Perversely expressed long noncoding RNAs can alter host response and viral proliferation in SARS-CoV-2 infection

Background: Regulatory roles of long noncoding RNAs (lncRNAs) during viral infection has become more evident in last decade, but are yet to be explored for SARS-CoV-2. Materials & methods: We analyzed RNA-seq dataset of SARS-CoV-2 infected lung epithelial cells to identify differentially expressed genes. Results: Our analyses uncover 21 differentially expressed lncRNAs broadly involved in cell survival and regulation of gene expression. These lncRNAs can directly interact with six differentially expressed protein-coding genes, and ten host genes that interact with SARS-CoV-2 proteins. Also, they can block the suppressive effect of nine microRNAs induced in viral infections. Conclusion: Our investigation determines that deregulated lncRNAs in SARS-CoV-2 infection are involved in viral proliferation, cellular survival, and immune response, ultimately determining disease outcome.

Family with sequence similarity 106   Analyzing the RNA-seq data, nine lncRNAs were found to be upregulated, while twelve were downregulated. Gene expressions are presented in log2 fold change (compared with uninfected control cells) value color coded heatmap. Color toward red indicates more upregulation and color toward green indicates further downregulation, while yellow color indicates absence of differential expression.
Identification of virally-induced miRNAs miRwayDB [40] provided information about human miRNAs that were previously identified as involved in viralmediated diseases. Additionally, information about host miRNAs involved in viral infection as identified by Girardi et al. [41] and miRNAs involved in viral acute respiratory infections as described by Leon-Icaza et al. [42] were retrieved. These miRNAs were manually curated for their involvement in viral pathogenesis and possible connection to SARS-CoV-2.
Extraction of microRNA targets Gene targets for the curated set of miRNAs were retrieved from experimentally validated miRNA-gene interactions from miRTarBase [43] and filtered for the DE protein-coding genes. DIANA-LncBase v3 [44] was used to retrieve miRNAs that target the DE lncRNAs, considering only high-confidence interactions. Combining the lists led to identification of the miRNAs that target both DE protein-coding genes and lncRNAs. The set of upregulated target genes for each miRNA were analyzed using NetworkAnalyst 3.0 [45] tools for functional over-representation and network enrichment. miRNAs that mostly targeted upregulated genes were finally selected.

Construction of biological networks
Networks depicting the interactions between DE lncRNAs, DE genes, viral proteins and curated miRNAs were built using Cytoscape v3.7.2 [37]. Gradient coloring scheme was used to depict the change in expression of DE genes.

lncRNAs differentially modulated in SARS-CoV-2 infection
To investigate the probable deregulation of lncRNAs in SARS-CoV-2 infection, first we have analyzed the 24 h postinfection transcriptome data of SARS-CoV-2 infected NHBE cells. Analyzing the RNA-seq data led to identification of 687 DE genes (Supplementary file 1). Intriguingly among those DE genes, we discovered 21 lncRNA genes, nine of them upregulated while 12 were downregulated ( Figure 1).
future science group www.futuremedicine.com Table 1. Differentially expressed genes, their encoded protein's functions and associated interacting long non-coding RNAs.

Gene name Protein function lncRNA interactor Interaction Type
Ref.
ADAR ADAR works as a regulator in RIG-I/MDA5 mediated induction of IFN-␣/␤ pathways and controls innate immune response against dsRNA in the cell. It exerts an antiviral effect on HCV, but works in favor of VSV, MV, HDV and type 1 HIV-1.

EDN1
Endothelin-1 is an endothelium-derived vasoconstrictor peptide that belongs to the endothelin/sarafotoxin family. The peptide works as a potent vasoconstrictor and its cognate receptors are therapeutic targets in the treatment of pulmonary arterial hypertension. It is involved in downstream GPCR-controlled signaling.

