaffold for RTC assembly;nsp5 is a 3CLpro (Mpro). Mpro plays a key role in the processingof pp1a and pp1ab polyproteins, cleaving the centraland C-proximal regions of pp1ab at 11 highly conserved sites,which releases mature nsp4–nsp16 proteins . Mpro isonly active as a dimer. Self-elimination of MERS-CoV Mproat the nsp4/nsp5 and nsp5/nsp6 sites occurs as a result of theligand-induced formation of an “immature dimer” during theconvergence of Mpro III domains within two polyproteins. Structural analysis of the SARS-CoV-2Mpro complexes with known antiviral inhibitors, Boceprevir(peptidomimetic NS3/4A protease of hepatitis C virus) and GC376 (inhibitor of CoV replication), revealed atomic-levelinteractions between Mpro and these inhibitors. Such studiesare important for the optimization and design of effectivedrugs against CoVs ;nsp6 interacts with nsp2, nsp8, and nsp9 in the Y2H system. Six of the predicted hydrophobic domainsof MHV nsp6 and SARS-CoV nsp6 are transmembrane domains. MHV nsp6 and SARS-CoV nsp6are localized in the membranes of the endoplasmic reticulum(ER) and induce the formation of autophagosomes from ERmembranes and activate autophagy .Co-transfection of nsp3, nsp4, and nsp6 induces a change inthe internal membranes of the host cell through the formationof double-membrane vesicles (DMVs), similar to the DMVsinduced by SARS-CoV ;and nsp8 co-crystallize to form the nsp7/8 hexadecamericsupercomplex. The assembly of the supercomplex involvesthe formation of two different nsp7/8 heterodimers, D1 andD2, which differ in nsp8 conformation. D1 and D2 eachdimerize to form the heterotetramers T1 and T2. The interactionof two T1 with two T2, in the order T1–T2–T1′–T2′ andwith ring closure through the T1–T2′ interaction, leads to theconstruction of the full supercomplex. The supercomplex hasa unique architecture: 16 molecules (8 nsp7 molecules and8 nsp8 molecules) interact tightly with each other, forminga hollow cylindrical structure in which two nsp8 conformationscoexist. The positive charge of the inner channel of thecylinder and its diameter (30 Å) indicates the ability of thensp7/8 supercomplex to surround and interact with doublestrandedRNA (dsRNA) . The SARS-CoVnsp7/8 hexadecameric supercomplex can be formed in solutionat an equimolar nsp7: nsp8 ratio .In solution, hexadecameric SARS-CoV nsp7/8 associateswith dsRNA (Kd ~1.2 μM). The association of nsp7/8 withdsRNA is mediated by nsp8 and enhanced by nsp7 . On its own, SARS-CoV nsp8 possesses primerindependentRNA-dependent RNA polymerase (RdRp)activity and initiates, with low fidelity, the de novo synthesisof short (less than 6 nucleotides) complementary oligomers(primers) on single-stranded RNA (ssRNA) templates . SARS-CoV nsp8 and nsp7/8 complex also exhibitprimer-dependent RdRp activity . TheFCoV (feline coronavirus) nsp7/8 complex is a 2 : 1 heterotrimerformed by the association of two nsp7 molecules and onensp8 molecule. This complex does not form a hollow structure,either in crystalline form or in solution. FCoV nsp7/8has primer-independent RdRp activity ;nsp9 is an ssRNA-binding protein . Both monomeric and dimeric forms ofPDCoV (porcine δ coronavirus) nsp9 and PEDV (porcineepidemic diarrhoea virus related to α coronaviruses) nsp9, as well as the dimeric form of SARSCoVnsp9 , have been found in solutionduring in vitro experiments. Studies of the crystal structuresof SARS-CoV nsp9 ,PDCoV nsp9, and PEDV nsp9  haverevealed dimeric forms of nsp9. The monomer SARS-CoVnsp9 is characterized by a different structure compared toother proteins involved in the replicative complexes of RNAviruses, the features of which are similar to the structures ofoligosaccharide/oligonucleotide-binding proteins . Mutations affecting the dimerization of SARS-CoVnsp9 weaken its interaction with ssRNA, which is lethal forSARS-CoV replication in cell culture ;nsp10 interacts with dsRNA, dsDNA, and ssRNA withmicromolar affinity . Crystal structurestudies of SARS-CoV nsp10 showed that the monomerstructure includes two zinc fingers, a new discovery amongzinc finger protein structures. Motifs of zinc-binding nsp10sequences have been identified. Twelve identical monomersform a unique spherical dodecameric architecture, which ishypothesized to be the functional form of nsp10 . Through two-hybrid analysis in mammaliancells, interactions of SARS-CoV nsp10 with nsp14and nsp16 have been revealed ;nsp11 is a short peptide resulting from the cleavage of thepp1a polyprotein by the 3CLpro/Mpro proteinase at the nsp10/nsp11 site. Nsp11 is encoded by the region of genomic RNAwhere the translational reading frame shift occurs (ORF1a toORF1b). This shift results in the formation of nsp12–nsp16proteins from the pp1ab polyprotein. SARS-CoV-2 nsp11contains 13 amino acid residues and has a disordered conformation,the dynamics of which have been studied in thepresence of lipid-membrane mimetics. In the presence of SDSmicelles, the disordered conformation of nsp11 is transformedinto an α-helix ;nsp14 is bifunctional. The N-terminal domain of nsp14has 3′–5′ exoribonuclease activity (ExoN), and its C-terminaldomain has (guanine-N7) methyltransferase activity(N7-MTase). ExoN corrects the low fidelity of synthesis ofthe complementary RNA strand by viral RdRp nsp12 andcatalyzes the removal of 3′-terminal erroneous nucleotidesin dsRNA. N7-MTase catalyzes the methylation of the viralRNA cap at the N7 guanine position in the presence of S- adenosylmethionine(methyl group donor). N7-MTase has anS- adenosylmethionine binding motif which recognizes the capof viral GpppRNA and methylates guanine N7 GpppRNA toform 7MeGpppRNA (cap-0). Cap-0 plays an important role inblocking the degradation of viral RNA by 5′–3′ exoribonucleases,translation initiation, and immune system controlescape . Mutants of the catalyticmotif MERS-CoV ExoN and SARS-CoV-2 ExoN arenot viable in cell culture . SARS- CoVnsp10 associates with the ExoN domain of SARS-CoV nsp14,increasing the ExoN activity of nsp14 by more than 35 timeswithout affecting its N7-MTase activity .Structural studies of the SARS-CoV nsp10/14 complexshowed that one nsp10 molecule associates with the ExoNdomain of nsp14, stabilizing and enhancing the activity ofthe ExoN. The architecture of the nsp10/14 complex has beenstudied and has been found to include two regions of contactbetween the nsp10 molecule and the ExoN domain of nsp14;nsp16 has (nucleoside-2′O) methyltransferase activity(2′O-MTase). 2′O-MTase recognizes the cap-0 of viralRNA and catalyzes the transfer of the methyl group fromS-adenosylmethionine to the 2′OH group of the first nucleotide’sribose after N7-methylated guanine, resulting in the formation of 7MeGpppN2′OMe-RNA (conversion of cap-0 intocap-1). SARS-CoV nsp10 associates with nsp16 and stimulatesthe 2′O-MTase activity of nsp16 .Mutagenesis mapping of the surface amino acid residues ofSARS-CoV nsp10 involved in nsp10–nsp14 interaction andstructural analysis of the nsp10/16 complex revealed overlappingsurfaces of nsp10 interacting with nsp14 and nsp16.Nsp10 can serve as a platform that recruits nsp14 or nsp16to the RTC, stimulating the ExoN activity of nsp14 or the2′O- MTase activity of nsp16. Therefore, nsp10 is an importantregulator of the RTC. Mutations have been identified thatdisrupt the nsp10–nsp14 and nsp10–nsp16 interactions, someof which lead to a nonviable viral phenotype in cell culture;nsp12, an RdRp, catalyzes the synthesis of complementaryRNA strands on (+) and (–) viral RNA templates and is a keyCoV RTC enzyme. CoVs nsp12 initiates de novo synthesisfrom the 3′ end of the viral genome of the full-length (–) RNAstrand, as well as for subgenomic (–) RNA transcripts, whichhave differing 3′ end lengths. In turn, the full-length (–) RNAstrands and subgenomic (–) RNA strands serve as templatesfor the synthesis of the new RNA genome and subgenomic(+) RNA transcripts. Subgenomic (+) RNA is important forthe expression of structural and accessory proteins encodedby genes in the 3′ proximal region of the viral genome, whichis inaccessible to ribosomes that translate the viral genome. Full-length recombinant SARS-CoVnsp12 associates with short (20–30 nucleotides long) dsRNAand ssRNA , and initiatesprimer-dependent RNA synthesis on both homo- and heteropolymericRNA templates of the same length . However, the recombinant SARS-CoV nsp12 does notassociate with a primer-template that mimics the 3′-terminal40 nucleotides of the SARS-CoV UTR and does not exhibitRdRp activity on this primer-template .Cryo-electron microscopy determined the structure of themonomer SARS-CoV-2 nsp12, which includes the nidovirusspecificN-terminal domain of the RdRp-associated nucleotidyltransferase (NiRAN), the interface domain, and theC- terminal RdRp domain. The C-terminal RdRp domain hasa conserved right-hand-like architecture, which includes threesubdomains: fingers, palm, and thumb. The active centre isformed by conservative motifs of amino acid residues localizedin the palm subdomain. The motifs of the channel ofentry for nucleotide triphosphates and the primer-template andthe exit of the resulting RNA strand converge in the centralcavity,where these motifs carry out matrix-dependent RNAsynthesis have been determined .The association of the nsp7/8 complex with nsp12 leadsto the formation of the nsp7/8/12 complex. This complexpossesses high RNA-binding capacity, polymerase activity,and processivity. It is also capable of initiating de novo RNAsynthesis on the 3′ UTR template