Primary chicken embryo fibroblast cells (CEFs) were prepared from 10-day-old specific pathogen-free chicken embryos, following our approved protocol. The CEFs and DF-1 cells (ATCC and CRL-12203) were maintained in Dulbecco’s modified Eagle’s medium (GIBCO) supplemented with 10% fetal bovine serum (FBS; GIBCO), penicillin (100U/ml), and streptomycin (100μg/ml) at 37°C in a humidified 5% CO₂ incubator.

The ALV-J (JS09GY3 strain) and the monoclonal antibody JE9 against ALV-J Env (gp85) were kindly provided by Prof. Aijian Qin (Yangzhou University, China). The 50% tissue culture infective doses (TCID₅₀) of the ALV-J virus were measured in DF-1 cells using the Reed and Muench method. ALV-J RNA was quantified by qPCR using Env gene-specific primers, and the results were normalized to the expression levels of GAPDH.

Total RNA was extracted from cells or eight kinds of chicken normal tissues using TRIzol reagent (Takara). cDNA was synthesized using a Vazyme cDNA Synthesis Kit with gDNA Eraser Kit (Vazyme, Nanjing, China) according to the manufacturer’s protocol. qPCRs were performed with the ChamQ SYBR qPCR Master Mix Kit (Vazyme) on a BioRad CFX Connect Real-time PCR detection system (Bio-Rad). The relative expression levels were normalized with the GAPDH gene by the comparative Ct (2^−∆∆Ct) method. The primers sequences for qRT-PCR are listed in Supplementary Table S1.

5' RACE and 3' RACE were performed with SMARTer RACE5'/3' Kit (Takara) according to the manufacturer’s protocol. Briefly, total RNA from CEF cells was isolated by using TRIzol reagent (Takara) and was reverse-transcribed into the first-strand cDNA using 5' or 3' end primers. Then, the cDNA was used as template for PCR amplification to generate 5' or 3'-end of the clone. 5'- and 3'-specific primer are listed in Supplementary Table S1.

To generate pcDNA3.1-lnc-LTR5B, the full-length lnc-LTR5B was PCR-amplified and cloned into a pcDNA3.1 plasmid with the MultiF Seamless Assembly Mix (ABclonal) according to the manufacturer’s instructions. The different DNA fragments of lnc-LTR5B promoter were PCR-amplified using the following pairs of primers: FL-F and FL-F for FL region; T1-F and FL-F for T1 region; T2-F and FL-F for T2 region; and FL-F and T3-R for T3 region. Then, these DNA fragments were cloned into the pGL3-basic vector (Promega) to generate the reporter construct. The oligo sequences for the plasmid construct are listed in Supplementary Table S1.

2×10⁵ 293T cells were seeded in 24-well plates and cotransfected with a control plasmid pRL-TK (Promega) and indicated plasmids for luciferase activity. After 36h of incubation, the cells were harvested and luciferase activities were measured using the Dual Luciferase Reporter Assay Kit (Vazyme), according to the manufacturer’s instructions. Relative luciferase signals (firefly/Renilla) were normalized against that of the pGL3-basic empty vector.

The specific targeting lnc-LTR5B (ASO-1 and ASO-2) or non-targeting control ASO (ASO-NC) were designed and synthesized by RiboBio Company. ASO transfection was performed in 12-well plates with three technical replicates for each sample. Briefly, DF-1 cells were transfected with a final concentration of 50nM of each ASO until cell density reached ~50% confluence by riboFECT^™ CP reagent (RiboBio) according to the manufacturer’s instruction. The ASO targeting sites are listed in Supplementary Table S1.

Cells were lysed in a lysis buffer (Cell Signaling Technology) supplemented with the complete protease inhibitor cocktail (Roche) and incubated on ice for 15min. Protein concentrations were measured by the BCA Protein Assay kit (Beyotime). A total of 25 to 50μg of protein were separated by SDS-PAGE gel and transferred to a nitrocellulose membrane (Cytiva). The membranes were incubated at 4°C overnight with the appropriate primary antibodies: anti-BiP (HuaBio), anti-Bcl2 (Proteintech), anti-cleaved-caspase-3 (Proteintech), anti-GAPDH (Abcam), and anti-β-actin (Abcam). The images were captured with a FlourChem Q imaging system (ProteinSimple). The relative intensities of the different protein bands were analyzed using Image J program.

