Endogenous Retrovirus-Derived lnc-ALVE1-AS1 Exerts Antiviral Defense Against ALV-J Infection in Chicken Macrophages


 Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections dating back many millions of years, and their derived transcripts with viral signatures are important sources of long noncoding RNAs (lncRNAs). We have previously shown that the chicken ERV-derived lncRNA lnc-ALVE1-AS1 exerts antiviral innate immunity in chicken embryo fibroblasts. However, it is not clear whether this endogenous retroviral RNA has a similar function in immune cells. Here, we found that lnc-ALVE1-AS1 was persistently inhibited in chicken macrophages after avian leukosis virus subgroup J (ALV-J) infection. Furthermore, overexpression of lnc-ALVE1-AS1 significantly inhibited the proliferation of exogenous ALV-J, whereas knockdown of lnc-ALVE1-AS1 promoted the proliferation of ALV-J in chicken macrophages. This phenomenon is attributed to the induction of antiviral innate immunity by lnc-ALVE1-AS1 in macrophages, whereas knockdown of lnc-ALVE1-AS1 had the opposite effect. Mechanistically, lnc-ALVE1-AS1 can be sensed by the cytosolic pattern recognition receptor TLR3 and trigger the type I interferons response. The present study provides novel insights into the antiviral defense of ERV-derived lncRNAs in macrophages and offers new strategies for future antiviral solutions.


Introduction
Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections dating back many millions of years, and they comprise nearly 8% of the human genome (Stoye 2012) and 3% of the chicken genome (Mason et al. 2016). Retroviral integration is an essential part of the endogenous retroviral lifestyle but is also a potential threat to the host. Recently, integrated chicken ERVs have been shown to have harmful impacts on poultry genetic traits and resistance (Mason et al. 2020). ERV activation has also been noted in various cancers, viral infections and autoimmunity, but whether ERVs contribute to these diseases is a highly controversial topic (Hayward and Katzourakis 2015). Because ERVs were once presumed to form much of the so-called redundant 'junk' DNA, without any function, or were recognized to have pathogenic potential and to be capable of causing harm to humans (Villarreal 2011), the biological effects of these endogenous retroviral elements bene cial to humans have largely been ignored and are currently not fully understood.
Recently, it has been shown that certain ERVs are activated in mammalian preimplantation embryos and embryonic stem cells (Liu et al. 2019;Xue et al. 2013;Yan et al. 2013). They can rewire the core regulatory network of human embryonic stem cells (Kunarso et al. 2010) and display essential roles in pluripotency (Fort et al. 2014; Macfarlan et al. 2012) and early mammalian embryonic development (Grow et al. 2015; Wang et al. 2014). Importantly, ERV activation can be sensed by innate immune receptors and trigger antiviral innate immunity (Grandi and Tramontano 2018;Hurst and Magiorkinis 2015). Surprisingly, ERVs not only promote T cell selection and improve the sensitivity with which T cells react to retroviral infection (Young et al. 2012) but also mobilize B cells to rapidly produce antibodies against pathogenic antigens (Grasset and Cerutti 2014;Zeng et al. 2014).
In general, ERVs have dual effects, harmful or bene cial, on host cell antiviral function. Only a few endogenous retroviral elements that have adapted to the evolution and plasticity of the host genome by positive selection have been adopted to regulate host gene expression and control cell function. These endogenous retroviral element-derived transcripts with viral signatures are important sources of noncoding RNAs (ncRNAs), which are sensed by innate immune receptors and trigger innate immunity. In mouse macrophages, a total of 1,278 full-length ERV-derived ncRNAs were identi ed, and among them, lnc-EPAV (ERV-derived lncRNA positively regulates antiviral responses) has been demonstrated to enhance host antiviral innate immunity (Zhou et al. 2019). The lnc-ALVE1-AS1 is transcribed from chicken ERVs ALVE1 was also shown to activate antiviral innate immunity and inhibit the proliferation of exogenous retrovirus ALV-J replication in nonimmune cells (Chen et al. 2019). However, it is not clear whether this endogenous retroviral RNA has a similar function in immune cells. Therefore, individual ERVderived lncRNAs with these immune functions need to be extensively investigated to reveal the impacts of these lncRNAs on antiviral innate immunity in host immune cells.
In this study, we explored the antiviral function of lncRNA lnc-ALVE1-AS1 derived from ALVE1 in macrophages. We found that lnc-ALVE1-AS1 was persistently inhibited in chicken macrophages after ALV-J infection. Overexpression of lnc-ALVE1-AS1 signi cantly inhibited the proliferation of ALV-J through the induction of antiviral innate immunity. Mechanistically, lnc-ALVE1-AS1 can be sensed by the cytosolic pattern recognition receptor TLR3 and trigger the type I interferons response. The present study provides novel insight into the antiviral defense of ERV-derived lncRNAs in macrophages. These results have important implications for the study of the antiviral function of ERVs and the development of new antiviral vaccines.