of the SARS-CoV genome,resulting in the elongation of the RNA product by over300 nucleotides. For nsp7/8/12 complex-mediated initiationof processive RNA synthesis, three amino acid residues fromnsp7  and one amino acid residue from nsp8(K58) are required for the interaction of nsp7/8/12 with theRNA template, while four amino acid residues from nsp8 interact with nsp12 . Moreover, nsp7/8/12 is able to associate with nsp14to form the nsp7/8/12/14 multicomplex. This ensemble ofnon-structural proteins possesses high RNA polymeraseactivity and is involved in 5′ RNA capping; however, it doesnot have ExoN activity . The structure ofthe SARS-CoV-2 and SARS-CoV nsp7/8/12 complexes wasdetermined by cryo-electron microscopy. These complexes,including the nsp12 monomer, nsp8 monomer, and nsp7/nsp8heterodimer, have similar architecture: the nsp8-1 subunitinteractswith the RdRp finger subdomain, while the nsp7and nsp8-2 subunits interact with the RdRp thumb subdomain;nsp13 possesses helicase and nucleoside-triphosphatase(NTPase) activities; nsp13 interacts with nsp7, nsp8, andnsp12 in the Y2H system .The crystal structure of MERS-CoV nsp13 was determined.The structure includes an N-terminal zinc-binding domain(ZBD) rich in Cys/His residues that coordinate three zinc ions,as well as C-terminal helicase RecA1 and RecA2 domainswhich contain parallel β-chains . The nsp13helicase separates dsRNA and dsDNA strands with overhanging(5–20 nucleotides long) 5′ ends; nsp13 interacts with thesingle-stranded 5′ end of the partial nucleic acid duplex ofdsRNA and dsDNA and unwinds it in the 5′–3′ direction, usingthe hydrolysis energy of the 5′-deoxy- and ribonucleotidetriphosphates .The RNA 5′-triphosphatase activity of nsp13 catalyzes thecleavage of the ϒ phosphate from the 5′-terminal nucleotideof RNA and is thought to be involved in the capping of viralRNA . Studies of SARS-CoV nsp13 haveshown that the unwinding of DNA duplexes occurs in discreteintervals of ~9.3 base pairs (bp) at a rate of 30 intervals persecond. Therefore, the unwinding speed of DNA duplexes isapproximately 280 bp/s.The helicase activity of nsp13 increases approximately2-fold when nsp13 interacts with nsp12, suggesting theinteractionof these proteins is involved in the function ofthe RTC . When SARS-CoV-2 nsp13interacts with the RTC, specifically with nsp7/2nsp8/nsp12:RNA, the stable complexes 2nsp13–RTC (67 %) and nsp13–RTC (20 %), as well as the dimer (2nsp13–RTC)2 (13 %) areformed. Using cryo-electron microscopy, the architecture ofthe dominant 2nsp13–RTC complex has been determined.This involves the interaction of the ZBD on the first nsp13molecule with the N-terminus of nsp8b and the nsp12 thumbsubdomain, as well as the interaction of the ZBD of the secondnsp13 molecule with the N-terminus of nsp8a. The catalyticRecA1 helicase domain of the first nsp13 molecule is fixedagainst nsp7 and the nsp8b head ;nsp15 is a nidovirus uridylate-specific endoribonuclease(NendoU). It cleaves RNA at the 3′ uridylate in unpaired,single-stranded, and looped regions . The crystal structures of SARS-CoV-2,SARS-CoV, and MERS-CoV nsp15 are homologous, functionallyactive hexamers formed by the dimerization of trimers.Each of the hexamer protomers includes three domains: theN- terminal, middle, and C-terminal catalytic NendoU domains.During trimer assembly, the N-terminal domain ofone protomer is packed into a gap between the central andC-terminal domains of the neighbouring protomer. During assembly of the hexamer, the N-terminal domains of the protomersof the two trimers are packed back-to-back and are inthe centre of the hexamer structure. The C-terminal domainscontaining active centres are located outward at the verticesof the cloverleaf. This architecture provides nsp15 with sixfunctionally active centres .The uridylate content in RNA, Mn+2 and, to a lesser extent,Mg+2 increase the affinity of nsp15 for RNA. The hexamericstructure of SARS-CoV and MERS-CoV nsp15 is criticalfor its substrate and catalytic activity. The study of a seriesof mono-, tri- and hexameric protein mutants revealed weakinteractions with RNA (rU16–20) and low catalytic activity inmonomers and trimers compared to hexamers and wild-typensp15 . Nsp8 andthe nsp7/8 complex interact with MERS-CoV nsp15, enhancingthe binding ability of the nsp15 hexamer for RNA and itscatalytic activity . The NendoU activity ofnsp15 is an antagonist of the IFN-induced cellular antiviralresponse and stimulates the initiation of viral RNA translation.A large number of non-structural subunits and their respectivecomplexes within the RTC is a defining feature of CoVs.An important feature is that some subunits have domains withdifferent enzymatic activities. For example, nsp14 has bothExoN and N7-MTase activity, while nsp13 has helicase andnucleoside triphosphatase activity. Non-structural proteinshave a set of activities universal for (+) RNA viruses: proteinases, RNA-dependent RNA polymerases (nsp12),RNA helicases (nsp13). There are also unique domains involvedin mRNA capping, cap modification .Some proteins possess 3′–5′ exoribonuclease activity (nsp14),which regulate the reliability of RNA genome replication,while some possess uridylate-specific endonuclease activity(nsp15). Many proteins serve as cofactors for importantenzymes  and affect cellular processes.In particular, some proteins suppress the antiviral cellularresponse . The nsp9 and nsp10structures are unique among the protein structures of the replicativecomplexes of RNA viruses. Many CoVs subunits andtheir complexes have a complex architecture. For example,nsp7 and nsp8 form a functional and unique hexadecamericsupercomplex, the nsp10 architecture includes 12 identicalsubunits, and the nsp15 architecture is a functionally activehexamer. The complex architecture of the RTC is defined bythe 2 nsp13/2 nsp8/1 nsp7/1 nsp12 model.The lack of effective drugs against the novel coronavirus infectionis a current global challenge. The RTC of CoVs replicates(+) RNA and determines the production of the virusin infected cells; however, the molecular mechanisms of thisremain unexplored. Currently, great efforts are being undertakenaimed at creating a structural or functional network ofinteractions within the CoV proteome, as well as its interactionswith the host cell. This would identify a large-scale panelof therapeutic targets. The determination of the structures ofindividual non-structural RTC subunits and their complexesand the identification of key interacting amino acid residuesand types of bonds between them will enable the design ofselective and effective inhibitors. Investigations employingbiochemical methods and mutational analyses can identifyfactors that affect the efficiency of viral genomic RNA productionand the virus in an infected cell, the multiple effects ofviral proteins on the host cell, and potential key drug targets.The authors declare no conflict of interest.Adedeji A.O., Lazarus H. Biochemical characterization of Middle Eastrespiratory syndrome coronavirus helicase. mSphere. 2016;1(5):e00235-16. DOI 10.1128/mSphere.00235-16.Adedeji A.O., Marchand B., Velthuis A.J.W., Snijder E.J., Weiss S.,Eoff R.L., Singh K., Sarafianos S.G. Mechanism of nucleic acid unwindingby SARS-CoV helicase. PLoS One. 2012;7(5):e36521. DOI10.1371/journal.pone.0036521.Angelini M.M., Akhlaghpour M., Neuman B.W., Buchmeier M.J.Severe acute respiratory syndrome coronavirus nonstructural proteins3, 4, and 6 induce double-membrane vesicles. mBio. 2013;4(4):e00524-13. DOI 10.1128/mBio.00524-13.Bhardwaj K., Sun J., Holzenburg A., Guarino L.A., Kao C.C. RNA recognitionand cleavage by the SARS coronavirus endoribonuclease.J. Mol. Biol. 2006;361(2):243-256. DOI 10.1016/j.jmb.2006.06.021.Bouvet M., Debarnot C., Imbert I., Selisko B., Snijder E.J., Canard B.,Decroly E. In vitro reconstitution of SARS-coronavirus mRNA capmethylation. PLoS Pathog. 2010;6(4):e1000863. DOI 10.1371/journal.ppat.1000863.Bouvet M., Imbert I., Subissi L., Gluais L., Canard B., Decroly E. RNA3′-end mismatch excision by the severe acute respiratory syndromecoronavirus nonstructural protein nsp10/nsp14 exoribonucleasecomplex. Proc. Natl. Acad. Sci. USA. 2012;109(24):9372-9377. DOI10.1073/pnas.1201130109.Bouvet M., Lugari A., Posthuma C.C., Zevenhoven J.C., Bernard S.,Betzi S., Imbert I., Canard B., Guillemot J.-C., Lécine P., PfefferleS., Drosten C., Snijder E.J., Decroly E., Morelli X. CoronavirusNsp10, a critical co-factor for activation of multiple replicative enzymes.J. Biol. Chem. 2014;289(37):25783-25796. DOI 10.1074/jbc.M114.577353Brunn A., Teepe C., Simpson J.C., Pepperkok R., Friedel C.C., ZimmerR., Roberts R., Baric R., Haas J. Analysis of intraviral proteinproteininteractions of the SARS coronavirus ORFeome. PLoS One.2007;2(5):e459. DOI 10.1371/journal.pone.0000459.Chen J., Malone B., Llewellyn E., Grasso M., Shelton P.M.M., OlinaresP.D.B., Maruthi K., Eng E.T., Vatandaslar H., Chait B.T., KapoorT.M., Darst S.A., Campbell E.A. Structural basis for helicasepolymerasecoupling in the SARS-CoV-2 replication-transcriptioncomplex. Cell. 2020;182(6):1560-1573.e13. DOI 10.1016/j.cell.2020.07.033Chen Y., Cai H., Pan J., Xiang N., Tien P., Ahola T., Guo D. Functionalscreen reveals SARS coronavirus nonstructural protein nsp14as a novel cap N7 methyltransferase. Proc. Natl. Acad. Sci. USA.2009;106(9):3484-3489. DOI 10.1073/pnas.0808790106.Cottam E.M., Maier H.J., Manifava M., Vaux L.C., Chandra-SchoenfelderP., Gerner W., Britton P., Ktistakis N.T., Wileman T. Coronavirusnsp6 proteins generate autophagosomes from the endoplasmicreticulum via an omegasome intermediate. Autophagy. 