Long noncoding RNA U90926 is crucial for herpes simplex virus type 1 proliferation in murine retinal photoreceptor cells

Long non-coding RNAs (lncRNAs) play vital roles in the pathogenesis of infectious diseases, but the role of lncRNAs in herpes simplex virus 1 (HSV-1) infection remains unknown. Using RNA sequencing analysis, we explored lncRNAs that were highly expressed in murine retinal photoreceptor cell-derived 661W cells infected with HSV-1. U90926 RNA (522 nucleotides) was the most upregulated lncRNA detected post HSV-1 infection. The level of U90926 RNA was continuously increased post HSV-1 infection, reaching a 100-fold increase at 24 h. Cellular fractionation showed that U90926 RNA was located in the nucleus post HSV-1 infection. Downregulation of U90926 expression by RNA interference markedly suppressed HSV-1 DNA replication (80% reduction at 12 h post infection) and HSV-1 proliferation (93% reduction at 12 h post infection) in 661W cells. The survival rates of U90926-knockdown cells were significantly increased compared to those of control cells (81% and 21%, respectively; p < 0.0001). Thus, lncRNA U90926 is crucial for HSV-1 proliferation in retinal photoreceptor cells and consequently leads to host cell death by promoting HSV-1 proliferation.

. Furthermore, cellular fractionation analysis revealed the nuclear localisation of U90926 RNA in HSV-1-infected 661W cells (Fig. 1c). While non-infected samples showed almost no U90926 transcripts (Fig. 1a). Vascular endothelial cells are an important target in ARN 16 . Therefore, we infected retinal microvascular endothelial cells with HSV-1 and quantified U90926 RNA and ICP27 (a HSV-1 gene) DNA at 2, 4, 8, 12 h post HSV-1 infection. Consequently, U90926 RNA was not detected before and after infection with HSV-1, although ICP27 DNA were upregulated after HSV-1 infection ( Supplementary Fig. 1). These results suggest that U90926 RNA induction by HSV-1 infection is specific to retinal photoreceptor cells.
Involvement of U90926 RNA in HSV-1 proliferation. First, we evaluated the effect of U90926 knockdown on HSV-1 replication and proliferation. We confirmed that the U90926 RNA levels in U90926-knockdown cells, which were transfected with two different small interfering RNAs (siRNAs), were less than 20% those of the control cells at all time points evaluated post HSV-1 infection (Fig. 1d, top). HSV-1 DNA replication and proliferation in U90926-knockdown cells were then investigated by measuring the HSV-1 genomic DNA and HSV-1 titre, respectively. The ICP-27 DNA level in U90926-knockdown cells was significantly lower than that in the control cells at 3 (p < 0.05), 6, 9 (p < 0.01), and 12 h (p < 0.001) post HSV-1 infection. At 12 h post HSV-1 infection, the HSV-1 DNA level in U90926-knockdown cells was decreased by about 80% compared with that in control cells (Fig. 1d, middle). In addition, the HSV-1 titre in U90926-knockdown cells was significantly (p < 0.0001) lower than that in control cells at 3, 6, 9, and 12 h post HSV-1 infection. At 12 h post HSV-1 infection, the HSV-1 titre in U90926-knockdown cells was decreased by about 93% compared with that in the control cells (Fig. 1d, bottom).
Role of U90926 RNA in the expression of HSV-1 genes. HSV-1 replication is stimulated by the expression of HSV-1 early genes 17 . Furthermore, the expression of HSV-1 early genes depends on the expression of HSV-1 immediate early genes 18 . Therefore, we analysed the expression of several HSV-1 immediate early genes (ICP0 and ICP4) in cells depleted of U90926 RNA and found that ICP0 and ICP4 RNA levels in U90926-knockdown cells were significantly (p < 0.0001) lower than those in control cells at 3, 6, 9, and 12 h after HSV-1 infection. At 24 h after HSV-1 infection, ICP0 and ICP4 RNA levels were approximately 88% lower in  www.nature.com/scientificreports/ control cells than in U90926-knockdown cells (Fig. 1g). Furthermore, in U90926-knockdown cells, the ICP-0 and ICP-4 proteins were not detected at 3, 6, 9, and 12 h after HSV-1 infection (Fig. 1h) (Supplementary Fig. 2).