Cells, virus, and plasmids
The chicken macrophage-like line HD11 were obtained from the Laboratory of Avian Preventive Medicine, Yangzhou University, China. HD11 cells are derived from chicken bone marrow and transformed with the avian myelocytomatosis virus MC29 (Beug et al. 1979). HD11 cells was cultured in Dulbecco's modi ed Eagle's medium (DMEM; Gibco) with 5% fetal bovine serum (FBS) at 41°C in 5% CO 2 and 95% humidity.
Primary chicken embryo broblasts (CEFs) were prepared from 10-day-old speci c pathogen-free (SPF) embryos of ALV-resistant G1 or ALV-susceptible G3 chickens obtained from Harbin Veterinary Research Institute (HVRI), the Chinese Academy of Agricultural Sciences. CEFs were cultured in DMEM with 5% FBS at 37°C in 5% CO 2 and 95% humidity. The JS09GY3 strain of ALV-J was obtained from the Laboratory of Avian Preventive Medicine, Yangzhou University, China. The plasmids pcDNA3.1-lnc-ALVE1-AS1 and pcDNA3.1-EGFP came from the plasmid bank in our laboratory. Viral infection HD11 cells were seeded into six-well plates and infected with the JS09GY3 strain of ALV-J at a multiplicity of infection (MOI) of 5. At 6, 12 and 24 h post infection (hpi), cells were collected for ALV-J proliferation analysis. CEFs were infected with the JS09GY3 strain of ALV-J at an MOI of 5 and then collected at 24 and 96 hpi for ALV-J proliferation analysis.
Plasmid transfection HD11 cells were transfected with the control or lnc-ALVE1-AS1 plasmid using Lipofectamine™ 3000 Transfection Reagent (Thermo Fisher Scienti c, USA) for 36 h, and then total RNA and protein were collected for gene expression analysis. For viral infection experiments, HD11 cells were rst infected with the ALV-J virus at a multiplicity of infection (MOI) of 5 for 12 h. Cells were then transfected with lnc-ALVE1-AS1 or the control plasmid using Lipofectamine™ 3000 Transfection Reagent (Thermo Fisher Scienti c, USA) for another 36 h and then collected for ALV-J proliferation analysis.  Table 1.

Cell treatment
After treatment with 100 µM Amlexanox (InvivoGen, California, USA) for 2 h, HD11 cells were then transfected with control or lnc-ALVE1-AS1 plasmid using Lipofectamine™ 3000 Transfection Reagent for another 36 h. Cell supernatant was then collected to detect the expression of the IFN-β gene, and total RNA was extracted to measure the expression of antiviral innate immunity-related genes. For TLR3 stimulation, HD11 cells were incubated for 24 h in medium containing TLR3 ligand (InvivoGen, California, USA), a synthetic analog of dsRNA poly (I:C) with a high molecular weight, and then collected for gene expression analysis.

Reverse transcription-quantitative PCR (RT-qPCR)
RT-qPCR assays were performed according to previous studies (Chen et al. 2019). Brie y, total RNA was extracted from chicken cells or tissues using TRIzol™ reagent (Thermo Fisher Scienti c, USA) according to the manufacturer's recommendations. The gDNA Eraser-treated RNA samples were reverse-transcribed with RT primers at 37°C for 15 minutes or strand-speci c RT primers at 42°C for 15 minutes with PrimeScript® Reverse Transcriptase (TaKaRa, Japan). Quantitative PCR was then performed with genespeci c primers and SYBR Green Master Mix (TaKaRa, Japan) on the CFX Connect™ Real-Time PCR Detection System (Bio-Rad, California, USA). GAPDH RNA levels were used as internal controls to normalize gene expression. The strand-speci c RT primers and gene-speci c primers are listed in Table 1.

Protein extraction and immunoblotting
Whole-cell lysates were prepared with Cell Lysis Buffer (Cell Signaling Technologies, USA), separated by 12% SDS-PAGE at 120 V for 90 min and transferred to polyvinylidene di uoride membranes at 50 V for 150 min. Membranes were blocked in TBST containing 5% nonfat dry milk (Bio-Rad, California, USA). Primary antibodies were incubated overnight at 4°C with agitation. The following antibodies were used to determine protein expression: rabbit anti-TLR3 (Novus Biologicals, USA), anti-GAPDH (Abcam, United Kingdom) and mouse monoclonal antibody JE9, which is speci c to the envelope protein of ALV-J. After washing extensively with TBST, secondary antibodies (anti-rabbit or anti-mouse horseradish peroxidase conjugate) were incubated for 1 h at room temperature. After washing extensively with TBST, blots were developed using enhanced chemiluminescent detection reagents on the FluorChem Q imaging system (Protein Simple, USA).