2011;7(11):1335-1347. DOI 10.4161/auto.7.11.16642.Daczkowski C.M., Dzimianski J.V., Clasman J.R., Goodwin O., MesecarA.D., Pegan S.D. Structural insights into the interaction ofcoronavirus papain-like proteases and interferon-stimulated geneproduct15 from different species. J. Mol. Biol. 2017;429(11):1661-1683. DOI 10.1016/j.jmb.2017.04.011.Deng X., Baker S.C. An “Old” protein with a new story: Coronavirusendoribonuclease is important for evading host antiviral defenses.Virology. 2018;517:157-163. DOI 10.1016/j.virol.2017.12.024.Deng X., Geelen A., Buckley A.C., O’Brien A., Pillatzki A., LagerK.M., Faaberg K.S., Baker S.C. Coronavirus endoribonucleaseactivity in porcine epidemic diarrhea virus suppresses type I andtype III interferon responses. J. Virol. 2019;93(8):e02000-18. DOI10.1128/JVI.02000-18.Egloff M.-P., Ferron F., Campanacci V., Longhi S., Rancurel C., DutartreH., Snijder E.J., Gorbalenya A.E., Cambillau C., Canard B. Thesevere acute respiratory syndrome-coronavirus replicative proteinnsp9 is a single-stranded RNA-binding subunit unique in the RNAvirus world. Proc. Natl. Acad. Sci. USA. 2004;101(11):3792-3796.DOI 10.1073/pnas.0307877101.Frieman M., Ratia K., Johnston R.E., Mesecar A.D., Baric R.S. Severeacute respiratory syndrome coronavirus papain-like proteaseubiquitin-like domain and catalytic domain regulate antagonism ofIRF3 and NF-kappaB signaling. J. Virol. 2009;83(13):6689-6705.DOI 10.1128/JVI.02220-08.Fu L., Ye F., Feng Y., Yu F., Wang Q., Wu Y., Zhao C., Sun H., Huang B.,Niu P., Song H., Shi Y., Li X., Tan W., Qi J., Gao G.F. Both Boceprevirand GC376 efficaciously inhibit SARS-CoV-2 by targetingits main protease. Nat. Commun. 2020;11(1):4417. DOI 10.1038/s41467-020-18233-x.Gadhave K., Kumar P., Kumar A., Bhardwaj T., Garg N., Giri R. Conformationaldynamics of 13 amino acids long NSP11 of SARS-CoV-2under membrane mimetics and different solvent conditions. Microb.Pathog. 2021;158:105041. DOI 10.1016/j.micpath.2021.105041.Gao Y., Yan L., Huang Y., Liu F., Zhao Y., Cao L., Wang T., Sun Q.,Ming Z., Zhang L., Ge J., Zheng L., Zhang Y., Wang H., Zhu Y.,Zhu C., Hu T., Hua T., Zhang B., Yang X., Li J., Yang H., Liu Z.,Xu W., Guddat L.W., Wang Q., Lou Z., Rao Z. Structure of theRNA-dependent RNA polymerase from COVID-19 virus. Science.2020;368(6492):779-782. DOI 10.1126/science.abb7498.Goyal B., Goyal D. Targeting the dimerization of the main protease ofcoronaviruses: a potential broad-spectrum therapeutic strategy. ACSComb. Sci. 2020;22(6):297-305. DOI 10.1021/acscombsci.0c00058.Graham R.L., Sims A.C., Brockway S.M., Baric R.S., Denison M.R.The nsp2 replicase proteins of murine hepatitis virus and severeacute respiratory syndrome coronavirus are dispensable for viralreplication. J. Virol. 2005;79(21):13399-13411. DOI 10.1128/JVI.79.21.13399-13411.2005.Hao W., Wojdyla J.A., Zhao R., Han R., Das R., Zlatev I., Manoharan M.,Wang M., Cui S. Crystal structure of Middle East respiratory syndromecoronavirus helicase. PLoS Pathog. 2017;13(6):e1006474.DOI 10.1371/journal.ppat.1006474.Hughes S.A., Bonilla P.J., Weiss S.R. Identification of the murinecoronavirus p28 cleavage site. J. Virol. 1995;69(2):809-813. DOI10.1128/JVI.69.2.809-813.1995.Imbert I., Guillemot J.-C., Bourhis J.-M., Bussetta C., Coutard B., EgloffM.-P., Ferron F., Gorbalenya A.E., Canard B. A second, noncanonicalRNA-dependent RNA polymerase in SARS coronavirus.EMBO J. 2006;25(20):4933-4942. DOI 10.1038/sj.emboj.7601368.Imbert I., Snijder E.J., Dimitrova M., Guillemot J.-C., Lécine P., CanardB. The SARS-Coronavirus PLnc domain of nsp3 as a replication/transcription scaffolding protein. Virus Res. 2008;133(2):136-148. DOI 10.1016/j.virusres.2007.11.017.Ivanov K.A., Thiel V., Dobbe J.C., Meer Y., Snijder E.J., Ziebuhr J.Multiple enzymatic activities associated with severe acute respiratorysyndrome coronavirus helicase. J. Virol. 2004;78(11):5619-5632.DOI 10.1128/JVI.78.11.5619-5632.2004Joseph J.S., Saikatendu K.S., Subramanian V., Neuman B.W., BroounA.,Griffith M., Moy K., Yadav M.K., Velasquez J., Buchmeier M.J.,Stevens R.C., Kuhn P. Crystal structure of nonstructural protein 10from the severe acute respiratory syndrome coronavirus reveals anovel fold with two zinc-binding motifs. J. Virol. 2006;80(16):7894-7901. DOI 10.1128/JVI.00467-06.Kamitani W., Narayanan K., Huang C., Lokugamage K., Ikegami T.,Ito N., Kubo H., Makino S. Severe acute respiratory syndromecoronavirus nsp1 protein suppresses host gene expression by promotinghost mRNA degradation. Proc. Natl. Acad. Sci. USA. 2006;103(34):12885-12890. DOI 10.1073/pnas.0603144103.Kanjanahaluethai A., Baker S.C. Identification of mouse hepatitis viruspapain-like proteinase 2 activity. J. Virol. 2000;74(17):7911-7921.DOI 10.1128/jvi.74.17.7911-7921.2000.Kim Y., Jedrzejczak R., Maltseva N.I., Wilamowski M., Endres M.,Godzik A., Michalska K., Joachimiak A. Crystal structure of Nsp15endoribonuclease NendoU from SARS-CoV-2. Protein Sci. 2020;29(7):1596-1605. DOI 10.1002/pro.3873.Lu R., Zhao X., Li J., Niu P., Yang B., Wu H., Wang W., Song H.,Huang B., Zhu N., Bi Y., Ma X., Zhan F., Wang L., Hu T., Zhou H.,Hu Z., Zhou W., Zhao L., Chen J., Meng Y., Wang J., Lin Y., Yuan J.,Xie Z., Ma J., Liu W.J., Wang D., Xu W., Holmes E.C., Gao G.F.,Wu G., Chen W., Shi W., Tan W. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus originsand receptor binding. Lancet. 2020;395(10224):565-574. DOI10.1016/S0140-6736(20)30251-8.Ma Y., Wu L., Shaw N., Gao Y., Wang J., Sun Y., Lou Z., Yan L.,Zhang R., Rao Z. Structural basis and functional analysis of theSARS coronavirus nsp14–nsp10 complex. Proc. Natl. Acad. Sci.USA. 2015;112(30):9436-9441. DOI 10.1073/pnas.1508686112.Ma-Lauer Y., Carbajo-Lozoya J., Hein M.Y., Müller M.A., Deng W.,Lei J., Meyer B., Kusov Y., von Brunn B., Bairad D.R., Hünten S.,Drosten C., Hermeking H., Leonhardt H., Mann M., Hilgenfeld R.,von Brunn A. p53 down-regulates SARS coronavirus replication andis targeted by the SARS-unique domain and PLpro via E3 ubiquitinligase RCHY1. Proc. Natl. Acad. Sci. USA. 2016;113(35):E5192-E5201. DOI 10.1073/pnas.1603435113.Malik Y.S., Sircar S., Bhat S., Sharun K., Dhama K., Dadar M., TiwariR., Chaicumpa W. Emerging novel coronavirus (2019-nCoV) –current scenario, evolutionary perspective based on genome analysisand recent developments. Vet. Q. 2020;40(1):68-76. DOI 10.1080/01652176.2020.1727993.Miknis Z.J., Donaldson E.F., Umland T.C., Rimmer R.A., Baric R.S.,Schultz L.W. Severe acute respiratory syndrome coronavirus nsp9dimerization is essential for efficient viral growth. J. Virol. 2009;83(7):3007-3018. DOI 10.1128/JVI.01505-08.Naqvi A.A.T., Fatima K., Mohammad T., Fatima U., Singh I.K.,Singh A., Atif S.M., Hariprasad G., Hasan G.M., Hassan M.I. Insightsinto SARS-CoV-2 genome, structure, evolution, pathogenesisand therapies: Structural genomics approach. Biochim. Biophys.Acta Mol. Basis Dis. 2020;1866(10):165878. DOI 10.1016/j.bbadis.2020.165878.Narayanan K., Huang C., Lokugamage K., Kamitani W., Ikegami T.,Tseng C.-T.K., Makino S. Severe acute respiratory syndrome coronavirusnsp1 suppresses host gene expression, including that oftype I interferon, in infected cells. J. Virol. 2008;82(9):4471-4479.DOI 10.1128/JVI.02472-07.Ogando N.S., Zevenhoven-Dobbe J.C., Meer Y., Bredenbeek P.J., PosthumaC.C., Snijder E.J. The enzymatic activity of the nsp14 exoribonucleaseis critical for replication of MERS-CoV and SARS-CoV-2.J. Virol. 2020;94(23):e01246-20. DOI 10.1128/JVI.01246-20.Oostra M., Hagemeijer M.C., Gent M., Bekker C.P.J., Lintelo E.G.,Rottier P.J.M., Haan C.A.M. Topology and membrane anchoring ofthe coronavirus replication complex: not all hydrophobic domainsof nsp3 and nsp6 are membrane spanning. J. Virol. 2008;82(24):12392-12405. DOI 10.1128/JVI.01219-08.Pan J., Peng X., Gao Y., Li Z., Lu X., Chen Y., Ishaq M., Liu D., DediegoM.L., Enjuanes L., Guo D. Genome-wide analysis of proteinproteininteractions and involvement of viral proteins in SARS-CoVreplication. PLoS One. 2008;3(10):e3299. DOI 10.1371/journal.pone.0003299.Pasternak A.O., Spaan W.J.M., Snijder E.J. Nidovirus transcription:how to make sense...? J. Gen. Virol. 2006;87(6):1403-1421. DOI10.1099/vir.0.81611-0.Rost B. Twilight zone of protein sequence alignments. Protein Eng.1999;12(2):85-94. DOI 10.1093/protein/12.2.85.Su D., Lou Z., Sun F., Zhai Y., Yang H., Zhang R., Joachimiak A.,Zhang X.C., Bartlam M., Rao1 Z. Dodecamer structure of severeacute respiratory syndrome coronavirus nonstructural protein nsp10.J. Virol. 2006;80(16):7902-7908. DOI 10.1128/JVI.00483-06.Subissi L., Posthuma C.C., Collet A., Zevenhoven-Dobbe J.C., GorbalenyaA.E., Decroly E., Snijder E.J., Canard B., Imbert I. One severeacute respiratory syndrome coronavirus protein complex integratesprocessive RNA polymerase and exonuclease activities. Proc.Natl. Acad. Sci. USA. 2014;111(37):E3900-E3909. DOI 10.1073/pnas.1323705111.Tahir M. Coronavirus genomic nsp14-ExoN, structure, role, mechanism,and potential application as a drug target. J. Med. Virol. 2021;93(7):4258-4264. DOI 10.1002/jmv.27009.Thiel V., Ivanov K.A., Putics Á., Hertzig T., Schelle B., Bayer S., WeißbrichB., Snijder E.J., Rabenau H., Doerr H.W., Gorbalenya A.E.,Ziebuhr J. Mechanisms and enzymes involved in SARS coronavirusgenome expression. J. Gen. Virol. 