SOCS3 Promotes ALV-J Virus Replication via Inhibiting JAK2/STAT3 Phosphorylation During Infection

Avian leukosis virus subgroup J (ALV-J) is an oncogenic retrovirus that causes immunosuppression and neoplastic diseases in poultry. Cytokine signal-transduction inhibitor molecule 3 (SOCS3) is an important negative regulator of the JAK2/STAT3 signaling pathway and plays certain roles in ALV-J infection. It is of significance to confirm the roles of SOCS3 in ALV-J infection and study how this gene affects ALV-J infection. In this study, we assessed the expression of the SOCS3 gene in vivo and in vitro, and investigated the roles of SOCS3 in ALV-J infection using overexpressed or interfered assays with the SOCS3 in DF-1 cells. The results showed that the SOCS3 expression of ALV-J infected chickens was different from uninfected chickens in the spleen, thymus and cecal tonsil. Further, SOCS3 is mainly expressed in the nucleus as determined by immunofluorescence assay. Overexpression of SOCS3 in DF-1 cells promoted the replication of ALV-J virus, and the expression of interferons (IFNα and INFβ), inflammatory factors (IL-6 and TNFα) along with interferon-stimulating genes (CH25H, MX1, OASL, and ZAP). Conversely, interference of SOCS3 showed the opposite results. We also observed that SOCS3 promoted ALV-J virus replication by inhibiting JAK2/STAT3 phosphorylation. In conclusion, SOCS3 promotes ALV-J replication via inhibiting the phosphorylation of the JAK2/STAT3 signaling pathway. These results would advance further understanding of the persistent infection and the viral immune evasion of the ALV-J virus.


INTRODUCTION
Avian leukemia virus subgroup J was first isolated and identified from commercial broilers in 1988, which mainly causes myeloid leukosis (Payne et al., 1991;Payne et al., 1992). Infection with ALV-J causes immunosuppression and decreases chickens production performance, leading to serious economic losses in the poultry industry. ALV-J virus can transmit vertically and horizontally, and the viral genome is highly variable. There are no treatments or vaccines to prevent ALV-J infection, and eradication of ALV-J can be achieved only by chicken population cleansing (Payne and Nair, 2012). Therefore, it is necessary to study the immune response mechanisms of hosts after ALV-J virus infection.
Previously, we performed RNA-seq on chicken primary monocyte-derived macrophages (MDM) cells infected with ALV-J. The results revealed that the cytokine signal transduction inhibitor 3 (SOCS3) was a differentially expressed gene in the JAK/STAT signaling pathway . Also, we further found that the SOCS3 gene was one of the differentially expressed genes based on the RNA-seq data (PRJNA552417) of spleen tissues from 7 days-old chicken infected with ALV-J (data unpublished). Furthermore, overexpression of SOCS3 in MDM cells promotes ALV-J virus replication . These observations imply that SOCS3 may play an important role in ALV-J virus replication in chickens.
SOCS3 is an inhibitor of signaling pathways initiating cytokines, which belongs to the SOCS family (Chen et al., 2000). It is mainly involved in the negative feedback regulation of the tyrosine-protein kinase/signal transduction pathway and transcriptional activator signal transduction pathway. Furthermore, SOCS3 is closely related to inflammatory response, oxidative stress, cell injury, and apoptosis (Nicholson et al., 2000;Rawlings et al., 2004;Kershaw et al., 2013). The critical role of SOCS3 is manifested by its binding to both the JAK kinase and the cytokine receptor, which further inhibits STAT3 phosphorylation (Harrison, 2012). The JAK/STAT pathway is an evolutionarily conserved signaling pathway that transduces signals from extracellular to the nucleus (Rawlings et al., 2004). JAK/STAT pathway activation stimulates cell proliferation, differentiation, cell migration, and immune challenge (Rawlings et al., 2004;Harrison, 2012;Coskun et al., 2013). The JAK/STATs signaling pathway consists of three main components: 1. tyrosine kinase associated receptor; 2. JAK kinases; 3. STAT proteins (Gao et al., 2018). In recent years, many studies have reported that the JAK/STAT signaling pathway plays an important role in viral infection (Cherng et al., 2021;Leite et al., 2021;Wang et al., 2021;Ye et al., 2021).
Newcastle disease virus (NDV) infection activates the expression of SOCS3 at the mRNA and protein level through a mechanism that depends on the MEK/ERK signaling pathway, which is conducive to the virus replication (Wang et al., 2019a). Similarly, overexpression of SOCS3 in DF-1 cells promotes infectious bursal disease (IBD) virus replication (Duan et al., 2020). Porcine reproductive and respiratory syndrome virus (PRRSV) infection also induces SOCS3 expression through p38/AP-1 signaling pathway to enhance PRRSV replication during infection (Luo et al., 2021). In addition, SOCS3 also plays an important role in bacterial infections, inflammatory responses, and cancer (Gao et al., 2018;Fukuta et al., 2021;Matsumura et al., 2021). However, the mechanism of SOCS3 on ALV-J virus replication remains unclear. Thus, in this study, we focused on analyzing the expression of SOCS3 after ALV-J infection and the mechanism of effects of SOCS3 on ALV-J replication.