Identification of U90926-regulated host genes in HSV-1-infected 661W cells.
To identify host genes regulated by U90926 during an HSV-1 infection, we compared the expression of host genes between HSV-1-infected 661W cells in the presence or absence of U90926 RNA. Finally, we identified upregulated differentially expressed genes upon HSV-1 infection and selected total 396 genes whose expression was completely suppressed www.nature.com/scientificreports/ in a U90926-dependent manner (Supplementary Data 2). Thereafter, we performed gene ontology enrichment analysis for the identified genes using PANTHER (https ://www.panth erdb.org). For molecular function terms, gene ontology analysis revealed that differentially upregulated genes had significantly enriched immune-related functions, including 'CXCR chemokine receptor binding' , 'chemokine receptor binding' , 'chemokine activity' , ' chemoattractant activity' , and 'cytokine activity' (Supplementary Fig. 3), suggesting that U90926 RNA activates the host immune response. Accordingly, we propose a model wherein HSV-1 infection triggers U90926 induction, thus upregulating HSV-1 immediate early genes and promoting HSV-1 DNA replication, ultimately supporting HSV-1 proliferation and activation of the host immune response.

Discussion
We found that the lncRNA U90926 was induced by HSV-1 infection in retinal photoreceptor cells and that it is a crucial factor for HSV-1 proliferation in these cells. A few reports have focused on the function of the lncRNA U90926. For example, U90926 expression was demonstrated to be upregulated by Toll-like receptor stimulation in murine macrophages as a bifunctional lncRNA, acting as a positive regulator of interleukin-10 induction and as a negative regulator of CD80 and CD86 19 . In addition, U90926 expression was reported to be downregulated during the differentiation of preadipocytes 20 . U90926 RNA is the first example of a lncRNA required for the proliferation of the viral DNA of HSV-1. Several lncRNAs are used to promote viral RNA proliferation in host cells, including ACOD1 for the vesicular stomatitis virus 21 ; IPAN, PANN, and VIN for type A influenza virus [22][23][24] ; and EGOT for hepatitis C virus 25 .
Two different siRNAs (19 nucleotides) targeting U90926 RNA efficiently suppressed the proliferation of HSV-1 in retinal photoreceptor cell-derived 661W cells and increased the survival rate of these cells. RNA interference therapy is considered useful for treating ophthalmological diseases, since the eye has a confined compartment and high accessibility, thus facilitating siRNA delivery 26 . However, siRNAs are generally unstable in the bloodstream and cannot efficiently cross the cell membrane; furthermore, they are immunogenic 27 . In eye, naked siRNA has been efficiently administrated by eye drop to the anterior segment or by intravitreal injection to the posterior segment of the eye 28 . For example, Fomivirsen (marketed as Vitravene) was the first Food and Drug Administration-approved antisense drug for cytomegalovirus retinitis with a 21-nucleotide sequence complementary to the immediate early region 2 of the cytomegalovirus mRNA, and administrated by intravitreal injection 29 . Considering these findings, lncRNA U90926 is a potential novel therapeutic target for nucleic acid medicine to treat HSV-1 retinitis as it appears to be crucial for HSV-1 proliferation in retinal photoreceptor cells.

Cell culture. Both the murine retinal photoreceptor cell line 661W and Vero cells were obtained from Osaka
Bioscience Institute. Both 661W and Vero cells were grown in Dulbecco's modified Eagle's medium (DMEM)/ Ham's F12 with l-glutamine and phenol red (661W) (Fujifilm Wako Pure Chemical Corporation) or high-glucose DMEM with l-glutamine, phenol red, HEPES, and sodium pyruvate (Vero) (Fujifilm Wako Pure Chemical Corporation), supplemented with 10% heat-inactivated foetal bovine serum (FBS) (Thermo Fisher Scientific) in a humidified incubator with 5% CO 2 . Retinal microvascular endothelial cells of BALB/c mice were obtained from Cell Biologics and were cultured in complete mouse endothelial cell medium/w Kit (Cell Biologics) supplemented with 10% heat inactivated FBS (Thermo Fisher Scientific) in a humidified incubator with 5% CO 2 .