2003;84(9):2305-2315. DOI10.1099/vir.0.19424-0.Tomar S., Johnston M.L., John S.E.St., Osswald H.L., Nyalapatla P.R.,Paul L.N., Ghosh A.K., Denison M.R., Mesecar A.D. Ligand-induceddimerization of Middle East respiratory syndrome (MERS)coronavirus nsp5 protease (3CLpro): implications for nsp5 regulationand the development of antivirals. J. Biol. Chem. 2015;290(32):19403-19422. DOI 10.1074/jbc.M115.651463.Velthuis A.J.W., Arnold J.J., Cameron C.E., Worm S.H.E., Snijder E.J.The RNA polymerase activity of SARS-coronavirus nsp12 is primerdependent. Nucleic Acids Res. 2010;38(1):203-214. DOI 10.1093/nar/gkp904.Velthuis A.J.W., Worm S.H.E., Snijder E.J. The SARS-coronavirusnsp7+nsp8 complex is a unique multimeric RNA polymerase capableof both de novo initiation and primer extension. Nucleic AcidsRes. 2012;40(4):1737-1747. DOI 10.1093/nar/gkr893Xiao Y., Ma Q., Restle T., Shang W., Svergun D.I., Ponnusamy R.,Sczakiel G., Hilgenfeld R. Nonstructural proteins 7 and 8 of felinecoronavirus form a 2:1 heterotrimer that exhibits primer-independentRNA polymerase activity. J. Virol. 2012;86(8):4444-4454. DOI10.1128/JVI.06635-11.Zeng Z., Deng F., Shi K., Ye G., Wang G., Fang L., Xiao S., Fu Z.,Peng G. Dimerization of coronavirus nsp9 with diverse modesenhances its nucleic acid binding affinity. J. Virol. 2018;92(17):e00692-18. DOI 10.1128/JVI.00692-18.Zhai Y., Sun F., Li X., Pang H., Xu X., Bartlam M., Rao Z. Insights intoSARS-CoV transcription and replication from the structure of thensp7–nsp8 hexadecamer. Nat. Struct. Mol. Biol. 2005;12(11):980-986. DOI 10.1038/nsmb999.Zhang L., Li L., Yan L., Ming Z., Jia Z., Lou Z., Rao Z. Structural andbiochemical characterization of endoribonuclease nsp15 encodedby Middle East respiratory syndrome coronavirus. J. Virol. 2018;92(22):e00893-18. DOI 10.1128/JVI.00893-18.Ziebuhr J., Snijder E.J., Gorbalenya A.E. Virus-encoded proteinasesand proteolytic processing in the Nidovirales. J. Gen. Virol. 2000;81(4):853-879. DOI 10.1099/0022-1317-81-4-853.


RNA editing contributes to transcriptome diversification through RNA modifications in relation to genome-encoded information . The deamination of Adenosine (A) to Inosine (I) or Cytidine (C) to Uridine (U) is the most common type of mammalian RNA editing. It occurs as a nuclear co- and/or post-transcriptional event catalyzed by ADARs (Adenosine deaminases acting on RNA) and APOBECs . RNA editing may modify the structure, stability, and processing of a transcript. This review focuses on RNA editing in psychiatric, neurological, neurodegenerative (NDs), and autoimmune brain disorders in humans and rodent models. We discuss targeted studies that focus on RNA editing in specific neuron-enriched transcripts with well-established functions in neuronal activity, and transcriptome-wide studies, enabled by recent technological advances. We provide comparative editome analyses between human disease and corresponding animal models. Data suggest RNA editing to be an emerging mechanism in disease development, displaying common and disease-specific patterns. Commonly edited RNAs represent potential disease-associated targets for therapeutic and diagnostic values. Currently available data are primarily descriptive, calling for additional research to expand global editing profiles and to provide disease mechanistic insights. The potential use of RNA editing events as disease biomarkers and available tools for RNA editing identification, classification, ranking, and functional characterization that are being developed will enable comprehensive analyses for a better understanding of disease(s) pathogenesis and potential cures. RNA editing refers to an epigenetic mechanism that contributes to transcriptome diversification through the introduction of alterations in RNA species relative to the corresponding genome-encoded information  .The major type of RNA editing in mammals is the chemical modification (deamination) of Adenosine (A) to Inosine (I) or Cytidine (C) to Uridine (U), resulting in A-I or C-U substitutions. It occurs as a nuclear and cytoplasmic co- and/or post-transcriptional event and is catalyzed by the ADAR (Adenosine deaminases acting on RNA) and APOBEC  enzyme families  Figure A,B.Depending on the RNA species undergoing editing and the edited position(s) within a transcript, RNA editing may result in an altered structure, stability, and transcript processing; strong evidence suggests that RNA editing is a major epigenetic gene expression regulator, acting both independently and in conjunction with RNA interference pathways, the latter of which may also be subjected to the dependent regulation of RNA editing. Furthermore, protein recoding  and immature stop codon introduction may occur as a result of non-synonymous changes introduced by RNA editing in coding regions C.RNA editing is highly regulated in a tissue- , developNF1 mRNA due to false C-U editing [As a result, several studies have associated RNA editing with disease conditions. Type I neurofibromatosis is associated with a dysfunctional protein resulting from the introduction of a premature stop codon in the  editing ; random  editing  and ADAR editing .Among other tissues, the brain displays the highest editing levels in both mice and humans ,14 and sIn this review, we focus on the role of RNA editing alterations in psychiatric, neurological, neurodegenerative (NDs) and autoimmune brain disorders in human cases and in related mouse models. We provide a comprehensive presentation of relevant data supporting RNA editing contribution in these disorders. We include studies focusing on editing events identified in selected, disease-associated targets, as well as recently published transcriptome-wide studies and discuss common editing patterns among different brain disorders. Furthermore, we attempt to identify commonly edited targets and affected cellular processes among human and mouse brain disorders, suggestive of common underlying molecular mechanisms and indicative of animal models’ utilization reliability. Subsequently, we discuss the significance of extending transcriptome-wide RNA editing studies at pre-clinical disease stages in relevant disease models, aiming to identify RNA editing alterations that may contribute to driver pathogenetic events, that could provide novel candidate intervention targets. Finally, we discuss future perspectives and challenges in the RNA editing study field, focusing on CNS disorders.Five independent reviewers conducted a systematic literature search on PubMed for suitable research published between 1995 and 2021, following preset methods and intending to identify studies related to RNA editing in brain disorders. The search terms were: “RNA editing” OR “RNA alterations” AND “neurological disorders” OR “neurodegenerative diseases” OR “neurodegenerative disorders” OR “neurodegeneration” OR “psychiatric disorders”.Studies were considered eligible if they met the following requirements: (1) Studies in humans and rodent models examining RNA editing events in relation to psychiatric, neurological, neurodegenerative, and autoimmune brain disorders; (2) genetically determined neurological and neurodegenerative disorders, influenced by environmental factors or caused by an injury; (3) studies in brain and/or spinal cord tissue material from either postmortem human material or animal disease models.Exclusion criteria were as follows: (1) non-original publications, such as letters to the editor, opinions, reviews, case reports, protocols, conference or meeting abstracts, comments, or meta-analyses; (2) studies unrelated to the subject; (3) studies with insufficient or unqualified data.Only articles written in English were considered. In addition, duplicate publications were excluded. The following information was gathered from all the studies that were included: basic information , disorder, species, brain area, study type, targets, methodology, validation method, differentially expressed targets compared to controls.The studies were evaluated using the criteria specified by the PRISMA  guidelines .The term ‘Neurological Disorder’ refers to a wide range of disorders concerning the nervous system, which are characterized by a variety of symptoms ranging from mild to severe and are either accompanied by neurodegeneration or not. Neurological disorders may be either inherited with genetic predisposition, associated with environmental factors or may occur as a result of injury. Neurological/neurodegenerative disorders, such as epilepsy, autism, Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), prion diseases, amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS), can be clustered together because they all involve the malfunction or damaging of the brain, spinal cord, and nerves. Nervous system degeneration affects neuronal communication resulting in behavior and emotional state disruptions, which also represent hallmarks of psychiatric disorders, such as schizophrenia (SCZ), chronic social defeat stress (CSDS), depression and suicide. Thus, despite their difference, neurological/neurodegenerative and psychiatric disorders display some degree of overlapping aspects and possibly common or similar pathogenetic mechanisms.These disorders have long attracted the interest of the scientific community due to a dramatic increase in cases and their economical as well as social impact. Studies on the effect of RNA editing within these disorders’ contexts first began in the 1990s. Gria2, Gria3 and Gria4 and by the Grik1 and Grik2 transcripts, respectively; the