ALV-J inhibits the expression of lnc-ALVE1-AS1 in macrophages
The influence 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 significantly 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 confirm the role of lnc-ALVE1-AS1 in antiviral defense, macrophages were transfected with lnc-ALVE1-AS1 after ALV-J infection, and then the influence of lnc-ALVE1-AS1 on ALV-J proliferation was assessed. Compared with the control group, transfection of lnc-ALVE-AS1 significantly inhibited the expression of the ALV-J env gene at both the mRNA and protein levels in HD11 cells (Fig. 2a and b). TCID50 and ELISA analysis results also showed that overexpression of lnc-ALVE-AS1 led to significant reductions in viral titers and viral protein in the culture medium of HD11 cells infected with ALV-J (Fig. 2c and d). The confocal immunofluorescence microscopy analysis results shown in Fig. 2e further confirmed 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 confirm the role of lnc-ALVE1-AS1 in antiviral defense. Conversely, knockdown of lnc-ALVE-AS1 significantly increased the expression of the ALV-J env gene at both the mRNA and protein levels in HD11 cells (Fig. 3a and b). TCID50 and ELISA analysis results also showed that knockdown of lnc-ALVE-AS1 led to significant 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 influence 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 confirmed 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 significant 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. Several studies in mammals have reported that endogenous retroviral RNAs can trigger signaling by the cytosolic pattern recognition receptors IFIH1 and TLR3 (Chiappinelli et al. 2015; Licht 2015; Yu et al. 2012), which recognize double-stranded RNA (dsRNA) associated with virus infection. 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 significant decrease in the expression levels of the TLR3 gene (Fig. 5c). Western blot results further confirmed 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 immunofluorescence further confirmed 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.
We next investigated whether lnc-ALVE1-AS1 affects the expression of antiviral innate immunity genes through TLR3 signaling. We observed that knockdown of TLR3 by RNAi significantly blunted the lnc-ALVE1-AS1-induced type I interferon (IFN-α and IFN-β) response in HD11 cells (Fig. 5g). Furthermore, inhibition of the TLR3-type I interferon signaling pathway with a TBK1/IKKε inhibitor (Amlexanox) also significantly blunted the expression of IFN-α and IFN-β induced by lnc-ALVE1-AS1 (Fig. 5h). In addition, activation of TLR3 by the TLR3 ligand induced lnc-ALVE1-AS1 expression in HD11 cells (Fig. 5i). These results indicate that lnc-ALVE1-AS1 can be sensed by the innate immune receptor TLR3 and trigger the TLR3-induced 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 significantly 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 significantly 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 significantly 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 confirmed 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.