https://orcid.org/0000-0002-0448-8467
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Figures
Abstract
Subgroup J avian leukosis virus (ALV-J), a retrovirus, elicits immunosuppression and persistent infections in chickens. Although it is widely acknowledged that ALV-J can evade the host’s innate immune defenses, the mechanisms behind this immune evasion remain elusive. N6-methyladenosine (m6A), the most prevalent internal RNA modification, plays a role in innate immune evasion. Our research identified ALV-J as an inefficient stimulator of innate immunity in vitro and in vivo, with its genomic RNA featuring m6A modifications predominantly in the envelope protein (Env) region and 3′ untranslated region (3′UTR). To elucidate the functional consequences of m6A modification, we subsequently generated m6A-deficient ALV-J through its culturing in the DF-1 overexpressing fat mass and obesity-associated protein (FTO) cells. The m6A-deficient ALV-J virus, or its RNAs significantly enhanced IFN-β production compared to the wild-type (wt) ALV-J, suggesting a pivotal regulatory function of m6A modifications in modulating innate immune response. Mechanistically, the m6A modification of the ALV-J genomic RNA directly impacted its recognition by MDA5, weakening its binding and ubiquitination and attenuating IFN-β activation. Moreover, m6A-deficient ALV-J, created by inducing mutations in m6A sites within Env and 3′UTR, exhibited reduced replication capacity and elevated IFN-β expression in host cells. Importantly, this phenomenon was abolished in MDA5-knockout DF-1 cells, further demonstrating the core role of MDA5. These data demonstrate that m6A modification of ALV-J genomic RNA dampens the host’s innate immune response through MDA5 signaling pathway.
Author summary
ALV-J has a long latency and can cause persistent infections with continuous viral shedding, leading to severe immunosuppression and tumor induction in chickens and consequently significant economic losses to the poultry industry. However, the mechanisms by which ALV-J escapes the host’s innate immune recognition and clearance and achieves persistent infection remain elusive. In this study, we systematically identified, for the first time, that the m6A modifications of ALV-J genomic RNA were primarily concentrated in the Env and 3′UTR regions and that this modification is crucial in the viral evasion of the host’s innate immunity. Furthermore, we parsed the in-depth mechanism that the m6A modifications of ALV-J RNA mediate the evasion of RNA recognition by the host’s innate immunity through the MDA5 signaling pathway to attenuate IFN-β expression. Our findings reveal the significance of m6A modifications in the immune evasion strategies of ALV-J and offer a novel perspective on the complex interactions between the virus and the host’s innate immune response.
Citation: Yu M, Zhang L, Wang Y, Wang S, Liu Y, Liu P, et al. (2025) N6-methyladenosine modification of the subgroup J avian leukosis viral RNAs attenuates host innate immunity via MDA5 signaling. PLoS Pathog 21(4): e1013064. https://doi.org/10.1371/journal.ppat.1013064
Editor: Richard A. Koup, National Institutes of Health-NIAID, UNITED STATES OF AMERICA
Received: September 26, 2024; Accepted: March 21, 2025; Published: April 8, 2025
Copyright: © 2025 Yu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data are in the manuscript and Supporting Information files.
Funding: This work was supported by National Natural Science Foundation of China (32230105) (YG), National Key R&D Program of China (2023YFE0106100) (SW), China Agriculture Research System (CARS-41) (YG), and National Major Agricultural Science and Technology Projects (NK2022100103) (YG). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Avian leukosis (AL) is a general term for contagious tumor diseases caused by the avian leukosis virus (ALV) [1]. The ALV has been classified into 11 subgroups (A–K) based on virus envelope genes and serological cross-neutralization experiment [2–4]. Among them, ALV-J has the strongest transmission ability and pathogenicity and induces the formation of tumors such as myelocytoma and hemangioma [3,5]. ALV-J, as a retrovirus, is primarily transmitted vertically. Young chicks are the most susceptible to ALV-J, however, most infected flocks do not have tumors until around the egg production stage. After infection, ALV-J has a long latency period, and there is persistent infection and uninterrupted shedding of the virus, causing critical damage to the production performance of infected chicks and threatening the healthy development and seed security of the poultry industry [5–7].
When the virus infects the host, it activates the host’s innate immunity, which is the first line of defense for the host against viral infections. The innate immune response primarily involves the host’s recognition of pathogens through various pattern recognition receptors (PRRs) [8] and the induction of the host to produce type I interferon or other antiviral factors, protecting the host [9–11]. To better survive and replicate in the host, the virus has evolved various strategies to evade the innate immune response of the host, such as targeting negative regulatory factors of interferon signaling pathways using viral structural or non-structural proteins, expressing viral miRNAs or regulating host miRNA expression to target host proteins and suppress host antiviral responses, or the special structure and epitranscriptomic modification of viral nucleic acids and proteins to PRRs [12–14]. Among them, the modification of viral nucleic acids has received significant attention recently [15].