serotonin receptor, encoded by the 5-HT2C transcript; and the KCNA1 encoded alpha subunit of the Kv1.1 potassium channel, which all have established roles in neuronal excitability [As shown in tability .Gria2 encoded subunit of the AMPA glutamate receptor seems to be the most significant, as the introduction of the Q/R recoding site in ADAR2-deficient mice prevents animal lethality [++ permeability. Given the prominent role of this recoding event, it is not surprising that Gria2 was selected as a study target in the context of several brain disorders, including HD, AD, ALS, SCZ and has been detected as under-edited in these disease conditions  also possess the Q/R RNA editing site, which also affects Ca++ permeability. Therefore, it was suggested that altered RNA editing in these sites might also contribute to epileptogenesis. KA-induced epilepsy leads to increased GluK1 Q/R site editing following seizures in the hippocampus in a rat model [Apart from the connection of RNA editing at the at model . Additioat model . In addiat model .Another RNA editing event that seems to be connected to epileptogenesis is the I/V site of the potassium channel Kv1.1. This ADAR2 catalyzed isoleucine (I) to valine (V) editing takes place in the pore domain of the channel, resulting in a rapid recovery from fast inactivation. This editing event increases fourfold in the entorhinal cortex of chronic epileptic rats compared to their healthy counterparts . Mutatio5-HT2C transcript has been selected as a study target in depression and suicide studies, as well as in autism, SCZ and AD. The 5-HT2C transcript is known to undergo A-I editing at five sites (A–E) within the region involved in G-protein coupling. Editing reduces the receptors’ G-protein coupling and thus its function. The differential representation of 5-HT2C isoforms resulting from the combination of editing at positions A-E was reported in these disorders and associated with disease progression. Additionally, reduced editing reported in the KCNA1 transcript in epilepsy was inversely correlated with disease duration [Moreover, given that abnormalities in serotonergic transmission are strongly associated with mood and psychiatric disorders, it is plausible that the serotonin receptor duration .PDE8A is expressed mainly in oligodendrocytes ) and encodes a phosphodiesterase involved in signal transduction affecting inflammatory responses, memory and cognition. PDE8A was studied in terms of RNA editing alterations in depression and suicide. Similar to 5-HT2C, several editing sites were reported in PDE8A and differential representation of the corresponding edited transcripts was detected in depression and suicide, providing novel targets for further functional characterization [EAAT, another glia-enriched transcript, expressed mainly in astrocytes. This editing alteration, detected in AD, occurs in intron 7 and affects intron retention, as shown by in vitro functional evidence [Apart from neuronal transcripts, a few studies have focused on glial targets. rization . Editingevidence .The previously mentioned, strictly focused, studies were extended by studies focusing on transcripts involved in known disease-related molecular pathways ,44,53, pAdvances in high-throughput transcriptomic studies, achieved through the development of efficient experimental technologies and robust bioinformatics approaches for data analysis, significantly contributed to RNA editing research. Indeed, several ADAR- and APOBEC-mediated editing events were reported in different organisms, and corresponding databases , thus providing some functional molecular links between RNA editing alterations and ASD. Interestingly, significantly overlapped patterns of editome alterations were observed in autism spectrum disorder (ASD) and fragile X syndrome (FXS), suggesting RNA editing as a link between ASD and FXS. FXS is caused by the silencing (methylation) of the FMR1 gene that is often present in features of ASD and accounts for about 5% of ASD cases [A link between RNA hypoediting and the neurodevelopmental condition of autism has been recently revealed through a transcriptome-wide study . The autSD cases .Another transcriptome-wide analysis aimed to identify differentially edited sites in the hippocampus of a mouse model with temporal lobe epilepsy relative to healthy controls . The autSOD2, MCUR1, PFKP and transcripts involved in fatty acid and lipid metabolism, such as HSDL2). In addition, by utilizing multi-omics  integration approaches, the authors highlight a negative correlation of RNA editing in the 3′UTR of SYT11 with SYT11 protein levels, and a correlation of RNA editing at the 3′-UTR of ORAI2 with aggregation of hyperphosphorylated tau and with neuritic amyloid plaque burden, suggesting functional molecular links between RNA editing alterations and AD [Finally, a systematic transcriptome-wide evaluation in a large dataset from the human cortex and cerebellum has uncovered a significant connection between RNA editing and Alzheimer’s disease. Through a correlation analysis and RNA editing ranking, the study highlights several RNA editing events highly associated with AD . Interestingly, these targets are enriched in biological processes related to synaptic transmission and signaling, the regulation of cell communication, pattern recognition receptor signaling, response to thyroid signaling and protein catabolism  or significantly advanced (mice) disease stages, possibly reflect common end-stage disease consequences rather than disease promoting events. When similar studies would be conducted at earlier disease stages will significantly aid towards the identification of disease driver/promoting RNA editing events. Thus, pre-clinical studies utilizing animal models are highly needed. To our knowledge, there is currently only one published study reporting pre-clinical disease alterations of RNA editing patterns . This stAlu regions in the brain of individuals with glioblastoma were suggested as a prognostic factor in a gender-dependent manner and decreases in global A-I editing profiles were reported as an efficient patient stratification approach in this disease [Altered RNA editing profiles within  disease . Moreove disease .AluSx+ sequence, which is in the Ctss 3′UTR and affects Ctss expression, was reported in peripheral blood mononuclear cells of rheumatoid arthritis (RA) patients [Peripheral blood analysis from healthy individuals, detected more than 1000 RNA editing sites . Moreovepatients . The sampatients . Furtherpatients .PDE8A RNA editing patterns in the blood of depressed patients and suicide attempters with major depression, suggesting its diagnostic potential [The above data suggest that RNA editing profile studies in body fluids may enhance disease diagnosis as well as patient responses to treatments. Indeed, a recent study reports characteristic otential . In addiotential . Furtherotential . This fuThe existing literature indicates RNA editing as an emerging mechanism that contributes to disease progression and highlights common and disease-specific editing patterns.The advent of transcriptome-wide studies has enabled a comprehensive profiling of RNA editing alterations in health and disease conditions. Further studies focusing on different disorders are expected to extend the so-far-limited data on transcriptome-wide and descriptive studies of RNA editing. A significant challenge in this field refers to the accurate identification of true RNA editing events, which is based on comparative analyses of RNA-seq data aligned against the corresponding reference genome for the detection of RNA:DNA differences (RDDs). Both experimental setup and data analysis pipelines affect the accuracy and efficiency of identifying RNA editing events. Accurate alignment in the reference genome was identified as a critical step influencing the correct calling of RNA editing, and several alignment approaches were reported (some of them reviewed in ). False-Importantly, and considering that RNA editing is tissue- and cell-type-specific, it is plausible to assume that refined studies focusing on specific cell sub-types or even on single-cell analyses, will help to unravel cell-type-specific, disease-related differences that may be undetectable in bulk tissue analyses. In this direction, few recent studies have reported cell-type-specific editing patterns in the human brain , murine Further improvements of existing pipelines to allow for a more efficient analysis of editing events residing in long non-coding RNAs , and theFunctional studies to delineate factors leading to altered editing and the estimation of RNA editing effects are also required. For the study of the functional effects of RNA editing, appropriate in vivo  and In summary, RNA editing studies provide significant insights in terms of CNS disease-related molecular mechanisms, suggesting a protective role in disease progression, which could be exploited in subsequent diagnostic/prognostic and even therapeutic approaches. The currently available data are mostly descriptive and further studies are required to extend the global editing profiles for different CNS disorders. Comprehensive analyses through the development of more efficient tools for RNA editing identification, classification, ranking, and functional characterization are being developed and are expected to allow for a better understanding of the role of RNA editing in these disorders.