Immuno uorescence confocal microscopy
Cells were xed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature, permeabilized with 0.25% Triton X-100 for 5 min, and blocked with 2% BSA for 30 min. Cells were then incubated with the mouse anti-ALV-J envelope protein (JE9 antibody) at room temperature for 1 h, followed by incubation with goat anti-mouse IgG conjugated with Alexa Fluor 488 dye (

Statistical analyses
The statistical analysis was performed with the Statistical Package for the Social Sciences (version 16.0) software. Statistical signi cance was assessed using a two-tailed unpaired Student's t-test with a P value threshold of < 0.05.

ALV-J inhibits the expression of lnc-ALVE1-AS1 in macrophages
The in uence of exogenous virus ALV-J infection on the expression of lnc-ALVE1-AS1 in macrophages was measured by RT-qPCR. As shown in Fig. 1, the expression of lnc-ALVE1-AS1 in the chicken macrophage cell line HD11 was persistently downregulated from 6 to 48 hpi and signi cantly downregulated at 24 h and 48 h after ALV-J infection. These results suggest that lnc-ALVE1-AS1 might be involved in the antiviral response to ALV-J infection in chicken macrophages.

Inhibition of ALV-J proliferation by lnc-ALVE1-AS1 in macrophages
To con rm the role of lnc-ALVE1-AS1 in antiviral defense, macrophages were transfected with lnc-ALVE1-AS1 after ALV-J infection, and then the in uence of lnc-ALVE1-AS1 on ALV-J proliferation was assessed. Compared with the control group, transfection of lnc-ALVE-AS1 signi cantly inhibited the expression of the ALV-J env gene at both the mRNA and protein levels in HD11 cells ( Fig. 2a and b). TCID 50 and ELISA analysis results also showed that overexpression of lnc-ALVE-AS1 led to signi cant reductions in viral titers and viral protein in the culture medium of HD11 cells infected with ALV-J ( Fig. 2c and d). The confocal immuno uorescence microscopy analysis results shown in Fig. 2e further con rmed the inhibition of lnc-ALVE-AS1 on ALV-J proliferation in chicken macrophages.
Knockdown of lnc-ALVE1-AS1 can promote the replication of ALV-J in macrophages Knockdown of lnc-ALVE-AS1 by RNAi in macrophages was performed to further con rm the role of lnc-ALVE1-AS1 in antiviral defense. Conversely, knockdown of lnc-ALVE-AS1 signi cantly increased the expression of the ALV-J env gene at both the mRNA and protein levels in HD11 cells ( Fig. 3a and b). TCID 50 and ELISA analysis results also showed that knockdown of lnc-ALVE-AS1 led to signi cant increases in viral titers and viral protein levels in the culture medium of HD11 cells infected with ALV-J ( Fig. 3c and d). Our data collectively indicate that lnc-ALVE1-AS1 may possess a function in antiviral defense in chicken macrophages.
