Study On Function And Mechanism Of LncRNA NEAT1 In Host Anti-hantaviral Innate Immune Responses

BackgroundHantaan virus(HTNV),belonging to Bunyavirales and Hantaviridae,is the major causative agent for severe hemorrhagic fever with renal syndrome(HFRS)in China.Since there is no effective therapeutic medicine against HTNV infection,the fatality rate of HFRS can reach 15%.The innate immune system,characterized by interferon(IFN)responses,provides the initial defense against viral invasion.Cellular pathogen recognition receptors(PRRs),including Toll like receptors(TLRs)and RIG-I like receptors(RLR),can detect distinct pathogen-associated molecular patterns(PAMPs)and trigger IFN responses.RIG-I has been shown to recognize hantaviral invasion,but its regulatory process remains unclear.Over the past decades,accumulating evidence suggests that long noncoding RNAs(lncRNAs)play key regulatory roles in innate immune system.However,the involvement of host lncRNAs in hantaviral control remains uncharacterized.In the present work,a new unexplored function of lncRNA NEAT1 in suppressing HTNV replication was found.NEAT1 promoted interferon(IFN)responses by acting as positive feedback for RIG-I signaling.ObjectiveOur study focus on the the function and mechanism of lncRNA in the host innate immune response against HTNV infection.The potential antiviral lncRNA was selected based on the digital gene expression(DGE)analysis,whose role during HTNV-host interaction was investigated by overexpressing and silencing experiments.We hope our study could provide new therapeutic targets for clinical treatment to limit the HTNV infection and promote the HFRS convalescence.Methods and Results 1.lncRNA NEAT1 suppresses HTNV infection by modulating type I IFN signaling 1.1 lncRNA NEAT1 is upregulated by HTNV in a time-and dose-dependent mannerTo study the relationship between HTNV infection and host lncRNA expression,HUVECs were infected with HTNV at an MOI 1,with the mock and Co60-inactivated virus(Co60-HTNV)infection as control groups.DGE was applied at 24 hpi to analyze the differentially expressed genes.DGE results indicate that there is a significant alteration of lncRNA expression after HTNV infection,including lncRNA NEAT1,GAS5 and IPW etc.The FISH and qRT-PCR results hint that lncRNA NEAT1 was indeed upregulated by HTNV at an MOI of 1 in HUVECs,HEK293,A549 and HeLa cells on a time-dependent manner,whose elevation at 72 hpi occurred in an HTNV dose-dependent manner.1.2 Alteration of NEAT1 expression affects HTNV replication by modulating IFN responses in human cellsIn order to clarify the role of NEAT1 in the process of HTNV infection,HUVECs were transfected with different small interfering RNAs(siRNAs)or plasmids targeted for NEAT1,and then infected with HTNV at an MOI of 0.1 or 1.Related experiments were performed at 48 hpi.The knockdown or overexpression efficiencies of NEAT1 with relevant siRNAs or plasmids were confirmed by qRT-PCR and FCM.The results of qRT-PCR and other experiments further demonstrate that NEAT1-2 knockdown suppressed IFN? expression but promoted the HTNV replication,whereas NEAT1 overexpression inhibited IFN? expression but promoted HTNV replication.Notably,the anti-hantaviral effects of NEAT1-2 could be blocked by applying type I IFN neutralizing antibodies.Additionally,qRT-PCR and ELISA results showed that silencing NEAT1-2 could promote IL-8 and CCL5 expression.1.3 NEAT1 silencing has profound effects on innate immune responses after HTNV infection in miceAlthough cell-based experiments revealed that NEAT1-2 is a crucial regulator of innate anti-hantaviral responses,its in vivo function remains unclear.In order to study the effect of NEAT1-2 on the innate immune response against HTNV infection in vivo,Si-NEAT1-2 was injected to C57BL/6J mice through the tail veins,and the NC group mice were treated with scrambled siRNAs(negative control,NC).Mice were challenged with HTNV by intramuscular injection on the second day after injection of siRNA.NEAT1-2 expression levels in liver,kidney and spleen were reduced at 2 dpi as measured by qRT-PCR after intravenously injecteting Si-NEAT1-2.Following experiments were performed at 3dpi to detect mice immune responses in different groups.Compared with NC+HTNV group,body weight loss of Si-NEAT1-2+ HTNV group was observed from 2 dpi to 5 dpi.Likewise,NEAT1-2 knockdown mice showed considerably lower serum IFN? production but higher HTNV levels in the liver,spleen and kidney at 3 dpi as assessed by qRT-PCR or ELISA.Moreover,the virus titers in related organs were higher in the NEAT1-2 silenced group compared with the NC group as detected by ELISA.In addition,reduced inflammatory cell filtration but anabatic tissue injury was found in NEAT1-2 knockdown mice during the early stage of infection by HE staining.Moreover,as FCM results shown,CD11b+F4/80+ M? and CD8+ IFN?