Penicillium digitatum, the pathogen of green mold in citrus fruits. This study examined the mechanism by which citronellal, a typical terpenoid of Cymbopogon nardus essential oil, acts on ergosterol to exhibit its antifungal activity against P. digitatum. We previously reported that citronellal inhibited the growth of P. digitatum with minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of 1.36 and 2.72 mg/mL, respectively. In citronellal-treated cells, the membrane integrity and ergosterol contents significantly decreased, whereas lanosterol, which serves as a precursor for ergosterol biosynthesis, massively accumulated. Addition of 150 mg/L of exogenous ergosterol decreased the inhibitory rate of citronellal, restoring the ergosterol content and hence the membrane structure to normal levels, and triggered expression of nearly all ERG genes. Based on our findings, we deduce that citronellal damages the cell membrane integrity of P. digitatum by down-regulating the ERG genes responsible for conversion of lanosterol to ergosterol, the key downregulated gene being ERG3, due to the observed accumulation of ergosta-7,22-dienol.Ergosterol (ERG) is a potential target for the development of antifungal agents against  Penicillium digitatum, is the major postharvest disease of citrus during harvesting and postharvest handling processes [Green mold, caused by the pathogen rocesses . Currentrocesses . As a coCymbopogon nardus essential oil, has a strong inhibitory effect against fungi [Penicillium strains including P. digitatum, P. italicum, and P. ulaiense, with the inhibition zone ranging from 31 to 43 mm2 [Penicillium species but also of Fusarium and Aspergillus species. Our previous study demonstrated that citronellal visibly inhibited the germination of P. digitatum spores at concentrations higher than 0.17 mg/mL and delayed the green mold decay of navel orange in a dose-dependent manner [P. digitatum, with minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of 1.36 and 2.72 mg/mL respectively. Combined with cinnamaldehyde, citronellal obviously reduced the green mold incidence in Satsuma mandarin citrus fruit and did not impair the fruit quality [Essential oils (EOs) are considered safer, highly active, and with low possibility to induce drug resistance ,4,5,6, tst fungi ,11. Prevo 43 mm2 . Recentlo 43 mm2  and Wango 43 mm2  confirmet manner . In addi quality . CitroneCandida albicans by affecting membrane integrity and arresting cell cycle [+ extrusion. Ergosterol, an integral component of the cell membrane that plays a key role in maintaining the integrity and fluidity of the membrane structure, is a key target of common chemical fungicides  against P. digitatum [P. digitatum, Aspergillus species, and Candida species, causing abnormal cell membrane function [P. digitatum by damaging the cell membrane integrity thereby leading to leakage of intracellular constituents [P. digitatum is still unknown. This study was therefore designed (i) to evaluate the effect of citronellal on the plasma membrane integrity of P. digitatum, (ii) to determine its effect on lanosterol and ergosterol contents and on sterol composition, and (iii) to analyze its effect on gene expression levels in ergosterol biosynthesis of P. digitatum.Existing literature suggests that cell membrane is the action target of citronellal against fungi. For example, citronellal inhibited the growth of ll cycle . In addill cycle  reportedigitatum ,19. Indefunction ,24,25,26tituents ,16, the Penicillium digitatum used in this study was isolated from infected Satsuma mandarin (Citrus unshiu Marc. cv. Miyagawa Wase), identified by morphological and molecular biology methods, and preserved at Xiangtan University [6 spores/mL using a hemocytometer.iversity . This stCitrus reticulata Blanco) were harvested on 21 December 2019 from an orchard in Luxi, Hunan, China. Healthy fruits of uniform size and without scars were selected for the experiments.Mature mandarin fruits (v:v) and topped up to the final volumes using distilled water. All the chemicals were of analytic grade.Citronellal (96%) was purchased from Aladdin Chemical Reagent Co., Ltd. , solutions prepared by dissolving the requisite amount in Tween-80  for 2 min, then washed with distilled water, wounded (depth of 3 mm and width of 3 mm) with a sterile needle, inoculated with 20 μL of P. digitatum spore suspension (105 spores/mL), and left to air-dry. The inoculated fruits were then immersed in in wax amended with citronellal at 1 × and 5 × MFC for 30 s. Fruits with wax and pathogen inoculation were used as control. Ten mandarin fruits constituted a single replicate, and each treatment was performed in triplicate. The incidence rate of disease (measured by counting the number of green mold-infected wounds) was calculated from the formula:Artificial inoculation of fruits with g et al.  with minThe diameter (mm) of lesion was measured using a G102-123-101 caliper .P. digitatum were tested in vitro by agar dilution method [Effects of addition of external ergosterol on the antifungal activity of citronellal against mycelial growth of n method  using fin method  to compaP. digitatum cells treated with citronellal (0 or 1/2 MIC) were analyzed by propidium iodide (PI) staining coupled with an ECLIPSE TS100 microscope  and the fluorescence value determined by an F97 PRO fluorescence spectrophotometer  as described in OuYang et al. [The plasma membrane integrity of g et al. . Each ofP. digitatum cells citronellal (1/2 MIC concentration) for 0, 30, and 60 min were determined by high performance liquid chromatography (HPLC) as we previous described [w/v) NaOH and 8 mL of absolute ethanol, then saponified at 85–90 °C for 2 h. The mixtures were extracted thrice with petroleum ether and washed twice in saturated NaCl solution. The upper organic layer was vacuum concentrated and each residue adjusted to 10 mL using ethanol. The detected wavelengths for lanosterol and ergosterol were set at 210 nm and 282 nm, respectively, calculating lanosterol and ergosterol contents from the standard calibration curve using lanosterol and ergosterol as standards, respectively. Control samples were not treated with citronellal, and the experiments were repeated with addition of 150 mg/L exogenous ergosterol.Total lanosterol and ergosterol contents of escribed  with minP. digitatum cells treated with 1/2 MIC citronellal for 0, 30, and 60 min were determined by GC-MS method [w/v) and absolute ethanol (8 mL) at 80 °C for 1 h. The mixtures were subsequently extracted in petroleum ether and washed using saturated NaCl solution and then the upper organic layer vacuum concentrated. Each concentrated sample was completely dissolved in 950 μL of methyltertbutylether and then the silylation reagent mixture  added and left at room temperature for at least 30 min to complete the silylation reaction followed by GC-MS analysis. Control samples were not treated with citronellal. Similar experiments were performed with addition of exogenous ergosterol (150 mg/L) to the P. digitatum culture medium.The composition of sterols in S method . The drym/z 50–700, scanning interval 0.5 s and scanning speed 1000 amu/s. The sterol TMS ethers were identified by comparison with commercial references, the NIST™ database, or data from literature [The analytical GC was carried out on a ShimadzuQP2010 plus gas chromatograph  equipped with flame ionization detector (FID). A non-polar cross-linked fused-silica capillary column, HP-5MS , was used. The oven temperature was held at 50 °C for 1 min, programmed at a rate of 50 °C/min to 260 °C, then increased to 300 °C at a rate of 4 °C/min where it remained for 10 min. The carrier gas was helium (1.3 mL/min). The injector temperature was 250 °C, detector temperature 310 °C and the volume injected was 2 μL. MS analysis was carried out on the same chromatograph equipped with a Shimadzu QP 2010 GC/MS system, ionization voltage 70 eV, ion source temperature 200 °C, mass range P. digitatum treated with citronellal (0 or 1/2 MIC concentration) for 0, 30, and 60 min was extracted using the Trizol reagent  following the manufacturer’s instructions. The expression levels of the ERG1, ERG2, ERG3, ERG4, ERG5, ERG6, ERG7, ERG9, ERG11 ERG24, ERG25, ERG26, and ERG27 genes were examined using RTFQ-PCR. RTFQ-PCR was performed through a BIO-RAD CFX Connect Thermal Cycler using FastStart Universal SYBR Green Master  with the following programs: 95 °C for 10 min followed by 40 cycles at 95 °C for 15 s, and at 60 °C for 1 min. Actin gene was used as internal reference, and the samples were quantified by 2−△△CT method [RNA of T method . All priAll data were expressed as their mean ± SD by measuring three independent replicates and analyzed using one-way ANOVA followed by Duncan’s test to test the significance of differences between means obtained among the treatments at the 5% level of significance using SPSS statistical software package release 16.0 .P. digitatum, the incidence rate in wax-treated fruits was 82%, a value much higher than the 10% observed in citrus fruits coated with citronellal (1 × and 5 × MFC)  effectively reduced the green mold decays of citrus fruits by the fifth day of inoculation, being able to alleviate the disease progression in inoculated fruits as shown in 5 × MFC) A. The av5 × MFC) B,C.P. digitatum are presented in P. digitatum were 1.36 and 2.72 mg/mL, respectively. After 4 days of treatment, the antifungal rate of citronellal (0.78 mg/mL = 1/2 MIC concentration) against P. digitatum was 59.60 ± 9.18%. This rate significantly reduced to 42.38 ± 7.16% following addition of external ergosterol (150 mg/L)  . This reMycelia treated with 1/2 MIC citronellal showed stronger red fluorescence after 30 min of treatment compared to the controls A. After P. digitatum samples was markedly higher than those of the control groups   B.P. digitatum  were repressed  in the test groups being significantly higher than in the control groups (p < 0.05) ( < 0.05) .Curcuma longa L. essential oil, citral, perillaldehyde, and thymol have an impact on the cell membrane integrity by targeting ergosterol metabolism [Ergosterol is the most abundant and main sterol component of fungal membranes , involvetabolism ,24,25,26P. digitatum both in vitro and in vivo. Our results show that treatment of P. digitatum with citronellal led to a significant inhibition of ergosterol biosynthesis and damage to the cell membrane. This observation corresponds to that reported by Singh et al. [C. albicans. Addition of 150 mg/L ergosterol to the growth medium greatly enhanced the tolerance of P. digitatum to citronellal, restoring the cell membrane integrity and ergosterol content to normal levels. This again is consistent with reports that the presence of exogenous ergosterol in growth medium promotes increased survival of some fungi under test agents stress [Saccharomyces cerevisiae. These findings confirm a direct correlation between ergosterol levels and cell membrane integrity and allude to the possibility that the antifungal effect of citronellal on P. digitatum is attributed to its inhibition of ergosterol biosynthesis.Citronellal is a promising antifungal compound with good inhibitory effects on h et al.  in whichs stress ,28,34,35s stress  and Liu ERG3, ERG5, ERG6, and ERG11 and acted on the ergosterol biosynthesis of P. digitatum by targeting the ERG11 gene [P. digitatum in which the expression of most of the ERG genes in citronellal-treated samples was markedly lower (p < 0.05) than in the control samples during the entire experimental time. In this pathway, catalyzing C14-demethylation of