Ethics Statement
All animal experiments in this study were conducted following the protocols approved by the Institutional Animal Care and Use Committee of South China Agriculture University (No: SCAU 2018c008) and following the Animal Protection Law of the People's Republic of China.

Tissue Samples Source
The 280 days-old chickens were from a local farm in Guangdong. They showed telltale symptoms of infection, such as a pale cockscomb, listlessness, a thin body, and obvious hemangiomas on their skin and digits. Zhang et al. (2021) have verified that the chickens were only infected with the ALV-J virus. We collected the spleen, thymus, and cecal tonsils from chickens infected with ALV-J (n = 3) and uninfected with ALV-J (n = 3), and stored them at −80°C till their later use.

RNA Isolation and cDNA Synthesis
Following the manufacturer's protocol, total RNA was extracted from tissues and DF-1 cells using the RNAiso reagent (Takara, Japan). RNA integrity and concentration were determined using 1% agarose gel electrophoresis and a Nanodrop 2000c spectrophotometer (Thermo, USA). cDNA was synthesized using MonAmp ™ RTIII All-in-One Mix (Monad, Guangzhou, China), following the manufacturer′s protocol. Synthesized cDNA was stored at -20°C until subsequent analysis using qRT-PCR.

Quantitative Real-Time PCR
qRT-PCR primers specific for SOCS3, JAK2, STAT3, IFNa, IFNb, IL-6, TNFa, ZAP, CH25H, MX1, and OASL were designed using Oligo 7.0 software. All primers were synthesized by Tsingke Biotech Technology Co., Ltd. (Guangzhou, China). The MonAmp ™ SYBR ® Green qPCR Mix (Monad, Guangzhou, China) was used for qPCR in an ABI 7500 Real-Time Detection instrument (Applied Biosystems, USA) following the manufacturer's protocol. Relative gene expression was measured by qRT-PCR three for each reaction, and the nuclear GAPDH gene was used as a control. The gp85 of ALV-J virus copy was performed as previously described (Dai et al., 2015). The primers used in qRT-PCR were shown in Table S1.

ELISA
According to the manufacturer's protocol, the p27 (the main protein component of the ALV viral capsid) level was measured using an avian leukosis virus antigen test kit (IDEXX, USA). The IFNa, IFNb, IL-6, and TNFa levels were measured using a Chicken Interferon a (IFNa) ELISA Kit (ML760024; mlbio, Shanghai, China), Chicken Interferon b (IFNb) ELISA Kit (ML760024; mlbio, Shanghai, China), Chicken Interleukin 6 (IL-6) ELISA Kit (ML760041; mlbio, Shanghai, China), and Chicken Tumor necrosis factor a (TNFa) ELISA Kit (ML760161; mlbio, Shanghai, China) according to the manufacturer′s protocol, respectively. In brief, cells and medium were collected from tissue culture dishes, subjected to three freeze-thaw cycles, and centrifuged at 3000 × g at 4°C for 10 min. The supernatants were collected and subjected to ELISA according to the manufacturer′s protocols.

Statistical Analysis
Statistical comparisons were performed using GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA, USA). The data are presented as means ± standard error of the mean (SEM). The date differences in data were evaluated by the Student′s t-test. A P value of < 0.05 was considered statistically significant (*P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001).

RESULTS
The Expression of SOCS3 in Immune Organs and DF-1 cells First, we evaluated the expression of SOCS3 in the spleen, thymus, and cecal tonsil of ALV-J infected and uninfected chickens by qRT-PCR. The chickens were only infected with ALV-J, as previously demonstrated by Zhang et al. (2021). The expression of SOCS3 in the cecal tonsil (P < 0.05) and thymus (P > 0.05) of ALV-J infected individuals was higher than that of uninfected individuals ( Figure 1A). Next, we collected and detected DF-1 cells after ALV-J infection at different time points to investigate the SOCS3 expression in vitro. The expression of SOCS3 in DF-1 cells infected with ALV-J was higher than that of uninfected cells, except at 12 h post-infection (hpi) ( Figure 1B). Besides, immunofluorescence assay results showed that SOCS3 was expressed in the nucleus and cytoplasm, but mainly in the nucleus ( Figure 1C).

