Long non-coding RNA NONSUST006715.1 suppresses Japanese encephalitis virus proliferation in PK-15 cells


 Background: Japanese encephalitis virus is a mosquito-borne neurotropic flavivirus that causes acute viral encephalitis in humans. Pigs are crucial amplifier host of JEV. Recently, increasing evidences have shown that long non-coding RNAs (lncRNAs) play important roles in virus infection. Methods: The JEV proliferation was evaluated after overexpression or knockdown of lncRNA-NONSUST006715.1 using western blotting and reverse-transcription polymerase chain reaction (RT-PCR). C-C chemokine receptor type 1 (CCR1) was found to regulate the expression of lncRNA-NONSUST006715.1 by inhibitors screen. The expression of lncRNA-NONSUST006715.1 was detected using RT-PCR after overexpression or knockdown of transcription factor SP1. In addition, the enrichments of transcription factor SP1 on the promoter of lncRNA-NONSUST006715.1 were analyzed by chromatin immunoprecipitation. Results: In this study, we demonstrated that swine lncRNA-NONSUST006715.1 could suppress JEV proliferation in PK-15 cells. We also found that CCR1 inhibited the expression of lncRNA-NONSUST006715.1 via the transcription factor SP1. In addition, knockdown of CCR1 could upregulated the expression of SP1 and lncRNA-NONSUST006715.1, resulting in resistance to JEV proliferation. Conclusions: These findings illustrate the importance of lncRNAs in virus proliferation, and reveal how this virus regulates lncRNAs in host cells to promote its proliferation.

function non-coding RNAs, lncRNAs have received increasing attention in antiviral-related research.
LncRNA MEG3 has been reported to be inhibited by RSV infection, whereas MEG3 inhibits RSV infection of respiratory epithelial cells by inhibiting the TLR4-dependent p38 MAPK and NF-κB signaling pathways [7]. LncRNA also plays an important role in the natural immunity of pigs to blue ear virus [10], and can be used as a diagnostic marker and therapeutic target for liver damage caused by dengue virus infection [11,12]. Recently, certain viruses have been shown to inhibit cell metabolism-related enzymes, such as GOT2 (mainly enriched in mitochondria), by activating the NF-κB signaling pathway and thereby promoting viral replication and proliferation [13,14].
JEV typically invades the central nervous system after infection, and can trigger a wide range of natural immune responses via substantial viral replication, leading to nerve cell necrosis [15]. The neuroin ammation caused by JEV is mainly related to the loss of control of microglia, which release in ammation-related cytokines and chemokines such as IL-1β, IL-6, TNFα, and MCP1, causing an irreversible in ammatory response and leading to neuronal necrosis. Several studies have found that microglia can also serve as long-term JEV containers [16]. Although many studies have investigated the molecular mechanisms by which micro RNAs (miRNAs) regulate JEV replication and proliferation [17][18][19][20], the molecular mechanism of the effect of lncRNA on JEV proliferation remains to be explored. Recent studies have shown that lncRNA Malat1 was signi cantly upregulated in JEV-infected mouse Neuro2a cells via the PERK endoplasmic reticulum stress signaling pathway [21], and that silencing lncRNA E52329 and N54010 can regulate the in ammatory response in JEV-infected mouse microglia cells by reducing the phosphorylation levels of JNK and MKK4 [22].
In this study, we aimed to explore the role of lncRNA-NONSUST006715.1 in the proliferation of JEV. To this end, PK-15 cells were transduced with overexpression vector and antisense oligonucleotides (ASO) of lncRNA-NONSUST006715.1 to evaluate its potential role in the proliferation of JEV. We found that lncRNA-NONSUST006715.1 could inhibit the proliferation of JEV, and CCR1 as a key regulator of JEV proliferation was involved in the expression regulation of lncRNA-NONSUST006715.1 via transcript factor SP1.