lanosterol by ERG11 is critical for ergosterol biosynthesis. Obviously, citronellal induced the downregulated of ERG11 gene and then led the exclusive accumulation of lanosterol, indicating that the ergosterol synthesis was hindered. Meanwhile, the side chain of ergosterol biosynthesis was activated, inducing the production of eburicol in citronellal-treated samples. These results are consistent with those reported by Ottilie et al. [TcCyp51 inhibitor, can increase the accumulation of both lanosterol and eburicol. It should also be noted that the loss of the ERG3 led to the typical fungal sterol ergosterol and ergosta-5,7,22,24(28)-tetraenol depletion, activating another side chain of ergosterol biosynthesis hence inducing the generation of ergosta-7,22-dienol [P. digitatum might be between the ERG11 and ERG3.Ergosterol biosynthesis is tightly regulated by ERG genes . Our preG11 gene , and Xu G11 gene  also note et al.  that pos2-dienol . This imS. cerevisiae and C. glabrata [P. digitatum was altered after supplementation of exogenous ergosterol in the citronellal treatment samples. In the citronellal-treated cells supplemented with ergosterol, genes ERG1, ERG3, ERG5, and ERG7 responded quickly and in just 30 min had already been upregulated more than those in citronellal-treated samples. By 60 min of treatment, all the genes in the test samples supplemented with ergosterol had been upregulated more than those in only citronellal treatment. This corresponds to the result by Liu et al. [ERG3 and ERG5 genes led to a decrease in lanosterol content to the normal value and a decrease in eburicol (a branch chain product) content to the detection limit after 30 min of treatment with exogenous ergosterol. This implies that ERG3 is the vital regulatory gene.Exogenous ergosterol is significant in improving the synthetic ability of ergosterol in glabrata ,35. In tglabrata . We alsoP. digitatum cells by citronellal treatment occurs between ERG11 and ERG3 genes and that gene ERG3 in P. digitatum may be the key regulatory site gene in response to citronellal treatment. These findings provide new insights into the antifungal mechanism of citronellal.In summary, the present study has demonstrated that the suppression of ergosterol biosynthesis in


Streptococcus pneumoniae infection. Pathway analysis of plasma miRNA signatures unique to Influenza or Streptococcus pneumoniae infections showed enrichment for specific proteoglycan, cell cycle, and immunometabolic pathways.Community‐acquired pneumonia (CAP) is a major cause of sepsis. Despite several clinical trials targeting components of the inflammatory response, no specific treatment other than antimicrobial therapy has been approved. This argued for a deeper understanding of sepsis immunopathology, in particular factors that can modulate the host response. Small non‐coding RNA, for example, micro (mi)RNA, have been established as important modifiers of cellular phenotypes. Notably, miRNAs are not exclusive to the intracellular milieu but have also been detected extracellular in the circulation with functional consequences. Here, we sought to determine shifts in circulatory small RNA levels of critically ill patients with CAP‐associated sepsis and to determine the influence of clinical severity and causal pathogens on small RNA levels. Blood plasma was collected from 13 critically ill patients with sepsis caused by CAP on intensive care unit admission and from 5 non‐infectious control participants. Plasma small RNA‐sequencing identified significantly altered levels of primarily mature miRNAs in CAP relative to controls. Pathways analysis of high or low abundance miRNA identified various over‐represented cellular biological pathways. Analysis of small RNA levels against common clinical severity and inflammatory parameters indices showed direct and indirect correlations. Additionally, variance of plasma small RNA levels in CAP patients may be explained, at least in part, by differences in causal pathogens. Small nuclear RNA levels were specifically altered in CAP due to Influenza infection in contrast to  Significantly different plasma miRNA levels were analysed for biological pathway enrichment, revealing putative functional targets of paracrine and/or autocrine signalling.In this study, we sought to determine changes in CNAs, specifically small non‐coding RNAs, measured in blood plasma samples from patients with CAP‐associated sepsis on intensive care unit (ICU) admission. Levels of plasma small RNAs in CAP were firstly compared to non‐infectious control participants, as well as patients with blood culture positive 22.1S pneumoniae and PCR positive Influenza A or B infection. Sepsis diagnosis was described in detail previously.The study was performed within the context of the Molecular Diagnosis and Risk Stratification of Sepsis (MARS) project, a prospective observational cohort study in the two tertiary referral centres in the Netherlands  .2.2www.genomescan.nl) using Solexa sequencing technology (Illumina) as described by manufacturer.Whole blood (EDTA) collected from non‐infectious control participants and sepsis patients were centrifuged at 1200rpm for 20 minutes to obtain 300ul plasma, and stored in −80C. Plasma from sepsis patients was obtained within 6 hours of ICU admission. Prior to nucleic acid isolation, plasma samples were thawed over ice and centrifuged at 12,000rpm for 10 minutes. Small non‐coding RNA was isolated from plasma samples using the miRNeasy mini kit (Qiagen) and MinElute PCR purification kit (Qiagen) as described by the manufacturer. Next generation sequencing libraries of small RNA were prepared using the Illumina TruSeq Small RNA‐Seq sample preparation kit (Illumina). Small non‐coding RNA was isolated from plasma samples using the miRNeasy mini kit (Qiagen) and MinElute PCR purification kit (Qiagen) as described by the manufacturer. Quantity and quality of isolated small RNA were evaluated by means of bioanalysis  and Agilent RNA 6000 Pico chip kit (Agilent) according to manufacturer's specifications. Next generation sequencing libraries of small RNA were prepared using the Illumina TruSeq Small RNA‐Seq sample preparation kit (Illumina). After adaptor ligation and size selection, the excised product was used for PCR amplification with quality and yield measured with Agilent High Sensitivity DNA Chips (Agilent) in an Agilent 2100 bioanalyzer. Clustering and cDNA sequencing was performed using the Illumina cBot and HiSeq 2500 (Illumina). Library preparation and sequencing were done at GenomeScan, Leiden, the Netherlands (2.3http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Trimmomatic version 0.32P <.05) and fold change ≥ 1.5 or ≤ −1.5 defined significance. After annotation with featureCounts v1.5.0‐p1, filtered out genes where there were counts greater than or equal to 10 in at least one sample. MirPath v3.0Quality of raw sequencing data (.fastq files) was assessed using FastQC v0.11.5 (2.4https://www.R‐project.org/.). For correlation studies, we used the Spearman correlation method. The Hmisc R package was used to compute the significance levels for spearman correlations and corrplot R package was used to create the correlogram. Heat maps were generated using the pheatmap R method and principal component analysis (PCA) was done using the FactoMinerR R package. Venn diagrams were made using the VennDiagram R package.Statistical analysis was performed in the R statistical environment . R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL 33.1S pneumoniae infection  or PCR positive Influenza A or B infection . After pre‐processing, read quality filtering and alignment to the human genome, 180k and 695k unique reads in CAP and non‐infectious control participants were available for further analysis. In CAP, the proportion of reads that uniquely aligned to all different non‐coding RNA species showed preponderance of miRNAs (84.5%). Other small non‐coding RNA species included miscellaneous RNA (misc_RNA: 13.62%), small nucleolar RNA (snoRNA: 1.61%), ribosomal RNA (rRNA: 0.12%), small nuclear RNA (snRNA: 0.10%) and small cajal body RNA (scaRNA: 0.02%)  and 13 CAP patients were used to map circulatory small non‐coding RNA. Table ‐Q3: 51 [) Figure . In non‐Comparing CAP patients to non‐infectious control participants uncovered 44 significantly altered plasma small non‐coding RNA Figure . The lar3.2P =.011)  , MIR501    and SNORD94    against APACHE IV scores uncovered two significant inverse correlations Figure. , specifi) Figure  and SNOR) Figure . Signifi) Figure , includi) Figure  and RN7S) Figure . We also) Figure . Two sig) Figure  and one ) Figure . No corrS pneumoniae or Influenza PCR positive (A or B) infection. Considering adjusted p‐values < 0.05 and fold change thresholds (≥ 1.5 or ≤ −1.5), S pneumoniae infected patients had 38 (18 reduced and 20 elevated) significantly altered small RNAs relative to controls  infection. Notably, snRNAs were only altered in Influenza infected patients.In this study, small non‐coding RNA levels in plasma of CAP‐associated sepsis patients were measured and compared to those of non‐infectious control participants. Relative to controls, patients presented significantly altered levels of primarily mature miRNAs in plasma. Through pathway analysis we learned, that those circulating miRNA may influence cellular mechanisms attuned to cell fate, survival and cell metabolism. Inter‐individual variation in plasma small non‐coding RNA levels in patients can be explained by differences in clinical severity and causative pathogens. We identified circulatory small RNAs that may discriminate patients with In recent years, several studies have evaluated CNAs, in particular miRNAs, as potential diagnostic and prognostic biomarkers for sepsis,CNAs, in particular miRNA, have been shown as a potential novel type of intercellular communicator.Our study has limitations. This interpretation of the study is hindered by its small sample size. Despite this limitation, genome‐wide significant differences in small non‐coding RNA were detected. The choice of source material (plasma) precluded identification of circulating small RNA cells‐of‐origin, which would enhance the functional interpretability of the small RNA profiles herein delineated.In conclusion, we provide a comprehensive map of circulatory small non‐coding RNAs in sepsis caused by CAP relative to controls. Significantly altered circulating miRNA in patients were predicted to target various cellular signalling cascades, particularly immunometabolic, cell proliferation and survival pathways. Our findings prioritize specific circulating small RNAs for functional validation studies as well as potential biomarker studies to discriminate bacterial and viral infections in larger cohorts.Hina N. Khan: Data curation ; Formal analysis ; Writing‐original draft ; Writing‐review & editing . Aldo Jongejan: Formal analysis ; Writing‐review & editing . Lonneke A. van Vught: Data curation ; Writing‐review & editing . Janneke Horn: Data curation ; Writing‐review & editing . Marcus J. Schultz: Data curation ; Writing‐review & editing . Aeilko H Zwinderman: Formal analysis ; Writing‐review & editing . Olaf L. Cremer: Writing‐review & editing . Marc J. Bonten: Writing‐review & editing . Tom van der Poll: Conceptualization ; Funding acquisition ; Writing‐review & editing . Brendon P. Scicluna: Conceptualization ; Data curation ; Formal analysis ; Supervision ; Writing‐original draft ; Writing‐review & editing .The authors have no conflicts of interest to declare.Supplementary MaterialClick here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.