UGDH-AS1
RNA-RNA [55,56] KYNU KYNU gene encodes Kynureninase enzyme, which catalyzes the cleavage of L-kynurenine and L-3-hydroxykynurenine into anthranilic acid and 3-hydroxyanthranilic acid, respectively. Among its functional pathways are NAD metabolism and tryptophan utilization.  lncRNAs interact with several differentially modulated proteins in SARS-CoV-2 infected cells We now sought to elucidate the putative effects of these deregulated lncRNAs in SARS-CoV-2 infection. In order to achieve that, we built a network of the deregulated lncRNAs along with their interacting protein coding target genes. Among the differentially expressed protein-coding genes, six were found to interact with the DE lncRNAs (Table 1). Among them, there are direct RNA-RNA interactions for four genes and RNA-protein interactions for the rest (Figure 2). These lncRNA interacting protein coding genes have potential roles during the viral infections (Table 1). Upregulated ADAR interacts directly with downregulated KCNQ1OT1 and GAS6-AS1, and upregulated MEG3 and INHBA-AS1 (Figure 2A). ADAR protein also interacts with UPF1 and MOV10 -two proteins that are also interactors of other DE lncRNAs. EDN1 is found to be upregulated in SARS-CoV-2 infection, which interacts directly with downregulated UGDH-AS1 ( Figure 2B). EDN1 protein can also interact with PRKAR2A, a protein that interacts with KCNQ1OT1 and viral protein Nsp13. KYNU mRNA interacts directly with FAM230J, which was downregulated ( Figure 2C) [74]. MALL, a gene found upregulated in SARS-CoV-2 infection, interacts directly with AC006058.1, which was also upregulated ( Figure 2D). Upregulated TLR2 interacts directly with downregulated KCNQ1OT1 ( Figure 2E). YWHAG protein interacts directly with KCNQ1OT1, SNHG8, GAS6-AS1, all downregulated ( Figure 2F).
Protein interactors of SARS-CoV-2 viral proteins also interacted with DE lncRNAs Gordon et al. investigated possible protein-protein interactions (PPI) between SARS-CoV-2 proteins and host proteins and identified 332 high-confidence interactions [34]. We retrieved these interactions from BioGRID [35], which are provided in supplementary file 2. We wanted to link whether these interacting proteins can also interact with the DE lncRNAs in SARS-CoV-2 infection. Subsequently, we constructed a network with the DE lncRNAs and their interacting host proteins, which also bind with viral proteins. Among these 332 proteins, we found ten such proteins that also interact with the DE lncRNAs (Table 2). Among them, seven proteins interacted at the protein-RNA level, whereas the rest were RNA-RNA interactions. SARS-CoV-2 proteins M (Membrane), N (Nucleocapsid), Nsp8, Nsp12, Nsp13, and ORF8 were found to interact with these host proteins ( Figure 3).
Gene name Protein function SARS interactor protein lncRNA interactor Interaction type Ref.

AKAP8L
AKAP8L (A-kinase anchor protein 8-like) protein probably plays a role in CTE-mediated gene expression by association with DHX9 by increasing nuclear unspliced mRNA export. In EBV infected cells, it may target PRKACA to nuclear sites containing EBNA-LP (an EBV protein) to modulate transcription from specific promoters. In synergy with DHX9, it can activate the CTE-mediated gene expression of type D retroviruses. In case of HIV-1 infection, it is involved in the DHX9-promoted annealing of host tRNA (Lys3) to viral genomic RNA as a primer in reverse transcription.
EXOSC5 is a noncatalytic component of the RNA exosome complex, which has 3 to 5 exoribonuclease activity and participates in a multitude of cellular RNA processing and degradation events.
GDF15 is a member of the GDNF family which binds to GDNF family receptor ␣-like (GFRAL) protein, a transmembrane receptor exclusively expressed in the hind brain. The protein is expressed in a broad range of cell types, acts as a pleiotropic cytokine and is involved in the stress response program of cells after cellular injury. Increased protein levels are associated with disease states such as tissue hypoxia, inflammation, acute injury and oxidative stress.