Results
Overexpression of lncRNA-NONSUST006715.1 inhibited JEV proliferation In our previous studies, we screened four lncRNAs involved in innate immunity to determine the role of lncRNA in JEV proliferation [23]. In the present study, we focused on the effect of lncRNA-NONSUST006715.1 on JEV proliferation. We performed reverse-transcription polymerase chain reaction (RT-PCR) analysis to detect lncRNA-NONSUST006715.1 levels following JEV infection. We found that lncRNA-NONSUST006715.1 was signi cantly increased at 36 h post-infection, but signi cantly decreased at 48 h post-infection. LncRNA exhibited an obvious response to JEV infection (Fig. 1A). In the previous studies, we also found that 36-48 h is a crucial stage for the proliferation of JEV. To explore the function of lncRNA-NONSUST006715.1 in JEV proliferation, RT-PCR was performed to detect the effect of lncRNA-NONSUST006715.1 overexpression at 48 h after transfection with vectors, and found that it was signi cantly upregulated compared to the control group (Fig. 1B). Western blotting results clearly showed that lncRNA-NONSUST006715.1 overexpression suppressed JEV-NS3 protein levels at 48 h postinfection (Fig. 1C). RT-PCR results showed that lncRNA-NONSUST006715.1 overexpression suppressed the JEV mRNA levels at 24 h and 48 h post-infection (Fig. 1D).
Knockdown of lncRNA-NONSUST006715.1 promoted JEV proliferation To verify the function of lncRNA-NONSUST006715.1 in JEV proliferation, we used ASO to knock down lncRNA-NONSUST006715.1 expression. RT-PCR transcription analysis showed that lncRNA-NONSUST006715.1 transcript levels were signi cantly decreased at 24 h by transfection of ASO1, ASO2, and ASO3 into PK-15 cells ( Fig. 2A). ASO1 was more e cient for knocking down lncRNA-NONSUST006715.1 transcript levels; therefore, we used ASO1 in subsequent experiments. We performed RT-PCR analysis to examine the effects of ASO1 at different time points; lncRNA-NONSUST006715.1 transcript levels were signi cantly decreased at 24, 36 and 48 h after ASO1 transfection into PK-15 cells (Fig. 2B). Western blotting results showed that the level of JEV NS3 protein increased signi cantly 36 hours after knockout of lncRNA-NONSUST006715.1 gene, and the increase was about 3-fold that of the control group, which indicated that lncRNA-NONSUST006715.1 inhibited the proliferation of JEV in PK-15 cells (Fig. 2C). RT-PCR results showed that lncRNA-NONSUST006715.1 knockdown promoted the JEV mRNA levels at 36 h post-infection (Fig. 2D). inhibitor) suppressed lncRNA-NONSUST006715.1 transcript expression, whereas the inhibitor ZK811752 (10 μM, C-C chemokine receptor type 1 (CCR1) inhibitor) promoted its expression at 48 h after add the inhibitor (Fig. 3A). We then performed Western blotting, and found that JEV NS3 protein levels were decreased at 48 h after JEV infection by ZK811752, which was 0.16-fold that of beta-actin; this result con rmed that CCR1 inhibitor inhibited JEV proliferation (Fig. 3B). RT-PCR results showed that CCR1 inhibitor ZK811752 could suppress the JEV mRNA levels at 48 h post-infection (Fig. 3C). We suspect that CCR1 may play an important role in JEV proliferation, by suppressing lncRNA-NONSUST006715.1 expression. We performed RT-PCR to detect CCR1 expression after JEV infection, and found that CCR1 messenger RNA (mRNA) levels were signi cantly decreased at 24 and 36 h post-infection (Fig. 3D). To verify the effect of CCR1 on the regulation of lncRNA-NONSUST006715.1 expression, we transfected three CCR1 short interfering RNAs (siRNAs) into cells and performed RT-PCR. We found that siRNA-B was optimal for CCR1 expression knockdown (Fig. 3E). siRNA-B was then used to knockdown CCR1 mRNA levels, and RT-PCR showed that CCR1 knockdown upregulated lncRNA-NONSUST006715.1 expression at 48 h after transfection siRNA (Fig. 3F). Western blotting was also performed; found that JEV NS3 protein level was decreased by CCR1 knockdown at 48 h after transfection with siRNA-B (Fig. 3G). RT-PCR results showed that CCR1 knockdown could suppress the JEV mRNA levels at 48 h post-infection (Fig. 3H).

CCR1 regulated lncRNA-NONSUST006715.1 expression via transcription factor SP1
To study the mechanism by which CCR1 regulates lncRNA-NONSUST006715.1 expression, we used an online tool (http://gene-regulation.com/) to analyze the promoter region of lncRNA-NONSUST006715.1, and found that the transcription factors CEBP-A, GATA1, SP1, and NF1 may be involved in the regulation of lncRNA-NONSUST006715.1 expression. CCR1 siRNA was transfected into PK-15 cells; RT-PCR analysis showed that SP1 expression was signi cantly increased at 48 h after CCR1 knockdown, whereas no change was detected in the mRNA levels of transcription factors CEBP-A, GATA1, and SP1 (Fig. 4A). Western blotting analysis con rmed that SP1 protein levels were upregulated at 48 h by CCR1 knockdown (Fig. 4B). To verify the effect of transcription factor SP1 on the expression of lncRNA-NONSUST006715.1, we transfected the siRNA and overexpression vector of SP1 into cells and performed RT-PCR, found that the expression of lncRNA-NONSUST006715.1 was signi cant decreased at 48 h after SP1 knockdown and increased at 48 h after SP1 overexpression ( Fig. 4C and D). To determine whether the enrichment of transcription factor SP1 was affected by CCR1 in the promoter region of lncRNA-NONSUST006715.1, we performed chromatin immunoprecipitation (CHIP), and detected signi cant SP1 enrichment at 48 h after CCR1 knockdown of the -1,645 to -1,458 bp, -1,024 to -867 bp, and +282 to +378 bp lncRNA-NONSUST006715.1 promoter regions ( Fig. 5A-C), whereas no such changes were detected in the promoter regions of GAPDH and lncRNA-NONSUST006715.1 ( Fig. 5D and E); this indicated that CCR1 downregulated SP1 mRNA levels and reduced the recruitment of SP1 to the promoter regions of lncRNA-NONSUST006715.1.