+ T cells were reduced in the spleen of NEAT1-2 knockdown mice in comparison to the NC group at 3 dpi.2.Study on the interaction between NEAT1-2 and RIG-I signaling 2.1 RIG-I signaling is crucial for NEAT1 expression after HTNV infectionNEAT1 expression could suppress HTNV infection,while it is still opaque that how HTNV activated NEAT1 transcription.Interestingly,qRT-PCR results suggest overexpression of the S or M segment of HTNV in HUVECs failed to induce NEAT1-2 expression,suggesting that NEAT1 transcription was closely related to live viral replication.Meanwhile,NEAT1-2 expression could not be induced by stimulation with different types of IFNs or cytokines.Using RIG-I and TLR4 deficient cell lines,RIG-I was confirmed to be indispensable for NEAT1-2 induction after HTNV infection.Additionally,silencing IRF7 also inhibited the HTNV-induced NEAT1-2 transcription.2.2 NEAT1-2 regulates HTNV-induced IFN? production by promoting RIG-I and DDX60 expressionTo investigate the regulatory mechanisms of NEAT1-2 on IFN? expression,we examined the effects of NEAT1-2 knockdown on the expression of multiple PRRs(TLR1,TLR2,TLR3,TLR4,RIG-I,DDX60,MDA5)upon HTNV infection through qRT-PCR or Western Blot.Both qRT-PCR and Western Blot results indicate that NEAT1-2 knockdown or overexpression remarkably suppressed or promoted RIG-I and DDX60 expression,respectively.RIG-I and DDX60 were knocked downed or overexpressed in HUVECs,and then qRT-PCR and dual luciferase reporter assay were performed to assess IFN? mRNA transcrition and IFN? promoter activation after HTNV infection,respectively.Ectopic expression of either RIG-I or DDX60 promoted IFN? expression and inhibited viral replication,whereas overexpression of both resulted in superior antiviral effects as assessed by q RT-PCR,indicating that efficient anti-HTNV responses might depend on the interactive effects of DDX60 and RIG-I.More importantly,RIG-I or/and DDX60 overexpression enhanced HTNV-induced IFN? expression as measured by Western Blot or dual luciferase reporter assay,showing that they had synergistic effects on IFN? production.In addition,we designed a series of siRNAs targeting RIG-I and DDX60,and we selected the si-RIG-I-2 and siRNA-DDX60-1 with the highest knockdown efficiency by qRT-PCR in HUVECs.Silencing RIG-I or DDX60,or both of them could suppress IFN? and promote HTNV replication with similar experiments as foresaid.2.3 NEAT1-2 promotes RIG-I and DDX60 expression by relocating SFPQ after HTNV infectionNEAT1 was found to interact with SFPQ(the splicing factor proline-glutamine rich)by RIP after HTNV infection,indicating that the modulatory effects of NEAT1 might be involved in SFPQ.Interestingly,the protein level of SFPQ,as well as another paraspeckle-forming constituent,NONO,remained unchanged after HTNV infection or after NEAT1 overexpression and knockdown as detected by Western Blot.However,IFA results showed that SFPQ became centralized rather than diffused in the nucleus after HTNV infection.The Co-IP results indicated enhanced interaction of SFPQ and NONO,showing excess formation of paraspeckles in the nucleus and re-localization of SFPQ.The qRT-PCR results showed that SFPQ knockdown could inhibit HTNV replication,which might have been related to the increasement of RIG-I and DDX60.3.NEAT1-2 expression in patients' monocytes is inversely correlated with HFRS severityIn order to study the relationship between NEAT1-2 and HFRS disease process,we collected the blood samples from 86 HFRS patients of different disease stages or severity,with 24 healthy human blood samples as control.The NEAT1-2 expression levels in the peripheral blood mononuclear cells were measured through qRT-PCR.The qRT-PCR results showed that NEAT1 levels in the acute(febrile/ hypotensive/ oliguric)stage were overtly higher than those in the diuretic/convalescent stage or in healthy individuals.In the early disease stage,HFRS patients in the mild/moderate group maintained higher NEAT1 levels compared with those in the severe/critical group.The relationship between NEAT1-2 level and patients' serum viral loads,peak serum creatinine values,lowest platelet counts,and peripheral white blood cells were analyzed.Here,statistical analysis revealed significant negative associations of NEAT1 with the plasma viral load and peak creatine level.In addition,the NEAT1 level was clearly positively correlated with the lowest platelet count.These data suggested that patients with higher NEAT1 levels tended to have less tissue injury and better outcomes.Nevertheless,no relationship of NEAT1 levels with the peak white blood cell count was found.ConclusionIn this study,we identified the lncRNA,NEAT1,as a vital antiviral modulator.HTNV infection activated NEAT1 transcription through the RIG-I-IRF7 pathway,whereas NEAT1 removed the transcriptional inhibitory effects of SFPQ by relocating SFPQ to paraspeckles,thus promoting the expression of RIG-I and DDX60.RIG-I and DDX60 had synergic effects on IFN? production,inhibiting HTNV infection and replication.Additionally,IL-8 or CCL5 was also secreted in response to NEAT1,which influenced immunocyte activation after HTNV infection.Notably,NEAT1 levels in peripheral blood monocytes were closely related with disease development and severity.Taken together,our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection,providing another layer of information about the role of lncRNAs in controlling viral infections.