The membrane and cytoplasmic cell fractions were extracted using Membrane and Cytosol Protein Extraction Kit (Beyotime), according to the manufacturer’s protocol. Briefly, the treated cells were harvested and washed twice with PBS and then incubated with ice-cold Extraction Regent A containing protease inhibitor mixture on ice for 15min with gentle agitation. The supernatant (cytoplasmic fraction) was obtained by centrifugation at 700g at 4°C for 10min. And the sediment was resuspended with 200μl of ice-cold Extraction Regent B containing the protease inhibitor mixture. Finally, the supernatant was used as the membrane fraction to detect BiP protein expression.

The viral entry assay was performed as described in a previous publication (Guan et al., 2018) with modifications. The cells were transfected with pcDNA3.1 or pcDNA3.1-lnc-LTR5B as described. After 24h post-transfection, the cells were washed twice with PBS, followed by incubation with ALV-J (MOI=5) at 37°C. After 2-h incubation, the cells were washed three times with pre-warmed PBS and then cultured in fresh DMEM supplemented with 2% FBS at 37°C with 5% CO2. The virus entry levels were measured at 12h post-infection by qRT-PCR for detecting ALV-J RNA.

The DNA fragments of full-length lnc-LTR5B and its antisense were amplified with primers containing a T7 promoter sequences (listed in Supplementary Table S1). The PCR products were transcribed to RNA in vitro using the TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific). Then, the biotinylated RNAs were produced using the Pierce RNA 3′ Desthiobiotinylation Kit (Thermo Scientific) and purified with phenol/chloroform/isoamyl alcohol, followed by ethanol precipitation. Next, 50pmol of biotin-labeled RNA was mixed with Pierce nucleic-acid compatible streptavidin magnetic beads in Protein-RNA Binding Buffer (Thermo Scientific) followed by incubation with DF-1 cell lysates for 1h with gentle agitation. After washing, the RNA-protein mixtures were extracted and further subjected to 12% SDS-PAGE for silver staining or western blotting. The selected bands were cut and subjected to mass spectrometry analyses.

For RIP assay, 2×10⁷ DF-1 cells were washed twice with PBS and lysed in RIP lysis buffer (25mM Tris–HCl pH 7.4, 150mM KCl, 5mM EDTA, 0.5mM DTT, and 0.5% NP-40) supplemented with protease inhibitor cocktail and an RNase inhibitor, for 30min on ice. The lysates were then incubated with prewashed Protein A/G magnetic beads and coated with 5μg BiP (HuaBio) or normal Rabbit IgG (Abcam), with overnight shaking at 4°C. The next day, the beads were washed three times with ice-cold NT2 buffer (50mM Tris–HCl (pH 7.4), 150mM NaCl, 1mM MgCl2, and 0.05% NP-40) with protease inhibitor cocktail and an RNase inhibitor, to obtain the RNA-Protein complex. RNA was then isolated using TRIzol and detected by qRT-PCR.

DF-1 cells were grown on glass coverslips; after 24h, cells were washed with PBS three times. To visualize intracellular proteins, cells were fixed with 4% paraformaldehyde in PBS for 10min at room temperature (RT) and permeabilized with 0.25% Triton X-100 in PBS for 10min on ice. After blocking with 5% BSA and 2% donkey serum in PBS for 30min, the slides were then subjected to incubation with primary antibodies. To visualize cell surface proteins, cells were immediately fixed in 4% paraformaldehyde in PBS (4°C, 15min), followed by blocking with 5% BSA and 2% donkey serum in PBS (4°C, 30min). All primary antibodies diluted in PBS were incubated overnight at 4°C. The primary antibodies used were as follows: mouse anti-Env (1:300) and rabbit anti-BiP (1:200, HuaBio). The secondary antibodies used in this study were Alexa Fluor 488 donkey anti-rabbit antibody (1:1000; Invitrogen) and Alexa Fluor 594 donkey anti-mouse antibody (1,1,000; Invitrogen). The slides were washed three times with PBS, and nuclei were stained using DAPI (Sigma-Aldrich). Imaging was obtained using a Leica SP8 Confocal system with 63x oil objective and analyzed by the LAS X software version 3.0.2.16120.

The data are represented as mean±standard deviation (SD) of three independent experiments for each group and statistically analyzed by GraphPad Prism version 7.0 (GraphPad software). Statistical significance was assessed by Student’s t test or one-way ANOVA as shown in each figure legend. Differences with p values less than 0.05 were considered as statistically significant.

