key: cord-0841738-h1q0e4f5 authors: Laha, Sayantan; Saha, Chinmay; Dutta, Susmita; Basu, Madhurima; Chatterjee, Raghunath; Ghosh, Sujoy; Bhattacharyya, Nitai P. title: In silico analysis of altered expression of long non-coding RNA in SARS-CoV-2 infected cells and their possible regulation by STAT1, STAT3 and interferon regulatory factors date: 2021-02-27 journal: Heliyon DOI: 10.1016/j.heliyon.2021.e06395 sha: ef79d4dbc8e288c864ce66dc325184484db28bd4 doc_id: 841738 cord_uid: h1q0e4f5 Altered expression of long noncoding RNA (lncRNA), longer than 200 nucleotides without potential for coding protein, has been observed in diverse human diseases including viral diseases. It is largely unknown whether lncRNA would deregulate in SARS-CoV-2 infection, causing ongoing pandemic COVID-19. To identify, if lncRNA was deregulated in SARS-CoV-2 infected cells, we analyzed in silico the data in GSE147507. It was revealed that expression of 20 lncRNA like MALAT1, NEAT1 was increased and 4 lncRNA like PART1, TP53TG1 was decreased in at least two independent cell lines infected with SARS-CoV-2. Expression of NEAT1 was also increased in lungs tissue of COVID-19 patients. The deregulated lncRNA could interact with more than 2800 genes/proteins and 422 microRNAs as revealed from the database that catalogs experimentally determined interactions. Analysis with the interacting gene/protein partners of deregulated lncRNAs revealed that these genes/proteins were associated with many pathways related to viral infection, inflammation and immune functions. To find out whether these lncRNAs could be regulated by STATs and interferon regulatory factors (IRFs), we used ChIPBase v2.0 that catalogs experimentally determined binding from ChIP-seq data. It was revealed that any one of the transcription factors IRF1, IRF4, STAT1, STAT3 and STAT5A had experimentally determined binding at regions within -5kb to +1kb of the deregulated lncRNAs in at least 2 independent cell lines/conditions. Our analysis revealed that several lncRNAs could be regulated by IRF1, IRF4 STAT1 and STAT3 in response to SARS-CoV-2 infection and lncRNAs might be involved in antiviral response. However, these in silico observations are necessary to be validated experimentally. Several current reviews are available to show that deregulated lncRNAs are involved in antiviral activity [25, 33, 38] . LncRNAs are observed to modulate (i) viral replication and growth, and (ii) expression of IFNs, and ISGs, possibly by interacting with transcription factors like STAT1, STAT3, NFκB and IRFs. Expressions of several lncRNAs are modulated by treatment with IFNs. Interferon (IFN)-stimulated lncRNA ISR [24] and MIR155HG [22] , IVRPIE [21] suppress IAV replication and growth while LOC100506319/LINC01988/LncRNA-PAAN [26] , TSPOAP1-AS1 [28] , NRAV/ DYNLL1-AS1 (Reviewed in [33] ) and PSMB8-AS1 [23] enhance IAV replication and growth. EGOT and CFAP58-DT/lncITPRIP-1inhibit replication and growth of HCV (Reviewed in [33] ); NEAT1 inhibits replication and growth of HIV [29] and HTNV [39] . LncRNA32/LUARIS inhibits the replication of EMCV, HBV and HCV [40] . NRIR/lncCMPK2 suppresses HCV replication [41] J o u r n a l P r e -p r o o f these findings suggest that IVRPIE is a critical regulator of host antiviral response [21] . Several evidences are available to show that altered expression of the lncRNA in different viral infected cells might involve in modulation ISGs. For examples, CFAP58-DT/lncITPRIP-1 suppresses HCV replication by increasing ISGs [42] . Expressions of several ISGs like IFIT2, IFIT3, IFITM3, OASL, and MxA were reduced in cells overexpressing NRAV/DYNLL1-AS1 [43] . LncRNA32/LUARIS inhibits the replication of EMCV, HBV and HCV, associated with hnRNP U and activates transcription factor ATF2 modulates expression of ISGs [40] . LincRNA-cox2/Ptgs2os2, induced by TLR ligands depending on MyD88 and NF-κB, reduced the expression of some ISGs, like IRF7 and Rasd2. LincRNA-Cox-2/Ptgs2os2 interacts with HnRNP-A/B and hnRNP-A2/B1 to regulate expression of ISGs [44] . TLR ligands suppress the expression of TTC39A-AS1/lincRNA-EPS and enhance expression of ISGs like IFIT2, RASD2, OAS1 and GPB5 by recruiting hnRNPL [45] . In IFN-stimulated hepatocytes, knocked down of NRIR/lncCMPK2 resulted in the transcriptional up-regulation of many ISGs like CMPK2, RASD2, ISG15, CXCL10, IFIT3, and IFITM1 [41] . Expression of several ISGs, including GBP1, ISG15, MXA, BST2, ISG56, IFI6, and IFITM1 was increased in EGOT knockdown cells with or without HCV infection. This result shows that EGOT may promote viral replication by blocking the IFN antiviral response [46] . Many lncRNAs are differentially expressed following IFN stimulation in high throughput studies; specific role of the identified lncRNAs in viral infection has been identified only for a small fraction and reviewed [38] . Expression