HECTD1
HECTD1 is an E3 ubiquitin-protein ligase which accepts ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfers the ubiquitin to targeted substrates and mediates 'Lys-63'-linked polyubiquitination of HSP90AA1, which leads to its intracellular localization and reduced secretion. The protein is involved in class I MHC mediated antigen processing and presentation and innate immune system.
LARP4B encodes a La-module containing factor that can bind AU-rich RNA sequence directly and promotes mRNA accumulation and translation. It was deemed a candidate tumor suppressor gene in glioma, as it was consistently decreased in human glioma stem cells and cell lines compared with normal neural stem cells. LARP4B overexpression strongly inhibited cell proliferation by inducing mitotic arrest and apoptosis and CDKN1A and BAX were upregulated. Nsp12

UGDH-AS1 SNHG8
Protein-RNA [85,86] LARP7 LARP7 works as a negative transcriptional regulator of polymerase II genes, acting by means of the 7SK RNP system. This snRNP complex inhibits a cyclin-dependent kinase, positive transcription elongation factor b, which is required for paused RNA polymerase II at a promoter to begin transcription elongation. Nsp8
UPF1 protein, an RNA-dependent helicase and ATPase, is required for NMD of mRNAs containing premature stop codons. It is recruited to mRNAs upon translation termination by release factors to stalled ribosomes together with the SMG1C protein kinase complex to form the transient SURF MOV10 is a 5 to 3 RNA helicase contributing to UPF1 mRNA target degradation by translocation along 3 UTRs and is involved in miRNA-mediated gene silencing by the RISC. It plays a role for both miRNA-mediated translational repression and miRNA-mediated cleavage of complementary mRNAs by RISC. UPF1-MOV10 is involved in antiviral activity through both NMD pathway and IFN induction.
Protein-RNA [93][94][95][96][97][98] PRKAR2A PRKAR2A is the cAMP-dependent protein kinase type II-alpha regulatory subunit, which works as the regulatory subunit of the cAMP-dependent protein kinases involved in cAMP signaling in cells. Upregulated targets of virally-induced microRNAs might be protected by competing lncRNAs ceRNA hypothesis illustrates that lncRNAs and other RNA molecules harboring miRNA response elements can suppress the expression and biological function of each other by competing for miRNAs that can bind to the complementary regions, thus regulating miRNA-mediated gene silencing of the target genes [17,99]. To reveal if such networks exists in SARS-CoV-2 infected cells, a list of miRNAs induced in viral infection was curated from miRwayDB database [40], Girardi et al. [41], and Leon-Icaza et al. [42]. Among them, few were found to target genes upregulated in the cells, which were apparently not targeted as DE lncRNAs were targeted simultaneously ( Figure 4). The names and functions of all identified target genes of the selected miRNAs that were differentially expressed in the infected cells are provided in supplementary file 3.