lnc-ALVE1-AS1 triggers antiviral innate immunity in macrophages Since lnc-ALVE1-AS1 can block the proliferation of ALV-J in macrophages, we next investigated the in uence of lnc-ALVE1-AS1 on the expression of host genes involved in antiviral innate immunity. In chicken macrophages, lnc-ALVE1-AS1 triggered an interferon response, which included type I interferons (IFN-α and IFN-β) and a panel of interferon-stimulated genes (ISGs; MX1, OASL and IFITM3) ( Fig. 4a and  b). Each ISG functions predominantly in antiviral innate immunity. Generally, the key upstream gene IRF7 in the type I interferon pathway was also upregulated by lnc-ALVE1-AS1 overexpression in macrophages. ELISA analysis further con rmed that the expression of IFN-α and IFN-β was induced by overexpression of lnc-ALVE1-AS1 (Fig. 4c). However, knockdown of lnc-ALVE1-AS1 by RNAi led to signi cant decreases in the expression levels of IFN-α, IFN-β, IRF7, MX1, OASL and IFITM3 (Fig. 4d and e). Knockdown of lnc-ALVE1-AS1 also decreased the concentrations of IFN-α and IFN-β in macrophages (Fig. 4f). These results suggested that lnc-ALVE1-AS1 is involved in antiviral innate immunity, especially the antiviral interferon response.
lnc-ALVE1-AS1 is involved in antiviral innate immunity by inducing TLR3 signaling We then sought to determine the molecular mechanism by which lnc-ALVE1-AS1 induced an antiviral interferon response. In HD11 cells, we found that lnc-ALVE1-AS1 only increased the transcript levels of cytosolic sensors for dsRNA (TLR3) but not IFIH1, MB21D1 or TLR7 ( Fig. 5a and b). Consistently, knockdown of lnc-ALVE1-AS1 caused a signi cant decrease in the expression levels of the TLR3 gene (Fig. 5c). Western blot results further con rmed that overexpression of lnc-ALVE1-AS1 induced the expression of TLR3 protein in HD11 cells (Fig. 5d), whereas knockdown of lnc-ALVE1-AS1 had the opposite effect (Fig. 5e). RNA FISH combined with confocal immuno uorescence further con rmed the colocalization of lnc-ALVE1-AS1 and TLR3 in HD11 cells (Fig. 5f). Thus, TLR3 could be an important dsRNA recognition receptor for lnc-ALVE1-AS1 to induce an interferon response.
lnc-ALVE1-AS1 exerts innate immune resistance to ALV-J in chickens Next, we investigated the antiviral innate immune function of lnc-ALVE1-AS1 in chickens. First, we found that the expression level of lnc-ALVE-AS1 in ALV-resistant (G1) chicken immune organs (thymus, spleen, and bursa of Fabricius) was signi cantly higher than that in ALV-susceptible (G3) chickens (Fig. 6a). Viral infection experiments showed that the ALV-J replication level in CEFs of ALV-resistant G1 chickens at 24 and 96 hpi was signi cantly lower than that in CEFs of ALV-susceptible G3 chickens, whereas the lnc-ALVE1-AS1 expression level showed the opposite results ( Fig. 6b and c). Furthermore, the expression of lnc-ALVE1-AS1 was signi cantly downregulated in ALV-resistant G1 and ALV-susceptible G3 chicken CEFs infected with ALV-J at 24 and 96 hpi ( Fig. 6d and e). However, the decrease in lnc-ALVE1-AS1 expression was more obvious in ALV-susceptible G3 chicken CEFs. It was further con rmed that overexpression of lnc-ALVE1-AS1 could inhibit ALV-J replication in ALV-susceptible G3 chicken CEFs (Fig. 6f).
Overexpression of lnc-ALVE1-AS1 also increased the expression levels of the dsRNA recognition receptor TLR3 and antiviral innate immune genes, including IFN-α, IFN-β and IFITM3 (Fig. 6g-i). These results suggested that high-level expression of lnc-ALVE1-AS1 in ALV-resistant chickens is associated with host viral resistance.

