MA Yu-chen,CHEN Hua-yuan,GAO Shen-yan,ZHANG Xiao-zhan,Ll Yong-tao,YANG Xia,ZHAO Jun,WANG Zeng#

1 College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,P.R.China

2 College of Veterinary Medicine,Henan University of Animal Husbandry and Economy,Zhengzhou 450046,P.R.China

Abstract Type I interferon (IFN-I) provides an important first line to protect avian species against pathogens invasion. IFN regulatory factor 7 (IRF7) has been identified as the most important regulator for both DNA and RNA virus-induced IFN-I production in chickens. Although four splicing variants of IRF7 have been identified in mammals,it is still unclear whether alternative splicing patterns and the function of IRF7 isoform(s) exist in chickens. In this study,we reported a novel short transcript isoform of chicken IRF7 (chIRF7),termed chIRF7-iso,which contained an intact N-terminal DNAbinding domain (DBD) and 14 amino acids different from chIRF7 in the C-terminal. Overexpression of chIRF7 in chicken leghorn male hepatocellular (LMH) cells activated the IFN-β promoter and significantly inhibited Newcastle disease virus(NDV) and fowl adenovirus serotype 4 (FAdV-4) replication. Conversely,overexpression of chIRF7-iso blocked the IFN-β promoter activity and was favorable for NDV and FAdV-4 replication in vitro. Collectively,our results confirm that a novel chIRF7 isoform-mediated negative regulates IFN-β production,which will contribute to understanding the role of chIRF7 in innate antiviral response in chicken.

Keywords: chIRF7,chIRF7 isoform,negative regulation,IFN-β

1.lntroduction

Virus infection initiates a cascade of intracellular signaling events to activate host immune system. Type I interferon(IFN-I) plays substantial roles in inducing cell-intrinsic antimicrobial states in infected and neighboring cells,and activating the adaptive immune system to promote the development of T-and B-cells (Ivashkiv and Donlin 2014). Four major classes of cellular pattern recognition receptors (PRRs),including retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs),Toll-like receptors(TLRs),nucleotide oligomerization domain (NOD)-like receptors (NLRs),and C-type lectin-like receptors(CLRs),are crucial to the induction of IFN-I (Neerukonda and Katneni 2020). When activated by corresponding pathogen-associated molecular patterns (PAMPs),these receptors recruit specific adaptor proteins to trigger signal transduction and finally converge on the activation of IFN regulatory factors (IRFs) (Zhaoet al.2016). The activated IRFs are then phosphorylated,form dimers,and translocate into nucleus to initiate IFN-I expression,which leading to the expression of hundreds of IFN stimulated genes (ISGs) thus establishing antiviral state in cells(Hayden and Ghosh 2004).

In mammals,10 cellular transcriptional factors,IRF1,IRF2,IRF3,IRF4 (also known as PIP or ICSAT),IRF5,IRF6,IRF7,IRF8 (also known as ICSBP),IRF9 (also known as ISGF3γ),and IRF10 have been classified into IRF family (Mamaneet al.1999;Taniguchiet al.2001;Alsamman and El-Masry 2018). All IRFs carry a wellconserved N-terminal DNA-binding domain (DBD),which binds to a consensus or similar DNA sequence with conserved 5′-GAAA-3′ repeats (Zhang and Pagano 2002),and a variable C-terminal region,which contains an IRF association domain (IAD) to interact with other transcriptional factors and defines multiple biological functions (Hondaet al.2006). IRF3 and IRF7,sharing the greatest structural and functional homology among all the IRFs,play essential roles in IFN-β production in mammals(Zhaoet al.2015). However,IRF3 is absent in chickens,and IRF7 appears to be selected to play a conserved role in regulating antiviral response (Campbell and Magor 2020). Chicken IRF7 (chIRF7) has been confirmed to be involved in both chicken stimulator of interferon genes (chSTING)-and chicken mitochondrial anti-viral signaling protein (chMAVS)-mediated IFN-β expression in responding to DNA and RNA viral infections,respectively(Chenget al.2019). Knocking down chIRF7 by specific siRNA significantly promotes Newcastle disease virus(NDV) replication in chicken embryo fibroblasts (CEFs)cells (Wanget al.2014). Knocking out chIRF7 by CRISPR/Cas9 system straightly inhibits IFN-β production and increases the replication of H6N2 avian influenza virus (AIV) in chicken embryonic fibroblast (DF1) cells(Kimet al.2020). Us3 facilitated Marek’s disease virus(MDV) replication in CEF cells through suppressing IRF7 dimerization and further nuclear transportation (Duet al.2022). The results demonstrate that chIRF7-mediated innate immune response is indispensable for chickens to antagonist virus infection.