Involvement of lncRNA may modulate important cellular pathways
Cell survival can be beneficial for continued viral replication and growth, but apoptosis is also necessary for further spread. Pathways related to cell survival are targeted by virally-induced microRNAs. IL-6/JAK/STAT3 signaling acts in favor of cell survival [100]. Upregulation of IL-6 during certain viral infections may promote virus survival and/or exacerbation of clinical disease [101]. Among let-7c-5p targets and let-7f-5p targets, four upregulated genes, IL6, MYC, PDGFB and STAT2 belong to JAK-STAT signaling pathway. KCNQ1OT1 can act as a sponge for MYC gene, a target of miR-185-5p along with these miRNAs ( Figure 4C [102]). Also, both let-7c and let-7f target IL-6, and absence of this interaction may lead to cell transformation progressing from initial inflammation, implying cell survival [103].
Wnt signaling pathway is known to regulate apoptosis through a variety of mechanisms [104] and it can respond to viral infections through modulation of β-catenin stabilization [105]. MYC, CSNK1E and FOSL1 belong to Wnt signaling pathway and are targeted by miR-185-5p. FOSL1 is also targeted by miR-574-5p ( Figure 4I).
Activation of PI3K-Akt signaling is a known viral strategy to delay apoptosis and prolong viral replication, as seen in acute and persistent infection [106]. Three upregulated genes, EFNA1, MYC and VEGFA, targeted by miR-185-5p, and two upregulated genes targeted by miR-222-5p, ITGA5 and TGFA, belong to PI3K-Akt signaling pathway ( Figure 4). Also, miR-185 was found to inhibit cell proliferation and induce apoptosis by targeting VEGFA directly in von Hippel-Lindau-inactivated clear cell renal cell carcinoma [107].
NF-κB signaling pathway is involved in upregulating various pro-inflammatory genes that encode cytokines and chemokines [108]. It is a major regulator of antiviral response, and NF-κB activation pathways are manipulated by viruses to avoid cellular mechanisms that eliminate the infection [109,110]. Two upregulated gene targets of let-7c-5p and let-7f-5p, CXCL8 and LYN, and two upregulated targets of miR-574-5p, TRAF3 and TGFBR2 is involved in NF-κB signaling pathway (Figure 4). MEG3 can act as a sponge to promote upregulation of TRAF3 [111] and TGFBR2 [112]. BCL3, a target of miR-197-5p, is involved in regulation of cell proliferation and participates in NF-κB signaling pathway ( Figure 4D).