To identify the host lncRNAs involved in ALV-J replication, we used RNA-seq to perform transcriptional analysis of CEF that were infected with ALV-J (JS09GY3 strain) or were mock-infected. The analysis data were presented in our previous work (Hu et al., 2018). Based on the idea that host lncRNAs targeted by viruses may participate in cellular antiviral responses, we focused on these downregulated lncRNAs during ALV-J infection. One of these lncRNAs, lnc-LTR5B (NCBI accession number: MG066622.1), was robustly downregulated in CEFs following ALV-J infection. RepeatMasker analysis showed that the MG066622.1 transcript has higher similarity with LTR5B elements, suggesting that lnc-LTR5B is an ERV-associated lncRNA. Considering that LTR contains a cis-regulatory region, the potential function of lnc-LTR5B in ALV-J infection requires urgent investigation.

We first examined lnc-LTR5B expression during ALV-J infection using reverse transcription quantitative PCR (qRT-PCR) and confirmed its downregulation upon ALV-J infection of primary CEFs (Figure 1A). Subsequently, we examined the expression of lnc-LTR5B in DF-1 cells infected with ALV-J. qRT-PCR and semi-quantitative RT-PCR showed that lnc-LTR5B was downregulated during ALV-J infection (Figures 1B,C). To identify the full-length sequence of lnc-LTR5B, we performed 5' and 3' rapid amplification of cDNA ends (RACE; Figure 1D) and the sequencing result show that the full-length transcript was 590 nucleotides (Supplementary Table S2). Subsequently, its protein-coding potential was assessed using the Coding Potential Calculator (CPC) program. The data showed that the CPC score for lnc-LTR5B was −0.980, which was similar to that of the validated lncRNA XIST (−0.945), suggesting that lnc-LTR5B lacks coding potential (Supplementary Figure S1). We further measured the relative abundance of lnc-LTR5B in several chicken tissues. Our results suggest that lnc-LTR5B is detectable in the brain, heart, liver, lung, spleen, kidney, intestines, and bursa. In addition, lnc-LTR5B was highly expressed in the lung and liver tissues, with a relatively low expression level in the brain tissues (Figure 1E).

BLAST analysis of the chicken genome from the UCSC Genome Browser¹ database showed that lnc-LTR5B is located on chicken chromosome 17 and has two exons and is flanked by the upstream and downstream protein-coding genes TUBB4B and ENTPD2L (Figure 2A). Interestingly, the first exon of lnc-LTR5B was shown to be derived from an LTR5B element, which belongs to the ERV-L LTR family. Next, a dual luciferase assay was performed to verify whether lnc-LTR5B is an LTR-activated lncRNA. We inserted the full-length (from −2000 to +198) lnc-LTR5B promoter and truncated promoter fragments into a luciferase reporter vector. The luciferase activity of the T2 promoter region (from −341 to +198) was higher than that of the other promoter fragments (Figure 2B). The results indicate that lnc-LTR5B is an ERV LTR-derived lncRNA and is a member of the ERV-L lncRNA family. Next, we analyzed the subcellular localization of lnc-LTR5B by performing RNA fluorescence in situ hybridization (FISH). The results indicated that lnc-LTR5B is localized in both cytoplasm and nucleus, with distinct perinuclear aggregation (Figure 2C). Altogether, we identified that lnc-LTR5B is a novel LTR-derived lncRNA and its expression is suppressed in chicken cells infected with ALV-J.

To investigate the role of lnc-LTR5B in ALV-J replication, we transfected DF-1 cells with either negative control vector pcDNA3.1 or the lnc-LTR5B expression vector and infected them with ALV-J at 12h post-transfection. At 48 hpi, the cells were harvested for immunoblotting and qRT-PCR analysis. With increasing levels of lnc-LTR5B (Figure 3A), ALV-J RNA levels and Env protein expression decreased in a dose-dependent manner (Figures 3B,C). Next, the impact of lnc-LTR5B silencing on viral replication was assessed. Two different sets (ASO-1 and ASO-2) of antisense oligonucleotides (ASO) targeting lnc-LTR5B or ASO-NC as a control were used. The results of qRT-PCR showed that DF-1 cells transfected with ASO-1 achieved the highest silencing efficiency (Figure 3D). Consistent with this, the levels of ALV-J RNA and protein Env expression were both significantly increased in the DF-1 cells transfected with ASO-1 compared to those in the ASO-2 and ASO-NC groups at 36hpi (Figures 3E,F). In addition, our data showed that overexpression of lnc-LTR5B did not have a significant effect on the activation of IFN signaling and its downstream gene, MX1 (Supplementary Figure S2A). Furthermore, the expression of lnc-LTR5B was not affected by poly(I:C)-induced IFN signaling (Supplementary Figure S2B). Together, these data suggest that lnc-LTR5B-mediated inhibition of ALV-J replication might not involve the activation of the innate immune response.