Granulomatous changes were significantly increased in patients with iCIs-related hepatitis compared with DILI and AIH patients (p < 0.05). The ratios of inflammatory cells CD4/CD8, and CD138/CD3 in ICIs-related hepatitis were significantly lower than those in AIH or DILI patients (p < 0.05). We demonstrated that the pre-existing ANA and characteristic liver histology including CD8+ cells dominancy and granulomatous hepatitis could be biomarkers for the diagnosis of iCIs-related hepatitis in the NSCLC with anti-PD-1 therapy.It has been reported that various kinds of immune checkpoint inhibitors (iCIs) could induce immune-related liver damage. We should focus on the programmed cell death-receptor-1 (PD-1) antibody and non-small cell lung cancer (NSCLC) to analyze the characteristics of hepatitis related to iCIs and find factors that could be useful biomarkers for the diagnosis. A single-center retrospective study of 252 NSCLC patients who received PD-1 antibody (nivolumab or pembrolizumab). Some of the biochemical markers and immunological markers were analyzed during PD-1-antibody treatment with or without ALT elevation. Histopathological features were reviewed by a single expert of hepatic pathology focusing on the following features: fibrosis, portal inflammation, lobular inflammation, lobular necrosis. The formation of macro- and micro-granulomas was also evaluated. The frequency of liver damage induced by nivolumab including grade 1 to 4 (ALT) was 41.9% (78/186 patients). The positive rate of anti-nuclear antibody in the nivolumab group with iCIs-related hepatitis was significantly higher than that in the nivolumab group without iCIs-related hepatitis ( Among them, anti-programmed cell death protein 1 (anti-PD-1) is widely used. However, it has been reported that various kinds of immune-related adverse events (irAEs) might be induced by dysregulating the immune system. The Department of Pulmonary Medicine in our institute has reported various kinds of irAEs in advanced non-small cell lung cancers patients treated with anti-PD-12. However, the detailed characteristics of iCIs-related hepatitis induced by treatment with anti-PD-1 have not been clarified. Some groups described that a characteristic liver histology could be observed in patients with liver injury during the treatment with iCIs5. An analysis of immune cell subsets such as CD8 positive cells and CD4 positive cells was carried out in iCIs-related liver injury7. However, the characteristics of liver histopathology and the subsets of immune cells from iCIs-related hepatitis have not been clarified due to the limited number of samples.It has been reported that the most common phenotype of iCIs-related liver damage was iCIs-related hepatitis8. Immune-related liver diseases such as auto-immune hepatitis (AIH) and primary biliary cholangitis (PBC) could be exacerbated by using iCIs. Therefore, an analysis of potential biomarkers to predict the onset of AIH or PBC such as anti-nuclear antibody (ANA) and anti-mitochondria antibody (AMA) might be useful for preventing liver injury induced by iCIs. Some groups including ours have reported that preexisting antibodies and rheumatoid factor were common among patients who developed irAEs9. However, the relation between preexisting antibodies and immune-related liver injury induced by iCIs has not been clarified. In this study, we tried to identify possible biomarkers to detect iCIs-related hepatitis induced by PD-1 antibody in lung cancer patients by analyzing the histopathology of liver and serum markers.In addition to the histopathology of iCIs-related hepatitis, we should consider possible biomarkers that might predict hepatitis after using iCIs. It has been reported that auto-immune disorders such as paraneoplastic syndromes could be potential biomarkers for predicting immune-related liver injury induced by treatment with iCIsThis study was approved by the Ethics Committee of Sendai Kousei Hospital following the ethical guidelines of the 1975 Declaration of Helsinki, and written informed consent was obtained from each individual.. Patients with advanced non-small cell lung cancer (NSCLC) who were treated with anti-PD-1 [nivolumab (3 mg/kg every 2 weeks) (186 patients) or pembrolizumab (200 mg every 3 weeks) (66 patients)] monotherapy at Sendai Kousei Hospital between January 2016 and June 2018 were enrolled in this study. We focused on the patients treated with monotherapy of PD-1 antibody (nivolumab or pembrolizumab) to exclude other factors potentially inducing liver damage such as molecular targeting agents, other iCIs and cell-killing anticancer drugs. In addition to the regimen of treatment, we focused on NSCLC patients to exclude the effects of primary cancer influencing the immune reactions.Patients with iCIs-related hepatitis were diagnosed by the following criteria. Alanine aminotransferase (ALT) elevations were consistent at least two weeks after using PD-1 antibody. The ALT elevations were dominant compared with aspartate aminotransferase (AST) and alkaline phosphatase (ALP) elevations. The patients were excluded if they had liver metastasis, disseminated intravascular coagulation, multiple organ failure, alcohol drinking (> 30 g/day), chronic hepatitis C. All HBsAg positive patients (3 patients) were inactive carriers. Age, sex and tissue type of lung cancer were compared between patients with iCIs-related hepatitis and those without iCIs-related hepatitis. The grade of liver damage was analyzed using common terminology criteria for adverse events (CTCAE) v5.0. We evaluated iCIs-related hepatitis together with ALT elevation using the grading of CTCAE v5.0. Some of the biochemical markers and immunological markers were analyzed during PD-1-antibody treatment with or without iCIs-related hepatitis. Overall survival with or without iCIs-related hepatitis was analyzed using the Kaplan–Meier method.10, portal inflammation (0–3), lobular inflammation (0–3), lobular necrosis (0–3). The formation of macro- and micro-granulomas was also evaluated.Liver biopsies were carried out immediately after consulting the Department of Hepatology about liver damage in some patients treated with PD-1 antibody. The selection of patients who should be undergo liver biopsy was evaluated by two hepatologists to exclude the risk of bleeding and the progression of lung cancer. All biopsy specimens were fixed in formalin and paraffin embedded (FFPE). Sections (3 µm) were then stained with hematoxylin and eosin. Histopathological features were reviewed by a single expert of hepatic pathology focusing on the following features: fibrosis according to Brunt’s criteria (G1–G4)Immunohistochemical staining for T-cell markers , B-cell marker (CD20), and plasma cell marker (CD138) was performed using an automatic immunostainer . The antibodies and sources were as follows: CD3 , CD4 , CD8 , CD20  and CD138 . Immunohistochemical analyses were performed in all of the patients and compared to those with typical auto-immune hepatitis (AIH) (n = 3) and classical drug-induced liver injury (DILI) (n = 3). We also performed double-staining immunohistochemistry, using DAB and fast-red staining, to visualize the proportion of CD4 and CD8 positive lymphocytes in case 17.2 test or independent t test was employed to compare the differences between the patients treated by nivolumab without liver damage and those treated by nivolumab with liver damage using JMP Pro 15.0  consisting of monoammonium glycyrrhizinate, glycine, aminoacetic acid and L-Cystein hydrochloride hydrate administration. However, some of the patients needed steroid or steroid with azathioprine. We could not determine the cell type of lung cancer that could easily induce liver damage by the administration of PD-1 antibody Tables .p = 0.537). The survival curves of HCC patients with or without iCIs-related hepatitis after treatment of pembrolizumab were almost the same .The overall survival with or without iCIs related hepatitis was analyzed using the Kaplan–Meier method and the analysis of log-rank test. The survival curves of HCC patients with or without iCIs-related hepatitis after treatment of nivolumab were almost the same Fig. C. The meame Fig. D. The MSp = 0.0112). Moroever, we could determine a significant difference in the positive rate of anti-nuclear antibody and age between pembrolizumab with iCIs-related hepatitis and that without iCIs-related hepatitis (p = 0.0013 and 0.0121). In addition to the univariate analysis, multivariate analysis was carried out to detect the possible risk factors for iCIs-related hepatitis before the PD-1 treatment. The positivity of anti-nuclear antibody could be detected as a risk factor for iCIs-related hepatitis among the patients treated with nivolumab by multivariate analysis (hazard ratio 2.133: 95%CI 1.085–4.194: p = 0.0281). In addition, the positivity of anti-nuclear antibody and age could be detected as the risk factors for iCIs-related hepatitis among the patients treated with pembrolizumab by multivariate analysis .We analyzed some immunological factors  to predict the susceptibility to liver damage induced by PD-1 antibody Table . The posp < 0.05). Granulomatous changes, especially large granulomatous change with necrosis . The ratios of inflammatory cells CD4/CD8, and CD138/CD3 in ICIs (0.63 and 0.044) were significantly lower than those in AIH (1.89 and 0.21) or DILI (1.08 and 0.39) patients   was seen in the liver biopsy specimens22. Dr. Okamura et al. analyzed how anti-PD-1 therapy could modify the cellular phenotypes of CD8+ T cells to destroy tumors and damage self-tissues23. They indicated that shifts in the cluster composition of autoreactive CD8+ T cells markedly reflected the severity of autoimmunity. Previously, another group reported that granulomatous hepatitis was observed in liver with grade ≥ 3 hepatitis caused by anti-PD-1/PD-L1 or CTLA-4 immunotherapies19. We were able to detect these characteristics of the liver histology when we carried out liver biopsy at the time of the G1 ALT elevation (5 patients / 7 patients). This finding could be helpful for managing patients with anti-PD1 treatment. It is difficult to decide on the discontinuation of PD-1 antibody and the use of steroid since such decisions might affect the survival of the patients.In addition to the topic of host factors that might induce iCIs-related hepatitis, the characteristics of the liver histology were important for the management of iCIs-related hepatitis30. In this study, we included 3 HBsAg positive inactive carrier. Recently, it has been reported that iCIs treatment might induce hepatitis in the HBsAg positive patients33. It has been reported that anti-PD-1 blockade with nivolumab with and without therapeutic vaccination could cause a decline in HBsAg by inducing HBsAg-specific T cells in some patients34. In contrast, the change of immune function, especially, an alteration of CD8+ T cells and Tregs function by the use of iCIs might induce re-activation of HBV36. We should distinguish between the re-activation of HBV and iCIs-related hepatitis. In this study, we did not have experience about the re-activation of HBV during iCIs treatment. Therefore, it has been difficult to determine the characteristics of iCIs-related hepatitis by analyzing HCC patients treated with iCIs, so far. We should analyze HCC patients with various background diseases case by case in the near future.ICIs-related hepatitis could be critical for patients of hepatocellular carcinoma (HCC) since many HCC patients could have limited liver function due to the liver cirrhosis. Previously, we reported that various kinds of factors including hepatitis B virus, hepatitis C virus, and the HCC microenvironment might affect the immune pathogenesis of liver diseases+ cells dominancy and granulomatous hepatitis could be biomarkers for the diagnosis of iCIs-related hepatitis in NSCLC with anti-PD-1 therapy. We should analyze the characteristics of iCIs-related hepatitis in various kinds of malignancies, and iCIs and combination therapy separately to understand the precise pathogenesis of iCIs-related hepatitis induced by specific treatment regimens of iCIs.In conclusion, we demonstrated that pre-existing ANA and a characteristic liver histology including CD8