Discussion
Long noncoding RNAs are regulators and modulators of complex cellular pathways and viral infection is no exception [19,20]. Dysregulation of lncRNA expression and interaction, either directly with viral RNA, or indirectly with host RNA, affect the progression of viral infection [12]. LncRNAs can work as a sponge for miRNA, bind protein as a competitive inhibitor, inhibit PPI, influence post-translational modification, or affect the activity of target protein. They can modulate viral life cycle, regulate innate immune response or assist adaptive immunity [18,21]. In our exploration of the deregulated lncRNA in SARS-CoV-2 infected NHBE cells, all of these functionalities come to fore. In our study, we find lncRNA-interacting DE protein-coding genes and SARS-CoV-2 protein interactors to be involved in various pathways. Both RNA-RNA and RNA-protein interactions were present, indicating a complex interplay between the components involved. Additionally, these lncRNAs may perform as sponges to protect upregulated genes that would otherwise be targeted by virally-induced miRNAs. Our findings shed light future science group www.futuremedicine.com on the possible role of DE lncRNAs in specific processes involved with viral infection, proliferation and cellular response. SARS-CoV-2 Nsp7-Nsp8-Nsp12 proteins interact to form a multi-subunit RNA-synthesis complex, where Nsp12 works as the RNA-dependent RNA polymerase. EXOSC5, interactor of Nsp8 protein, is a noncatalytic component of the RNA exosome complex. RNA exosome complex is involved in 3 processing of various stable RNA species and is crucial for RNA quality control in the nucleus [114], thus Nsp8 binding may be fundamental to diminishing the capacity of exosome to act against viral mRNAs. Antiviral drug remdesivir has been predicted to target and disassemble this complex [115,116]. Interaction of EXOSC5 protein with KCNQ1OT1 and SNHG8 can modulate this interaction. The putative RNA-RNA interaction between upregulated AC006058.1 and MALL mRNA, a gene involved in cholesterol homeostasis and membrane trafficking, can exert an influence in maturation of SARS-CoV-2, an enveloped virus. FAM230J, which has no reported function, is downregulated in the infected cells. Its absence may activate the upregulated KYNU, which is involved in metabolite biosynthesis pathways, through lack of RNA-RNA interaction.
Host RNA-binding proteins that interact with SARS-CoV-2 proteins are involved in regulating viral transcription and mRNA stability. This is evident through the interaction of SARS-CoV-2 nucleocapsid (N) protein with UPF1 and MOV10 proteins. In case for murine hepatitis virus, a model coronavirus, the N protein carries out this interaction to inhibit nonsense-mediated decay (NMD) of viral mRNAs containing multiple stop codons, thus favoring viral mRNA transcription. NMD pathways recognized cytoplasmic viral mRNAs as a substrate for degradation, but viral replication induced the inhibition of the NMD pathway through N protein [117]. In human T-lymphotropic virus type 1-infected cells, viral protein tax bound to components of NMD pathways, including UPF1, to inhibit the process [118]. SARS-CoV-2 Membrane (M) protein interacts with AKAP8L, which assists in viral infection progression through favoring transcription. Both LARP7 (nsp8 interactor) and LARP4B (nsp12 interactor) are RNA-binding proteins involved in RNA transcription regulation. Interaction of these RNA-binding proteins with the DE lncRNAs may be crucial for viral mRNA transcription and stability against cellular defense.
ADAR can be regarded as the 'Editor-in-Chief ' of innate immunity against viral infection [119]. MEG3 was found to act as a biomarker and regulate cell functions by targeting ADAR in colorectal cancer. The cells overexpressing MEG3 exhibited increased ADAR expression, and downregulation of MEG3 was found in colorectal cancer tissues, cell lines and serum [120]. In the infected cells, MEG3 upregulation may lead to ADAR overexpression, which could have been favorable to the virus, as evidenced in influenza A [121]. INHBA-AS1 upregulation in virus-infected cells may also contribute to cell survival through interaction with ADAR, as evidenced in gastric cancer [122] and oral squamous cell carcinoma [123]. As the role of ADAR as a controlling element in cellular response to viral infection is paramount, its regulation by MEG3 and interactions with lncRNAs in SARS-CoV-2 infected cells may influence progression of the disease.
Cell survival can be both beneficial to virus and be inhibitory. Viruses need the cell to survive for a certain period to undergo replication, but they also need apoptosis to exit the cell and infect others. Thus, cell survival and apoptosis becomes a key indicator of pathogenesis. Among lncRNAs, downregulated SNHG8 has possible link to apoptosis and cell death [124], as does KCNQ1OT1 [125][126][127][128]. Unlike both, downregulation of GAS6-AS1 may be linked with cell proliferation and survival [129,130]. All three interact with YWHAG, which is involved in signal transduction. IL-6/JAK/STAT3 signaling, Wnt signaling pathway, and PI3K-Akt signaling, involved in cell survival, are also protected by probable ceRNA function by lncRNA.
Cellular response to viral infection can make or break the progression of viral life cycle. Innate immune response is inevitable, but the complex interactions underlying its activation and effect may have been the target of lncRNAs. HECTD1 is an E3 ubiquitin-protein ligase that interacts with Nsp8. As it is involved in class I MHC mediated antigen processing and presentation and innate immune system, the binding may lead to modulation of that response. In case of HECTD1 mRNA, absence of RNA-RNA interaction with downregulated KCNQ1OT1 lncRNA can facilitate the response. PTGES2 gene was upregulated and also found to interact with SARS-CoV-2 Nsp7 protein. As it is involved in innate immune system and signaling pathways, this binding may exert an indirect influence. TLR2, as part of the innate immune response, has been connected to antiviral action against multiple viral infections [131][132][133][134]. KCNQ1OT1, the lncRNA having putative RNA-RNA interaction with TLR2, was found to attenuate sepsis-induced myocardial injury via regulating miR-192-5p/XIAP axis. Downregulation of the lncRNA advanced cardiac injury by allowing miR-192-5p to target XIAP [135]. XIAP functions in the inhibition of apoptosis, whereas TLR2 is known to promote apoptosis. The downregulation of KCNQ1OT1 in infected cells thus will promote apoptosis, in accordance with its interaction with overexpressed TLR2 mRNA. Virally-induced microRNAs can also be involved in targeting pathways related with inflammation and host defense. Response by NF-κB signaling pathway against the viral infection is probably protected by lncRNA against inhibition by these miRNAs.
DE lncRNAs also had probable involvement in regulating cellular processes, including signaling. The downregulation of UGDH-AS1 can be related to EDN1 overexpression, which can be crucial in vasoconstriction, leading to severe symptoms, as seen in SARS-CoV-2 infections [136]. SARS-CoV-2 Nsp13 protein works as a viral helicase and interacts with MIPOL1 and PRKAR2A. Function of MIPOL1 is unclear, whereas PRKAR2A is involved in cAMP-mediated signaling, which is the probable target for modulation by Nsp13. The RNA-RNA interactions of downregulated KCNQ1OT1 with the mRNAs of these two genes may have implications on viral infection progression. Additionally, MAPK pathway is probably regulated through lncRNA involvement.
Growing evidence indicates that the ceRNA regulation mechanism plays a role in disease progression and drug efficiency [137][138][139][140]. We checked if any of the DE lncRNAs in the infected cells were exerting such effects to ensure a particular miRNA does not downregulate a gene. In turn, the upregulated genes play a crucial role in viral replication, disease progression and immune response. Although some of these miRNAs also target a few downregulated genes, their decrease in expression may be controlled by separate mechanisms. There were previous instances where some of these miRNAs were blocked by a specific lncRNA that was deregulated in the infected cells. For instance, KCNQ1OT1 was found to regulate Rab14 expression, a target of miR-185-5p, in oral squamous cell carcinoma cells as a ceRNA [141]. MEG3 aggravated palmitate-induced insulin resistance by regulating miR-185-5p/Egr2 axis as a ceRNA in insulin-resistant hepatocytes [142]. Additionally, miRNA let-7c-5p was found to negatively regulate NLRC5 expression in ethanol-induced hepatic injury, where MEG3 functioned as a ceRNA against the miRNA [143]. MEG3 acted as an endogenous sponge to suppress the function of miR-223 and to increase NLRP3 expression and enhance endothelial cell pyroptosis in ox-LDL-treated human aortic endothelial cells [144]. KCNQ1OT1 plays the role of a ceRNA to regulate MYC, an upregulated target of let-7c-5p, let-7f-5p and miR-185-5p [102]. We can deduce that these lncRNAs can be involved in a similar role in infected cells.