Because lncRNAs can exert their functions through RNA-protein interactions to modulate target genes, we attempted to identify the proteins that bind lnc-LTR5B to explore the mechanism by which lnc-LTR5B restricts ALV-J replication. Therefore, we used RNA pull-down assays followed by silver staining and mass spectrometry (MS) to identify the protein partners of lnc-LTR5B (Figure 4A). MS analysis of the differentially displayed bands associated with lnc-LTR5B revealed that BiP is a potential protein that binds to lnc-LTR5B (Figure 4B). Subsequently, significant pulldown of BiP by the biotin-labeled sense lnc-LTR5B compared with the antisense sequence was confirmed by western blotting (Figure 4C). Next, the interactions between lnc-LTR5B and BiP were verified using RNA immunoprecipitation (RIP) assays (Figure 4D). Moreover, RNA FISH and immunofluorescence assays demonstrated the colocalization of lnc-LTR5B and BiP in the cytoplasm of DF-1 cells (Figure 4E).

Because the lnc-LTR5B interacting protein BiP was reported as an entry receptor for ALV-J, it must be located on the plasma membrane of DF-1 cells. However, the expression levels of cell surface BiP are generally considered to be low under normal physiological conditions. The translocation of BiP to the cell surface can be observed under conditions of cellular stress, including viral infection (Gonzalez-Gronow et al., 2021). To examine whether ALV-J infection induces the translocation of BiP to the DF-1 cell surface, we performed immunocytochemical staining to analyze the distribution patterns of BiP in both permeabilized and non-permeabilized cells (Figure 5). We observed colocalization of intracellular BiP and ALV-J Env, implying that BiP plays an important role in ALV-J infection through its direct interaction with the ALV-J protein. In addition, we found that BiP colocalized on the cell surface with ALV-J Env, and the colocalization was stronger at 24hpi compared to that at 4 hpi, suggesting that ALV-J infection induces BiP translocation to the cell surface during ALV-J infection.

Next, we sought to find an association between lnc-LTR5B and BiP. Therefore, we quantified the expression of the BiP protein in DF-1 cells during ALV-J infection. The results of western blotting revealed that the expression of BiP was unchanged during the ALV-J infection within 12h, followed by a slight increase at 24hpi (Figure 6A). However, the expression of lnc-LTR5B began to decrease at 12hpi, the time point at which we first detected the expression of lnc-LTR5B (Figure 1B). Meanwhile, the overexpression of lnc-LTR5B did not affect the protein expression of BiP (Figure 6B). These data suggest that lnc-LTR5B is not directly involved in the regulation of BiP expression.

Because lnc-LTR5B binds BiP in the cytoplasm, we deduced that lnc-LTR5B can affect the BiP action that may be involved in BiP translocation to the cell surface. Western blot analysis of membrane fractions was performed to assess the effect of lnc-LTR5B overexpression on the surface expression of BiP upon ALV-J infection. As presented in Figure 6C, the expression of BiP on the membrane was attenuated in DF-1 cells overexpressing lnc-LTR5B compared to that in the corresponding control. Following this observation, we examined ALV-J binding and entry in lnc-LTR5B overexpressed DF-1 cells. As expected, we found that the overexpression of lnc-LTR5B markedly reduced the entry of ALV-J (Figure 6D). In addition, cell surface BiP has been reported to promote cell survival, which is considered an important target for cancer treatment (Araujo et al., 2018; Samanta et al., 2021). We speculate that the reduction of cell surface BIP caused by lnc-LTR5B leads to increased apoptosis. To test this, we assessed the effect of lnc-LTR5B overexpression on cell apoptosis signaling following ALV-J infection. The data showed that lnc-LTR5B overexpression inhibited the expression of the anti-apoptotic protein Bcl-2 and increased caspase-3 activation (Figure 6E), suggesting that lnc-LTR5B is involved in the activation of apoptotic signaling pathways in ALV-J-infected cells.