Four splicing variants of IRF7,named IRF7A,IRF7B,IRF7C,and IRF7H,have been identified in humans (Auet al.1998). Comparing to IRF7A,IRF7B lacks 29 amino acids (aa) in the middle,IRF7C encodes a protein with 164 aa that contains the intact DBD and extra different 13 aa in the C-terminal,and IRF7H encodes a deduced 514-aa protein with the highest homology to IRF3. Studies demonstrated that IRF7A and IRF7B were predominantly expressed in spleen,thymus,and peripheral blood leukocytes (PBLs),IRF7C was only weakly expressed in PBLs,and IRF7H was restricted to lymphoid cells (Auet al.1998;Zhang and Pagano 2002). IRF7A played a crucial role in the second phase IFN inductionviaRIG-I/MAVS signal pathway during H1N1 influenza virus infection in BCi-NS1.1 human cell (Wuet al.2020). IRF7A,IRF7B,and IRF7C contributed to inactivating Epstein-Barr virus(EBV)BamHI Q promoter in type III latency (Zhang and Pagano 1997). Translocation of IRF7H from cytoplasm to nucleus promoted IFNs expression in NDV-infected L929 cells (Auet al.1998). All these results indicate that IRF7s are extensively involved in virus infection.

Although the mammalian IRF7s play important roles in innate antiviral immune response,it is still unclear whether alternative splicing patterns and the function of IRF7 isoform(s) exist in chickens. Here,we identified a human IRF7C-like short transcript isoform of chIRF7,designated chIRF7-iso,and performed a preliminary study on its function.

2.Materials and methods

2.1.Cell and virus

Leghorn male hepatocellular (LMH) cells were grown in DME/F-12 medium supplemented with 10% fetal bovine serum plus 1% antibiotics at 37°C 5% CO2. NDV LaSota strain was propagated in 10-day-old specificpathogen-free (SPF) embryonated chicken eggs. The recombinant fowl adenovirus serotype 4 (FAdV-4) strain rCH/HNJZ/2015-Δ1966/EGFP,with EGFP gene being inserted into 1 966-bp deletion on the right end region of viral genome,was constructed in the background of CH/HNJZ/2015 (GenBank accession no.KU558760) and preserved in our laboratory.

2.2.Plasmids construction

The chIRF7 (NM_205372.1) and chMDA5 (NM_001193 638.1) were amplified from cDNA of HD11 cells (a chicken macrophage cell line) and cloned into pCAGGS-HA vector through homologous recombination to study their function on virus infection. When positive clones were picked for plasmids extraction and sequencing,pCAGGS-HAchMDA5 was successfully constructed. However,bands with different sizes of chIRF7 were observed,and a novel short transcript isoform of chIRF7,named chIRF7-iso,was identified after being subjected to online BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi) with the obtained nucleotide sequence. To better understand its function,specific primers were used to amplify chIRF7-iso from cDNA of HD11 cells to construct pCAGGS-HAchIRF7-iso for further study. Additionally,the chicken IFN-β promoter EGFP reporter plasmid,pGL3-EGFPchIFN-β,was constructed and preserved in our laboratory.Briefly,the -158 to +14 of chicken IFN-β promoter was obtained from CEF genomic DNA using primers withNheI andBglII sites and inserted into a modified pGL3-Basic-EGFP vector,with luciferase being replaced by EGFP,to generate pGL3-EGFP-chIFN-β. Primers used in this study was shown in Table 1.

Table 1 Primers used for plasmid construction and identification

2.3.PCR

A 6-week-old SPF chick was euthanized,and the heart,lung,liver,spleen,bursa of fabricius,kidney,and brain were collected and used for RNA extraction. After being reversely transcribed into cDNA,chIRF7 and chIRF7-iso were amplified to detect their tissue distribution.