Discussion
After porcine is naturally infected by mosquitoes carrying JEV, the virus rst propagates in skin epithelial cells and lymph nodes, infects peripheral organs such as kidney, liver and spleen, and then invades, and then causes transient viremia. After that, the neurotropic virus spread to the central nervous system.
Porcine kidney epithelial cell line, PK-15 cells, has a similar susceptibility and function as skin epithelial cells. In addition, scientists have conducted a large number of studies on JEV in PK-15 cells. Therefore, PK-15 cells are a good model to evaluate the role of lncRNAs in host response to JEV infection.
LncRNAs regulate many biological processes including gene imprinting, cell growth, cell differentiation, apoptosis, immune responses, the p53 pathway, stem cell self-renewal, and DNA damage response [24][25][26][27][28][29]. LncRNA expression is usually tissue-speci c or affects speci c developmental stages [30][31][32]. SARS coronavirus-infected mice were found to contain 500 annotated lncRNAs and 1,000 non-annotated genomic regions [33]. LncRNA GAS5 has been found to suppress hepatitis C virus (HCV) replication via interaction with viral NS protein [34]. LncRNA NEAT1 is crucial for the nucleocytoplasmic transport of mRNA in response to stimuli [35]. Recent studies have also shown that virus lncRNA, or lncRNA produced during the viral life cycle, can regulate the host's antiviral immune response, thus playing an important role in promoting the replication and proliferation of the virus and packaging of the genome into the virions [36,37]. Cellular lncRNA and virus-encoded lncRNA can form chimeric lncRNA, which impacts virus infection [38,39]. Some studies have shown that lncRNAs regulate the host's innate immune response, including pathogen recognition receptor-related signaling and the production of interferons and cytokines [40,41].
In-depth study of lncRNAs has shown that they act as a medium for molecular scaffolds, guides, decoys, or signals in chromatin remodeling, transcription, post-transcription, or post-translational regulation [42,43]. lncRNAs exhibit both negative and positive functions for host's innate immunity and virus replication [44,45]. Different forms of miRNAs lead to mRNA degradation through base pairing to mRNA sequence motifs; thus, lncRNAs utilize speci c sequences or structural motifs to bind with DNA, RNA, or proteins, to modulate gene expression and protein activity including cis (impacting neighboring genes) and trans (impacting gene expression via chromosome conformation) functions [42,46].
In this study, we found that upregulation of lncRNA-NONSUST006715.1 transcription levels inhibited the expression of JEV nonstructural protein NS3 and JEV mRNA levels ( Fig. 1C and D), and knockdown of lncRNA-NONSUST006715.1 promoted JEV proliferation ( Fig. 2C and D). JEV-NS3 is a multifunctional protein consisting of 619 amino acid residues, one-third of which are n-terminal. The protein also has a catalytic domain of helicases, the activity of serine protease, nucleoside 5' -triphosphatase, and RNA triphosphatase active [47][48][49]. NS3 also play crucial roles in the replication and assembly of viruses, that has been con rmed in the Flaviviridae, such as Japanese encephalitis virus, dengue fever virus, yellow fever virus; Hepacivirus, such as hepatitis C [50][51][52]. We speculate that lncRNA-NONSUST006715.1 could suppress JEV proliferation by inhibiting NS3, but it is still unclear that the lncRNA-NONSUST006715.1 interacts with protein JEV-NS3 to inhibit the activity directly or affecting the NS3 activity via other process. In this study, we investigated the effects of lncRNA on anti-virus in PK-15 cells, but the neuroin ammation caused by JEV is mainly related to the loss of control of microglia cells [16], Furthermore, we will study the function of lncRNAs in microglial cell of swine. CCR1, also called CD191, is a G protein-coupled receptor that can serve a therapeutic target for the treatment of in ammatory diseases. Mouse homolog studies have suggested that this gene plays roles in host protection, including the in ammatory response and susceptibility to viruses and parasites [53]. CCR1 also directs leukocytes to in ammation sites [54]. CCR1 is mainly expressed in lymphocytes, neutrophils, and monocytes [55,56]; its known ligands include CCL3, CCL5, CCL7, and CCL23 [57]. In humans, CCR1 is highly expressed on monocytes, whereas in rodents, it is primarily expressed on neutrophils [54,58]. CCR1 recruits monocytes and type-1 T helper cells to activate in ammation after chronic HCV infection [59]. In rheumatoid arthritis, CCR1 regulates the expression of TNFα and IL-10, and is therefore an e cient therapeutic target [60]. Since lncRNA-NONSUST006715.1 suppresses JEV proliferation, CCR1 may play a positive role in promoting JEV proliferation post-infection. Downregulation of CCR1 expression has been reported after infection with Leishmania infantum or coronavirus [61, 62]. In the present study, we found that CCR1 expression was negatively correlated with lncRNA-NONSUST006715.1 expression after JEV infection, CCR1 expression was downregulated at 36 h after JEV infection, but recovered at 48 h (Fig. 3D). By contrast, lncRNA-NONSUST006715.1 expression was very high at 36 h after JEV infection, but decreased sharply at 48 h (Fig. 1A); therefore, we concluded that the regulation of lncRNA-NONSUST006715.1 expression by CCR1 is crucial for JEV proliferation. In this study, we found the transcript factor SP1 could regulate the expression of the lncRNA-NONSUST006715.1 (Fig 4C and D), and found that CCR1 inhibited lncRNA-NONSUST006715.1 expression via the transcript factor SP1 (Fig 4A and B, Fig. 5A-C); however, the mechanism by which CCR1 regulates SP1 remains unclear. Furthermore, the mechanism by which lncRNA-NONSUST006715.1 suppresses JEV proliferation requires further study.