Plasmid constructs.
To generate a U90926 overexpressing vector, we purchased a pTwist CMV vector (Twist Bioscience) harbouring a part of the U90926 cDNA sequence (498 bp) between the NotI and BamHI site. This vector was used as a template to amplify full-length U90926 cDNA using the forward 5ʹ-CAC ACA CAC ACA CAC ACA CAC ACA TAT ATA TAT ATA TGTT-3ʹ and reverse 5ʹ -TAA TGT AAG CTT TTT ATT GAC ACA TCA GGT AGGGA-3ʹ primers. A linear vector was amplified with the forward 5ʹ -AAA AAG CTT ACA TTA TCC GGA CTC AGA TCT CGAG-3ʹ and reverse primer, 5ʹ-GTG TGT GTG TGT GTG ACC GGT AGC GCT AGC GG-3ʹ using pEGFP-C1 vector as the template. The whole U90926 cDNA sequence was subsequently cloned into the linearized vector using In-Fusion HD Cloning Kit (Takara) in accordance with the manufacturer's instructions. www.nature.com/scientificreports/ Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). Total RNA from HSV-1-infected 661W cells was isolated using the NucleoSpin RNA kit (Macherey-Nagel) and reverse-transcribed into cDNA using Prime Script RT master Mix (Takara). cDNA was amplified using the primer sets listed in Supplementary Table S1 using SYBR Premix Ex Taq II (Takara) in accordance with the manufacturer's instructions. qPCR was then performed using a Thermal Cycler Dice Real Time System (Takara).

HSV-1 infection. HSV-1 (KOS strain) was propagated in monolayers of
Gapdh mRNA was used for transcript normalisation.

Subcellular fractionation.
Nuclear and cytoplasmic fractions were separated using a PARIS kit (Thermo Fisher Scientific) according to the manufacturer's instructions. The RNAs in the nucleus and cytoplasm of 661W cells were extracted, and the expression levels of U90926 in the nucleus and cytoplasm were examined by RT-qPCR. Neat1(v2) and β-actin were detected as fractionation indicators.
Transfection of siRNA and U90926 overexpressing vectors. All siRNAs were purchased from Thermo Fisher Scientific. The siRNA sequences were listed in Table S3. Both siRNAs and U90926 overexpressing vectors were transfected into 661W cells by electroporation using a 4D-Nucleofector X Unit ( Thereafter, the membrane was incubated with a 1:5000 dilution of anti-mouse immunoglobulins goat polyclonal antibody conjugated with horseradish peroxidase (HRP) (Dako, P0447) for 1 h at 25 °C. The secondary antibody was diluted with IMMUNO SHOT reagent 2 (Cosmobio). Lastly, the proteins were visualized using Immobilon Forte Western HRP substrate (Merck) and chemiluminescence signal was detected with a Luminescent Image Analyzer LAS-4000 mini (Fujifilm). . In all groups, FPKM values were determined and upregulated differentially expressed genes completely suppressed in a U90926-dependent manner were selected on the basis of the mean FPKM value in each group. If the mean FPKM value in group A was > 0, those genes meeting all following criteria were selected; the mean FPKM value in group B was > 1, the value that [(mean FPKM value in group B)/(mean FPKM value in group A)] was > 5, the value that [(mean FPKM value in group C)/(mean FPKM value in group B)] was < 0.2, and the value that [(mean FPKM value in group D)/(mean FPKM value in group B)] was < 0.2. While, if the mean FPKM value in group A was 0, those genes meeting all following criteria were selected; mean FPKM value in group B was > 1, the value that [(mean FPKM value in group C)/(mean