2.4.Antibody preparation and protein identification

Rabbit polyclonal antibody against chIRF7/chIRF7-iso was produced as follow. Briefly,a New Zealand White rabbit (2-4 kg) was immunized intramuscularly with 200 mg pCAGGS-HA-chIRF7 and pCAGGS-HA-chIRF7-iso plasmids (100 mg each) at day 1,14,and 28. The antisera were collected 2 weeks after the third immunization. 1×106LMH cells were lysed with 50 mL RIPA buffer (Thermo)containing 1×PMSF (Solarbio),and 20 and 30 mL lysates were separately subjected to 12% SDS-PAGE to detect the endogenous expression of chIRF7 and chIRF7-iso with the rabbit antisera as the first antibody.

2.5.Plasmid transfection and Western blot

LMH monolayer cells were seeded in 6-well plate and transfected with 2 μg pCAGGS-HA-chIRF7,pCAGGSHA-chIRF7-iso,or pCAGGS-HA vector as a control,respectively,by using Lipofectamine 2000 (Invitrogen,USA). At 24 and 48 h post-transfection,the cells were washed with chilled PBS and harvested for protein extraction. To detect the expression of chIRF7 and chIRF7-iso,the transfected cells were lysed in RIPA lysis buffer plus 1× PMSF to extract proteins,which were fractionated by using 12% SDS-PAGE and transferred to nitrocellulose membranes (Pall,USA). The proteins were then probed with either an anti-HA rabbit polyclonal antibody (Sino Biological Inc.,China) or an anti-β-actin rabbit monoclonal antibody (ABclonal,USA) for 2 h at room temperature. After hybridization with goat anti-rabbit secondary antibody,the membranes were visualized with enhanced chemiluminescence reagents (CWBIO,China).

2.6.Chicken lFN-β promoter activity assay

LMH monolayer cells seeded overnight in 24-well plate were co-transfected with 0.25 μg pGL3-EGFP-chIFN-β,and 1 μg pCAGGS-HA-chIRF7,pCAGGS-HA-chIRF7-iso,pCAGGS-HA-chMDA5 as a positive control,or pCAGGSHA as a blank control. Green fluorescence was observed at 24 and 48 h post-transfection to evaluate the level of chicken IFN-β promoter activity.

2.7.qRT-PCR

LMH monolayer cells seeded overnight in 24-well plate was individually transfected with 1 μg pCAGGS-HAchIRF7,pCAGGS-HA-chIRF7-iso,pCAGGS-HA-chMDA5 as a positive control,or pCAGGS-HA as a blank control.Cells were harvested at 24 and 48 h post-transfection,and RNA was extracted and reversely transcribed into cDNA. IFN-β was amplified using specific primers shown in Table 1. The relative expression of IFN-β wasnormalized to the expression of GAPDH and expressed as fold changes relative to the control group.

2.8.Viral replication in transfected LMH cells

To evaluate the effects of chIRF7 and chIRF7-iso on NDV and FAdV-4 replication,monolayers of LMH cells in 24-well plates were transfected with 1 μg pCAGGSHA-chIRF7,pCAGGS-HA-chIRF7-iso,or pCAGGSHA. 24 h post-infection (hpi),the cells were infected with LaSota or rCH/HNJZ/2015-Δ1966/EGFP at an MOI of 0.01 and maintained in Opti-MEM (Gibco) at 37°C.The intensity of green fluorescence in cells infected with rCH/HNJZ/2015-Δ1966/EGFP was regularly observed and aliquots of culture supernatants from cells infected with LaSota or rCH/HNJZ/2015-Δ1966/EGFP were respectively collected at various time points (hpi) for further virus titration.

2.9.Statistical analysis

Data analysis was performed by using Graphpad Prism 6.0 Software. Significant differences were determined using a two-tailed Student’st-test (＊,P＜0.05;＊＊,P＜0.01;＊＊＊,P＜0.001).