of BISPR/lncBST2 is increased by IFNA or IFNG J o u r n a l P r e -p r o o f treatment. HCV or HEV infected Huh7 cells and in liver of HCV-infected patients show similar result. Expression of both BISPR/lncBST2 and BST2 was STAT-dependent. Based on the inhibitory effect of BST2 on virion secretion, BISPR/ lncBST2 might be involved in regulating viral infection partially by increasing the expression of antiviral protein BST2 and reviewed [25] . NRIR/lncCMPK2 is stimulated by IFNA or IFNG and possibly regulated by STAT2. Expression of NRIR/ lncCMPK2 is increased in liver of chronic HCV infected patients [41] . IFNA treatment stimulates expression of EGOT. In response to HCV infection, EGOT is activated by NFκB [46] . IAV infection in mouse model enhances expression of interferon (IFN)-stimulated lncRNA (ISR) like ISR2, ISR8. Knockdown of the lncRNAs in cells resulted in increased IAV replication and over expression reduced the viral replication. IFNB treatment induces the lncRNA expression. Induction of the lncRNA by IAV infection was not observed in cells deficient of IFNAR1. LncRNA ISR is regulated by RIG-I-dependent signaling that regulates IFNB production during IAV infection, and has an inhibitory capacity in viral replication [24] . Expression of MALAT1 is increased in HIV infected cells. MALAT1 interacts with chromatin modulator polycomb repressive complex PRC2, releases the core component enhancer of zeste homolog 2 (EZH2) from binding with HIV-1 LTR promoter and removes PRC2 complexmediated methylation of histone H3 on lysine 27 (H3K27me3). Thus MALAT1 may relieve epigenetic silencing of HIV-1 transcription [47] . NRON, highly expressed in resting CD4+ T lymphocytes, involves in HIV latency inducing Tat protein degradation and potentially suppresses the viral transcription by decreasing the cellular abundance of viral transactivator protein Tat [48] . Increased expression of NEAT1 has been observed in HIV, IAV, HTNV, HSV, Japanese encephalitis and rabies virus-infected cells. Mechanism of actions of increased NEAT1 has been worked out in detail in HTNV infected cells. Silencing NEAT1 by siRNA in HTNV infected HUVEC human cells, enhances HTNV replication while exogenous over-expression of NEAT1 effectively inhibited the replication. NEAT1 overexpression increased IFNB production and inhibited HTNV replication. Similar inhibitory effect of NEAT1 on HTNV virus titers is also J o u r n a l P r e -p r o o f observed in animals [39] . This result shows that NEAT1 affects HTNV viral replication through IFNB. Besides, NEAT1 has been shown to regulate expression of IL8 by relocating SFPQ from its promoter and recruiting SFPQ into the paraspeckles [49] . Antiviral activity of NEAT1, observed in several experiments, could be mediated through formation of paraspeckles, a class of membraneless subnuclear bodies, observed in the interchromatin space of mammalian cells. Paraspeckles are RNA-protein structures formed by the interaction between NEAT1, an indispensable structural component, and different class of RNA and many RNA binding proteins. Paraspeckles could sequester paraspeckle-localizing proteins and RNA and modulates their functions outside the paraspeckles, thus acting as molecular sponges [50, 51] . It has been revealed that NEAT1 may promote IFN responses by acting as a positive feedback for RIG-I signaling. Increased NEAT1 by interacting with SFPQ, relocates SFPQ from the promoters of RIG-I and DDX60 to paraspeckles. Thus NEAT1 removes the transcriptional inhibitory effects of SFPQ on RIG-I and DDX60, resulting in increased expression of transcriptional factor IRF7, which in turn induced the expression of IFN and NEAT1 [39] . NEAT1 may also activate IRF3 through formation of multi-subunit complex with HEXIM1. This NEAT1-HEXIM1 complex interacts with cGAS sensor and its partner PQBP1, releases proteins from paraspeckle. Released proteins are recruited to STING and activates IRF3 producing type 1 IFN. These results indicate that NEAT1 has a critical role in the antiviral response of IFN through (i) RIG-I signaling and (ii) cGAS-STING-IRF3 pathway and reviewed [25] . Paraspeckles is induced by IAV, HSV [49] and HIV [29] . Altered expression of lncRNA in SARS-CoV-2 infected cells or tissues from COVID-19 has not been studied to the best of our knowledge. Reusing the RNA sequencing data in GSE147507, we observed that expression of several lncRNAs were altered in SARS-CoV-2 infected cell lines and lung tissue from COVID-19 patients. Interacting protein/gene partners of these deregulated lncRNAs were associated with pathways relevant for viral replication, inflammation and immune function indicating possible role of the lncRNA in SARS-CoV-2 infection. Signature of protein-coding genes in SARS-CoV-2 infected cells in vitro and lung tissue from COVID-19 patients has been reported [8] . In