Discussion
ERVs have adapted long-term evolutionary selection by the host and are important sources and evolutionary origins of various regulatory non-coding RNAs, including lncRNAs, microRNAs, and piRNAs. When these ERV-derived ncRNAs are activated by exogenous infections or other stimuli, they may activate the functions of immune cells, including B cells, T cells and macrophages. The long noncoding RNA lnc-ALVE1-AS1 is derived from chicken ERV ALVE1 (Chen et al. 2019), which contains LTR, gag, pol and env regions and is 7.5 kb in length. It is located on chromosome 1 (Tereba et al. 1979) and is regulated by DNA methylation (Groudine et al. 1981). Chicken ERV ALVE1 is highly homologous to exogenous retrovirus ALVs and thus provides a unique model for investigating the interaction between endogenous and exogenous retroviruses as well as the symbiotic relationship and interplay between In this study, we found that lnc-ALVE1-AS1 inhibited the proliferation of ALV-J by activating antiviral immunity in chicken macrophages. Inhibition of virus proliferation by lncRNAs derived from ERVs was also observed in mouse macrophages (Zhou et al. 2019). The piRNA derived from the chicken endogenous retrovirus ALVE may be involved in resistance to ALV ). Therefore, noncoding RNAs such as lncRNAs and piRNAs derived from the chicken endogenous retrovirus ALVE may be an important part of the cellular immune response to enhance the host's resistance to foreign viral infections.
Activation of the type I interferon response mediated by TLR3 signaling may be an important mechanism by which lnc-ALVE1-AS1 inhibits the proliferation of ALV-J in chicken macrophages (a working model is shown in Fig. 7). It has been shown that lnc-ALVE1-AS1 activates TLR3 signaling in chicken CEFs (Chen et al. 2019). In this study, we further found that lnc-ALVE1-AS1 signi cantly activates the expression of antiviral innate immune genes such as TLR3 and type I interferons (IFN-α and IFN-β) in chicken macrophages. However, the effect of lnc-ALVE1-AS1 is signi cantly reduced after interfering with TLR3 or suppressing TLR3 signaling. Confocal localization analysis showed that lnc-ALVE1-AS1 can directly bind to TLR3 protein in macrophages. In addition, studies have also found that TLR3 ligand stimulation can induce abnormal expression of a large number of lncRNAs (Wang et al. 2016), suggesting that lncRNAs could be an important signal for TLR3 recognition and a regulator of innate immunity (Murphy and Medvedev 2016). These results indicate that the induction of TLR3 signaling is an important mechanism by which lnc-ALVE1-AS1 activates antiviral innate immunity.
TLR3 is an important double-stranded RNA (dsRNA) recognition receptor and participates in the antiviral immune response (Beutler 2004 . We also found that the key dsRNA recognition receptor TLR3 was signi cantly upregulated in macrophages transfected with lnc-ALVE1-AS1, indicating that lnc-ALVE1-AS1 may form a dsRNA structure that can bind to TLR3. The long noncoding RNA lnc-ALVE1-AS1 is an antisense lncRNA that may form dsRNA with the sense RNA complementary to its sequences. In addition, some short dsRNA fragments may be formed in the secondary structure of lnc-ALVE1-AS1. These dsRNAs may be recognized by TLR3 and activate antiviral innate immunity. This phenomenon is very similar to the observation that the DNA methylation inhibitor 5-Aza-dC induces an interferon response by activating endogenous retroviral dsRNA (Chiappinelli et al. 2015; Roulois et al. 2015). In addition, TLR3 can also recognize the virus-derived single-stranded RNA segments harboring stem structures with bulge/internal loops (Tatematsu et al. 2014;Tatematsu et al. 2013).
However, it must be emphasized that the present study is not a simple repetition of past study although the results of lnc-ALVE1-AS1 inhibits ALV-J proliferation and activates TLR3 signaling in chicken macrophages are consistent with those in chicken CEFs (a non-immune cell). First, it is not clear whether this endogenous retroviral RNA has a similar function in immune cells before this study. Secondly, this study more comprehensively evaluated the resistance of lnc-ALVE1-AS1 to ALV-J in macrophages when compared with the single result of ALV-J inhibition by lnc-ALVE1-AS1 overexpression in chicken CEFs. Finally, the role of lnc-ALVE1-AS1 in the antiviral defense against ALV-J infection in vivo through ALVresistant/susceptible chickens was evaluated only in this study.
In conclusion, the present ndings collectively show that lnc-ALVE1-AS1 exerts antiviral protective roles by triggering TLR3-induced antiviral innate immunity in macrophages. Individual lncRNAs with these immune functions remain to be extensively revealed in future research. Code or data availability All data and materials are available for publication.
Ethics approval Animal experiment was performed in strict accordance with the recommendations provided in the Guide for the Care and Use of Laboratory Animals of Yangzhou University. The protocol was approved by the Committee on the Ethics of Animal Experiments of Yangzhou University (licence number: 06R015).
Consent to participate Not applicable.
Consent for publication Not Applicable.
Con icts of interest The authors declare that they have no competing interests.      hpi. (f) RT-qPCR analysis of ALV-J env gene expression in the ALV susceptible (G3) chicken CEF cells, which rstly infected with the ALV-J virus at MOI of 5 for 12 h and then transfected with lnc-ALVE1-AS1 or the control for another 36 h. RT-qPCR analysis of lnc-ALVE1-AS1 (g) and innate immunity genes (h) expression in the ALV susceptible (G3) chicken CEF cells transfected with lnc-ALVE1-AS1 or the control for 36 h. (i) ELISA analysis of IFN-α and IFN-β expression in the ALV susceptible (G3) chicken CEF cells transfected with lnc-ALVE1-AS1 for 36 h. Error bars represent the s.d., n=3. *P < 0.05 and **P < 0.01 (twotailed Student's t-test).

Figure 7
A working model of TLR3-mediated antiviral interferons response triggered by lnc-ALVE1-AS1 in macrophages. Chicken ERVs derived lncRNA lnc-ALVE1-AS1 are sensed by TLR3, which recruit TRIF (Toll-IL1 receptor domain-containing adaptor inducing IFN-β) protein to the TIR domain of the receptor. This is followed by activation of IRF7 and led to production of interferons, which further induces the expression of interferon stimulated genes (ISGs). Ultimately, signals from TLR3 sensor promote antiviral innate immunity and inhibit the proliferation of ALV-J in chicken macrophages.