3.Results

3.1.Chicken lRF7 undergoes alternative splicing

To explore the effects of chIRF7 and chMDA5 on virus infection,the pCAGGS-HA-chIRF7 and pCAGGS-HAchMDA5 were planned to be generated. After being singly picked and cultured overnight at 37°C,the colony PCR was performed with identification primers for chIRF7(Table 1),and the length of amplicon was 390 nucleotides(nt) in theory. However,bands with different sizes of chIRF7 were observed,and two of them were selected for sequence analysis (Fig.1-A). The smaller band represented the normal form of chIRF7,which is located on chromosome 5 and has a CDS length of 1 476 base pairs encoding 491 aa as described previously (Kim and Zhouet al.2015). The larger one contained extra 147 nt insertion at site between 457 and 604,with a TGA stop codon at site 499. BLAST analysis revealed that the inserted 147 nt shared 98.64% homology with the intron sequence at site between 1 789 and 1 935 of chIRF7 complete CDS (GenBank: AF268079.1),indicating that the larger band of chIRF7 includes an intact intron. Given the inserted 147 nt contained a TGA stop codon and a well conserved 5′-GT-AT-3′ motif for nearly all canonical splicing sites (Gaoet al.2019),the larger band was speculated to encode two proteins,the normal chIRF7 and an alternative transcript isoform chIRF7-iso. Further analysis showed that chIRF7-iso encoded 166 aa and shared the same 152 aa at the N-terminal with chIRF7(Fig.1-B).

Fig. 1 Characterization of a novel short transcript isoform of chIRF7. A,colony PCR with identification primers was performed to detect the positive clone of chIRF7,and bands with different sizes were observed. The arrow indicated ones were selected for sequence analysis. B,schematic diagram of the larger band and the speculated expression of chIRF7 and chIRF7-iso. Boxes represented the exons,solid bar indicated the inserted 147 nt,and the underlined TGA was the stop codon.

Fig. 3 Effect of chIRF7-iso on the regulation of IFN-β promoter activity. A,LMH cells were co-transfected with indicated plasmids and pGL3-EGFP-chIFN-β reporter,and green fluorescence was observed at 24 and 48 h posttransfection. B,LMH cells were individually transfected with indicated plasmids,and cells were collected at 24 and 48 h post-transfection for RNA extraction and realtime quantitative PCR to detect the expression of IFN-β.Data were expressed as mean±SD of three independent experiments. ＊＊,P＜0.01;＊＊＊,P＜0.001.

Fig. 4 chIRF7-iso positively regulated Fowl adenovirus serotype 4 (FAdV-4) and Newcastle disease virus (NDV) replication in vitro. LMH cells transfected with 1 mg pCAGGS-HA-chIRF7,pCAGGS-HA-chIRF7-iso,or pCAGGS-HA were inoculated with rCH/HNJZ/2015-Δ1966/EGFP or LaSota at an MOI of 0.01.Green fluorescence (A) was regularly observed and aliquots of culture supernatants were collected at 12,24,and 48 h post-infection for FAdV-4 (B) and NDV (C) titration. Data were expressed as mean±SD of three independent experiments. ＊,P＜0.05;＊＊,P＜0.01.

3.2.ldentification the expression of chlRF7-iso

To determine the tissue distribution of chIRF7-iso,PCR was performed using tissue cDNA as template. As shown in Fig.2-A,chIRF7 was clearly detected in lung,spleen,and bursa of fabricius. Although specific bands of chIRF7-iso were observed in the same tissues as chIRF7,its expression level was significantly lower than that of chIRF7. Previous study showed that chIRF7 expressed at low levels in many cells and was strongly induced by IFN-I signaling (Satoet al.1998). Therefore,whether the expression of chIRF7-iso was regulated by upstream signal and the function of chIRF7 in the process still need to be further illustrated.

To confirm the existence of chIRF7-iso at the protein level,pCAGGS-HA-chIRF7 and pCAGGS-HA-chIRF7-iso were constructed and used to generate rabbit polyclonal antibody to detect the endogenous chIRF7-iso in LMH cells through Western blot. As shown in Fig.2-B,a clear band above 50 kDa was observed,demonstrating the expression of chIRF7. Apart from the expected band,an additional smaller one with size of approximately 18 kDa was obtained. All the results directly demonstrated the existence of chIRF7-iso.

3.3.chlRF7-iso negatively regulates lFN-β promoter activity

IRF7 is a critical transcription factor for chickens involved in the induction of IFN-β triggered by pathogen. To better understand the effect of chIRF7-iso on antiviral immunity,pCAGGS-HA-chIRF7,pCAGGS-HA-chIRF7-iso,or pCAGGS-HA as a control,was individually transfected into LMH cells,and proteins were extracted and subjected to 12% SDS-PAGE to detect chIRF7 and chIRF7-iso.As shown in Fig.2-C,both the two proteins were well recognized by anti-HA antibody at 24 and 48 h posttransfection,suggesting that the two plasmids can be used for further study.