Overexpression and Knockdown of SOCS3 Could Influence ALV-J Virus Replication
To explore the functions of SOCS3, we overexpressed and interfered with SOCS3 in DF-1 cells to evaluate its effect on ALV-J virus replication. The gp85, the major viral envelope protein, is the most variable of the structural proteins in the genome of ALV-J and exhibits high diversity (Venugopal et al., 1998;Pan et al., 2012). The capsid protein ALV p27 is encoded by the gag gene and acts as a major group-specific antigen (Weiss, 2006;Yun et al., 2013). Therefore, we evaluate the effect of SOCS3 on ALV-J virus replication by qRT-PCR (gp85) and ELISA (p27). The qRT-PCR, ELISA, and WB results showed that the overexpression of SOCS3 in DF-1 cells promoted the expression of gp85 and p27, suggesting that the SOCS3 benefits the replication of ALV-J virus (Figures 2A-C).
In contrast, knockdown of SOCS3 in DF-1 cells inhibited the ALV-J virus replication ( Figures 2D-F).

SOCS3 Affects the Phosphorylation of the JAK2 and STAT3
Since SOCS3 plays an important role via the JAK/STAT signaling pathway (Chen et al., 2000;Rawlings et al., 2004), we further evaluated the expression of JAK2 and STAT3 genes in ALV-J infected and uninfected tissues and cells to study how SOCS3 promotes ALV-J virus replication. The expression of JAK2 was higher in the cecal tonsil but not in the thymus, since the difference was not significant in the thymus after ALV-J infection compared with uninfected chickens. In contrast, the expression of JAK2 in the spleen was lower than in the uninfected chickens ( Figure 3A). The expression of STAT3 was higher in the cecal tonsil but lower in the spleens and thymus in chickens infected with ALV-J, compared to uninfected chickens ( Figure 3B). Furthermore, after the DF-1 cells were infected with ALV-J, the expression of JAK2 and STAT3 were increased and higher than that of uninfected cells from 12 hpi to 96 hpi ( Figures 3C, D).
Based on the above results, we speculated that SOCS3 might play an important role in ALV-J infection through JAK2/STAT3. Therefore, we analyzed the effect of SOCS3 on the JAK2/STAT3 signaling pathway. Overexpression of SOCS3 in DF-1 cells significantly increased the JAK2 and STAT3 mRNA expression, and significantly decreased the phosphorylation levels of JAK2 and STAT3 (Figures 4A-C). In contrast, knockdown of SOCS3 in DF-1 cells significantly decreased the JAK2 and STAT3 mRNA expression and significantly increased the phosphorylation levels of JAK2 and STAT3 (Figures 4D-F).
Besides, we further verified the effect of JAK2/STAT3 on SOCS3. Knockdown of JAK2 in DF-1 cells significantly reduced the SOCS3 and STAT3 expression and the phosphorylation levels of JAK2 and STAT3 ( Figures 5A-D). Also, knockdown of STAT3 in DF-1 cells significantly reduced the SOCS3 expression and the phosphorylation of STAT3, but did not affect the JAK2 expression and phosphorylation of JAK2 ( Figures 5E-H). The above experimental data demonstrated that SOCS3 is a negative regulator of the JAK2/STAT3 signaling pathway.

SOCS3 Affects ALV-J Virus Replication by Inhibiting JAK2/STAT3 Phosphorylation
To investigate how SOCS3 affects ALV-J virus replication, we conducted co-transfection experiments with pcDNA3.1-SOCS3, si-JAK2, and si-STAT3, respectively. After the knockdown of JAK2, the STAT3 expression decreased while the ALV-J virus content increased compared to the control. In pcDNA3.1-SOCS3 + si-JAK2 groups, the STAT3 expression was significantly increased, but the phosphorylation level of STAT3 was decreased. Both the qRT-PCR and ELISA results showed that the content of the ALV-J virus was significantly increased ( Figures 6A-D). After the knockdown of STAT3, there was no significant difference in JAK2 expression, but the ALV-J virus's content significantly increased compared to the control. In pcDNA3.1-SOCS3 + si-STAT3 groups, the STAT3 expression and ALV-J virus were significantly increased, but the phosphorylation level of JAK2 was decreased ( Figures 6E-H).
These results above indicated that SOCS3 inhibits the phosphorylation of JAK2 and STAT3, thereby affecting the replication of the ALV-J virus.