this study, cell lines A549 and Calu3 cells were Besides, sequencing data for RNA samples from lungs tissues from COVID-19 patients and control was also analyzed. We downloaded the raw RNA sequencing data from Gene Expression Omnibus (GSE147507). The data contained the raw read counts obtained for different experimental conditions, divided into distinct series. Each series had two or more replicates in which mock-treated cells served as control. We carried out differential expression analysis between the mock-treated samples and the infected samples as contained in the respective series using Bioconductor package edgeR. Low abundant genes were filtered out from the dataset before carrying out the analysis. We downloaded experimentally determined and curated physical interaction data of noncoding RNA with DNA, mRNA, microRNA, proteins and others from NPInter v4.0 (http://bigdata.ibp.ac.cn/npinter4) [52] . The database catalogues experimentally derived interactions, collected manually from publications in peer-reviewed journals and annotated using other databases like NONCODE, miRBase and UniProt. Annotation of genes in NPInter database was further annotated using NCBI database (ftp://ftp.ncbi.nih.gov/gene/DATA/GENE_INFO/Mammalia/). To identify possible functional implication of the deregulated lncRNAs, we analyzed the interacting protein and mRNA partners of the deregulated lncRNA by enrichment analysis. Enrichment analysis provides information about the overrepresentation of the given genes in particular pathway. We carried out enrichment analysis using online facility at Enrichr at https://amp.pharm.mssm.edu/Enrichr/ [53] . Enrichr is an integrative web-based software application for analysis of a gene-set comparing with various gene-set libraries. Given an input list of genes, it provides enrichment for different libraries like BioPlanet pathways and Gene Ontology (GO) terms for biological process. The online facility uses pathways catalogued in various databases; we have chosen the BioPlanet pathways for comprehensive coverage of different disease conditions including infection disease [54] . To find binding abilities of transcription factors at the putative promoters (-5Kb upstream to +1kb downstream of transcription start sites of the lncRNAs), we utilized the searchable J o u r n a l P r e -p r o o f experimental data from chromatin immune-precipitation followed by sequencing (ChIP-seq) datasets [55] . Binding sites for transcription factors (TFs) IRF1-IRF5, IRF8, IRF9 and STAT1 Table 2 . Binding of different TFs at the putative promoters of MALAT1 and NEAT1 in more than one independent experiment is shown in Figure 5A . Altered expression of the lncRNAs and their possible regulation by transcription factors associated with viral infections [58, 59] indicates that these lncRNAs might be involved in pathogenesis of SARS-CoV-2 infection Figure 6 of the referred paper). LncRNA interacting genes/proteins are associated with many pathways. Among these, 20 MALT1 and NEAT interacting gene/protein was associated and over represented with interleukin-6 signaling pathway. Excessive production of interleukin-6, popularly called J o u r n a l P r e -p r o o f "cytokine storm" has been associated with severe COVID-19 resulting in multi-organ failure [63] . Role of MALAT1 and NEAT1 in this processes, inferred from our in silico analysis, has yet to be validated. Mechanisms by which lncRNA including MALAT1 and NEAT1 can modulate IL-6 and NLRP3 inflammasome have recently been reviewed ( [64] ). Interleukin-2 signaling pathway was significantly enriched with 148 interacting partners of 15 deregulated lncRNA. Decrease in CD8 + T cell and lymphocyte of COVID-19 patients has been shown to mediate through this pathway [65] . It is difficult to infer with high confidence the role of deregulated lncRNAs in IFN response in SARS-CoV-2 infected cells from the in silico analysis. Reduced levels of Type I and III interferon response have been observed in these cell lines, animal models and human lung tissue from the COVID-19 patients. However, a subset of ISGs is induced in these cells [8] . [66]LINC00842 [67] , NEAT1 [56] in high throughput assays could contribute to the ISGs levels as has been observed with LncRNA32/LUARIS [40] . Similar interactions of PART1, TP53TG1, EPB41L4A-AS1, HIF1A-AS2, LINC00174, LINC00662, MALAT1, RMRP [66] and NEAT1 [56] may also modulate ISGs as has been observed with TTC39A-AS1/lincRNA-EPS [45] . It is unknown whether paraspeckles is induced in response to SARS-CoV-2 infection; although expression of NEAT1, the indispensible component of paraspeckles was increased in our analysis as well as various viral infections including IAV, HIV and HTNV. Thus similar mechanism might be operative in SARS-CoV-2 infected cells to increase ISGs. 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