Real-time quantitative PCR analysis
Primers for lncRNA-NONSUST006715.1, CCR1, SP1, NF1, GATA1 and CEBP-alpha were designed using the Primer 5 software; Primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as an internal control. Total RNA was extracted from cells using TRIzol® Reagent (Invitrogen) according to the manufacturer's protocol. The reverse transcription of total RNA (1 μg) was performed using a RevertAid™ RT Reagent Kit (RR036A, Takara) in a 20 μl reaction volume according to the manufacturer. Primer information for the Real-time quantitative PCR is also available in the Supplemental information (Table  S1).
Ten micrograms of total protein per sample was loaded onto sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) at 80 V for 3-4 h and transferred to PVDF membrane at 350mA for 90 min (Version8, Roche, USA) using an electro-blotting method. After incubating in blocking buffer (PBST with 1% (w/v) BSA (A7030, Sigma)) for 1 h, membranes were incubated with rabbit polyclonal antibody for NS3 (GTX125868, Genetex, USA), rabbit polyclonal antibody for SP1 (ab13370, Abcam, USA) at 4 °C for 12 h. After primary antibodies were used, the membranes were washed before Horseradish Peroxidase (HRP)-conjugated Goat anti-rabbit IgG second-antibody (sc-2030, Santa Cruz, USA) was added for 1 h at room temperature and washed again. The membranes were visualized with an ECL Western blot detection kit (NC15080, Thermo). The β-actin (#4970, Cell Signalling Technology, USA) protein level was also examined as an internal control. The chemiluminescence intensity of each protein band was quanti ed using the Image J, and then protein levels were normalized by the amount of β-actin protein.

Chromatin immunoprecipitation assay
Formaldehyde was added at a nal concentration of 1% directly to media of PK-15 cells. Fixation proceeded at room temperature for 10 min and was stopped by the addition of glycine to a nal concentration of 0.125M for 15 min. Cells were centrifuged and rinsed 3 times in cold PBS with 1mMPMSF. Then, cell nuclei were collected according to the manufacturer's protocol, SimpleChIP Enzymatic CHIP Kit (#9002, Cell Signalling Technology, USA). Samples were sonicated on ice with an Ultrasonics sonicator at setting 5 for six 10 s pulses to an average chromatin length of approximately 400 to 800 bp. For the immunoprecipitation, 2 μg rabbit polyclonal antibody for SP1 (ab13370, Abcam, USA) in a nal volume of 500 μl immunoprecipitation (IP) buffer were added in combination to the nuclear sonicate. After the immunoprecipitation, the IP was eluted and the DNA was recovered. DNA obtained from IP samples were quanti ed by real-time PCR and normalized to input DNA control samples. Primer information for the ChIP assay is available in the Supplemental information (Table S1).

Statistics
Data are presented as means ± SEM. Signi cant differences were analyzed by Mann-Whitney test or oneway analysis of variance (ANOVA) using SPSS software (ver, 20.0, SPAA Inc, USA). P-values < 0.05 were considered to be statistically signi cant.

Declarations
Ethics approval and consent to participate Not applicable.

Consent to publication
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Availability of data and material
To whom requests for materials should be addressed (email: zay503@zafu.edu.cn).

Competing interests
The authors declare no competing nancial interests.