To explore the regulatory role of chIRF7-iso in IFN-β production,a chicken IFN-β promoter reporter activity assay was performed. As shown in Fig.3-A,the overexpression of chIRF7 in LMH cells strongly activated the IFN-β promoter at 24 and 48 h post-transfection,with the intensity of green fluorescence only a little bit lower than that of positive control chMDA5. Like the control group,however,the overexpression of chIRF7-iso inhibited the activation of IFN-β promoter,without green fluorescence being observed at 24 and 48 h posttransfection. Consistent with previous data,chIRF7 and chMDA5 overexpression significantly induced the production of IFN-β at 24 and 48 h post-transfection,compared to those of control groups. Strikingly,chIRF7-iso overexpression had no obvious effect on the induction of IFN-β,and its relative mRNA expression levels were sharply lower than those of cells transfected with chIRF7 and chMDA5 (Fig.3-B). The results indicated that chIRF7-iso negatively regulated the IFN-β promoter activation.

3.4.chlRF7-iso is conducive for FAdV-4 and NDV replication in vitro

Since chIRF7-iso negatively regulated IFN-β promoter activity,its antiviral activity was further evaluated.For FAdV-4,green fluorescence intensity of chIRF7-transfected LMH cells was lower than those of chIRF7-iso-and pCAGGS-HA-transfected ones (Fig.4-A),and its viral titer only reached 4.2 log10TCID50/100 μL at 48 h post-infection,which was significantly lower than the corresponding counterparts,with 5.2 and 4.9 log10TCID50/100 μL for those of chIRF7-iso-and pCAGGSHA-transfected cells,respectively (Fig.4-B). Besides,similar results were observed for NDV. The viral titers in chIRF7-iso-transfected cells were dramatically higher than those in chIRF7-transfected ones at 24 and 48 h postinfection (Fig.4-C). These results indicated that chIRF7-iso positively regulated FAdV-4 and NDV replication in LMH cells through blocking IFN-β production.

4.Discussion

Alternative splicing of precursor mRNA is an essential mechanism to increase the complexity of gene expression,and spliced isoforms of many immune related proteins,such as STING (Wanget al.2018),TRIM9 (Qinet al.2016),TRAF3 (Weiet al.2018),and MAVS (Lakhdariet al.2016),have been fully described(Ming and Jie 2017). Four IRF7 isoforms have been reported in human,among which only IRF7C contains the complete DBD with unique 13 aa at the C-terminus.In this study,we identified an IRF7C-like isoform in chicken,termed chIRF7-iso,which was composed of the N-terminal DBD plus 14 aa different from chIRF7 in the C-terminal. Western blot analysis directly demonstrated the expression of chIRF7-iso in LMH cells at protein level.However,the distribution and expression level of chIRF7-iso in different tissues remain unknown.

It is well-documented that IRF7C plays various roles in EBV infection. Zhaoet al.(2010) reported that IRF7C was upregulated at RNA and protein levels in EBV type III latency cell lines,and importantly,IRF7C repressed the transactivation of IRF7-mediated IFN promoterviaDBD to constrain the spontaneous IFN production during EBV transformation (Zhaoet al.2010). Data indicated that EBV facilitated the expression of IRF7C,which might competitively bind to IFN promoter to facilitate virus induced transformation process. Herein,we found that chIRF7-iso,like IRF7C,was able to block the activation of IFN-β promoter and further supported NDV replication in LMH cells. However,the functional discrepancy of chIRF7-iso during DNA and RNA virus infection and the exact molecular mechanism are worth exploring. Besides IRF7A and IRF7C,both IRF7B and IRF7H play vital roles in establishing antiviral state. And whether counterparts of IRF7B and IRF7H exist in chickens and their functional roles in immune response also deserve further studies.

5.Conclusion

In this study,chIRF7-iso generated by alternative splicing was identified as a novel short transcript isoform of chIRF7. And chIRF7-iso acted as an effective negative regulator of IFN-β promoter and supported FAdV-4 and NDV replicationinvitro. All the research will further improve our understanding of chicken immune system and be conducive to illustrating the molecular mechanism of addressing pathogens infection.

Acknowledgements

This work was supported by the grants from the National Natural Science Foundation of China (32002259) and the Natural Science Foundation of Henan Province,China(202300410198).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Ethical approval

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People’s Republic of China. All experiments were performed in a biosafety-level 2 facility at Henan Agricultural University,China.