Conclusion
Our findings explore the molecular footprint of SARS-CoV-2 infection from the lens of lncRNAs. The integrated analysis has linked multiple actors in the complex interplay of molecules in exacerbating the infection and identified possible drug targets. We trace the involvement of lncRNAs with cellular behavior in this situation and illuminate their role in cell survival, viral replication and immune defense. The lethality and swift transmission of this virus is entwined with the deregulated cellular environment, and lncRNA regulation is crucial for understanding its parameters. Our results could provide insights for scheming some novel RNA therapeutics during the lacking of effective cure option.
Future perspective SARS-CoV-2 has wreaked havoc across the world within a short period. This virus may persist in the population and assume a seasonal nature. The dysregulation in cellular level is ultimately responsible for the disease, and lncRNAs are crucial pieces of the puzzle. The lncRNAs can control cellular response and determine disease outcome. Our elucidation of these molecular connections can deepen our understanding of the virus and the nature of its infection. Effective treatment methods of the future will certainly rely on this knowledge.
• These lncRNAs are involved in cell survival and regulation of gene expression.
• Six differentially expressed genes interact with these lncRNAs. • Ten proteins that interact with SARS-CoV-2 viral proteins also interact with these lncRNAs. • These protein-coding genes function in cellular signaling, metabolism, immune response and RNA homeostasis.
• The lncRNAs can act as competing endogenous RNAs for virally induced microRNAs.
• Effect of these microRNAs center on host defense and cell survival.

Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.