SOCS3 Affects the Expression of Interferon, Inflammatory Factors, and Interferon-Stimulated Genes
In the present study, the ALV-J virus significantly increased the mRNA expression levels of IFNa, IFNb, IL-6, and TNFa after overexpression of SOCS3 in DF-1 cells (Figures 7A-D). Furthermore, overexpression of SOCS3 significantly promoted the mRNA expression of interferon-stimulated genes ZAP, CH25H, Mx1, and OASL ( Figures 7E-H). After three freezethaw cycles, DF-1 cells were detected by ELISA. The results were similar to those of qRT-PCR. Overexpression of SOCS3 significantly increased the content of IFNa, IFNb, IL-6, and TNFa ( Figures 7I-L). However, the expression of IFNa, IFNb, IL-6, TNFa, ZAP, CH25H, MX1, and OASL genes significantly decreased after SOCS knockdown in DF-1 cells (Figure 8). It may be associated with ALV-J persistent infection.
Furthermore, SOCS3 inhibits the catalytic activity of JAK2 by occupying the receptor or blocking substrate association. SOCS3 uses a short motif (the kinase-inhibitory region, KIR) to restrain signaling transmission by directly inhibiting the catalytic activity of JAKs. Moreover, SOCS3, through binding to gp130, inhibits STAT3 phosphorylation, and it also regulates the response to cytokines and growth factors (Nicholson et al., 2000;Yoshimura et al., 2007;Gao et al., 2018). Studies indicate that the tyrosine phosphorylation of SOCS3 accelerates the degradation of SOCS3 protein, thereby regulating feedback inhibition of JAK/STAT signaling (Haan et al., 2003;Ke and Liu, 2003). The JAK/STAT pathway constitutes the fulcrum in many important cellular processes, including growth, differentiation, proliferation, and immune functions (Coskun et al., 2013). The binding of a cytokine to its cell-surface receptor results in receptor dimerization, resulting in the activation of JAKs via crossphosphorylation. Specific tyrosine residues on the receptor are then phosphorylated by activated JAKs and serve as docking sites for a family of latent cytoplasmic transcription factors known as STATs. STATs are phosphorylated by JAKs, then dimerize and subsequently leave the receptor and translocate to the nucleus, where they activate gene transcription (Ke and Liu, 2003;Gao et al., 2018). In addition, JKA proteins can bind to the multichain receptor, such as the IL-2R family (IL-2, IL-4, IL-7, IL-9), the IL-3R family (IL-3, IL-5), IL-6R family , and IFN-R family (IFNs, IL-10, IL-19, IL-20) (Coskun et al., 2013).
Genetic knockout studies have shown that JAKs and STATs have highly specific functions in controlling various immune responses (Ke and Liu, 2003). The mice experimental study showed that JAK-or STAT-deficiency is lethal or immunodeficiency (Coskun et al., 2013). Viruses can evade the host immune system by inactivating different adaptors of the IFN-activated JAK/STAT signaling pathway (Ke and Liu, 2003). Under normal conditions, STAT3 is low-expressed or not expressed in the signaling pathway. However, STAT3 is activated when the host is stimulated. It was found that overactivated STAT3 protein promotes viral replication (Koeberlein et al., 2010;Okemoto et al., 2013).
ALV infection can result immunological tolerance, intermittent viremia, and persistent viremia (Dai et al., 2019;Yu et al., 2019). In the present study, we found that when the amount of ALV-J virus increased, the expression of JAK2 and STAT3 was also increased, indicating that JAK2 and STAT3 may have a positive effect on ALV-J persistent infection. Furthermore, when SOCS3 was co-transfected with si-JAK2 or si-STAT3, SOCS3 affected JAK2 or STAT3 and promoted ALV-J virus replication. In addition, we also interfered with the expression of JAK2 and STAT3, and found that both reduced SOCS3 expression. These results verified that SOCS3 is a negative regulator in JAK/STAT signaling pathway.
Interferon, inflammatory factors, and interferon-stimulating genes play an important role in the host's innate immune response to antiviral infection. After overexpression of SOCS3, NDV, DHAV-1 and Hepatitis C virus (HCV) can reduce interferon and interferon-stimulated genes to evade IFNmediated antiviral responses (Collins et al., 2014;Wang et al., 2019a;Xie et al., 2019). However, in this study, overexpression of SOCS3 promoted the expression of those genes, while knockdown of SOCS3 inhibited the expression of those genes, which may result from the ALV-J infection. Compared with other viruses, the clinicopathological changes of ALV-J virus infection require a longer period to appear under certain factors. Besides, some individuals may even be infected for life without disease clinicopathological changes, suggesting that ALV-J infection may be more complex than previously thought. Altogether, these results suggest that SOCS3 promotes ALV-J replication via inhibiting JAK2/STAT3 phosphorylation. We conclude that SOCS3 is an important negative regulator of the chicken innate immune signaling pathway. The finding was representing a substantial increase in our understanding of the mechanisms of persistent ALV-J infection.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

ETHICS STATEMENT
The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of South China Agriculture University.  After three freeze-thaw cycles, the levels of the content of (I) IFNa, (J) IFNb, (K) IL-6, and (L) TNFa in cells were detected by ELISA assay. These experiments were performed independently at least three times with similar results. Differences in data were evaluated by the Student's t-test. The error bars are the standard error of the mean (SEMs) (*P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001).