RNA has over 160 modification types, among which N6-methyladenosine (m6A) modification is the most common mRNA epitranscriptomic modification, with over 25% of mammalian transcripts exhibiting m6A modification [16–18]. m6A modification is reversible and is primarily regulated by two classes of intracellular proteins. The “writers” protein of m6A methylation catalyzes the methylation of m6A on specific motifs RRACH and primarily comprises two subunits, namely METTL3 (catalytic enzyme) and METTL14 (an allosteric activator) [19–21]. The “erasers” of m6A modification include RNA demethylase FTO and ALKBH5 [22–24], which can selectively remove m6A modification. The m6A modification is primarily recognized by m6A “reader” proteins (YTHDF1, YTHDF2, YTHDF3, and YTHDC1) [25,26]. The binding of m6A “reader” proteins to m6A modification markedly influences RNA transport, stability, localization, and translation [26–28]. Viruses are strictly intracellular parasites that can acquire m6A modification within the host. Since the 1970s, m6A modifications have been found in the genomes of influenza A virus, simian virus 40, human immunodeficiency virus (HIV), and adenovirus with nuclear replication [29–31]. m6A modification can mask their nucleic acids to evade the monitoring by the host’s innate immunity and achieve persistent infection [32–34].
Similarly, ALV-J is an intracellular nucleus-replicating avian virus that evades the host’s immune surveillance, establishes persistent infection in the host, and induces tumor formation [12,35]. Recent reports indicated that the RNA m6A modification is essential in regulating the immune system. However, data confirming the m6A modification of ALV-J genomic RNA and explaining its mechanism in the innate immune response to ALV-J are lacking. In this study, we found that the envelope protein (Env) and 3′untranslated region (3′UTR) regions of the ALV-J genome are enriched with m6A modifications, which can reduce PRRs sensing and IFN-β production. We also observed that the m6A modification of viral RNA primarily inhibits IFN-β expression by weakening MDA5 recognition of viral RNA, diminishing the binding between MDA5 and viral RNA and reducing MDA5 ubiquitination. In addition, m6A-deficient ALV mutants induced a stronger IFN response in vivo and in vitro, attenuating the replication ability.
Results
ALV-J is an inefficient stimulator of innate immunity
To understand the innate immune response triggered by ALV-J in vivo and in vitro, we initially inoculated ALV-J strain HLJ09SH05 into DF-1 cell lines and chicken primary macrophages. Real-time quantitative polymerase chain reaction (RT-qPCR) results showed that HLJ09SH05 successfully infected and was replicated in both cell types (Fig 1A and 1B). However, it did not stimulate Interferon-β (IFN-β) (Fig 1C and 1D), IFN-α-stimulated gene 1 (ISG12–1) (Fig 1E and 1F), IFN-induced transmembrane protein 3 (IFITM3) (Fig 1G and 1H), Zinc-finger antiviral protein (ZAP) (Fig 1I and 1J), or single-copy antiviral gene (Mx-1) (Fig 1K and 1L) production. This was in contrast to the robust expression induced by transfection with poly (I:C) at 1ug/ml in both cell types. Furthermore, we assessed the IFN-β expression of chickens infected with the ALV-J strain HLJ09SH05. The serum from 1 to 7 days post-inoculation (dpi) was collected to be detected virus viremia and IFN-β induction. These results indicated that HLJ09SH05 successfully replicated in the chicken, with viremia detectable on 3 dpi (Fig 1M), however, HLJ09SH05 induced no significant increase in IFN-β production compared with that in the control group (Fig 1N). Taken together, these results suggest that ALV-J, while capable of replication, does not effectively trigger a robust innate immune response in vivo and in vitro, suggesting that ALV-J is a poor inducer of innate immune responses.
(A-L) Following infection with the prevalent virus strain HLJ09SH05, DF-1 cells and primary macrophages were assessed the mRNA expression levels of p27 in DF-1 cells (A) and chicken primary macrophages (B), IFNβ in DF-1 cells (C) and chicken primary macrophages (D), ISG12-1 in DF-1 cells (E) and chicken primary macrophages (F), chIFITM3 in DF-1 cells (G) and chicken primary macrophages (H), ZAP in DF-1 cells (I) and chicken primary macrophages (J), and Mx-1 in DF-1 cells (K) and chicken primary macrophages (L) using the RT-qPCR at 3 to 72 hpi. (M and N) Similarly, 1-day-old SPF chickens were intraperitoneally inoculated with 104 TCID50 HLJ09SH05 strain, and viral loads in whole-blood samples were measured using RT-qPCR (M), and the IFN-β was also evaluated using chicken IFN-β ELISA kit. The relative amounts of p27, IFN-β ISG12-1, IFITM3, ZAP, and Mx-1 mRNA were normalized to the GAPDH mRNA levels in each sample, and the fold differences were compared with those in the mock samples. *:p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001; ns: no significant difference.
ALV-J genomic RNA is modified by m6A methylation
To systematically determine whether the ALV-J genome exhibits m6A modifications and their possible locations in the genome, the mRNAs from HLJ09SH05-infected DF-1 cells were subjected to m6A immunoprecipitation, followed by high-throughput sequencing (MeRIP-seq). Sequencing results indicated that there were seven m6A peaks in the HLJ09SH05 genome, most of which were at the Env and 3′UTR regions (Fig 2A and 2B). Sequence analysis revealed that 87.5% (14/16) of the predicted m6A motifs within the Env and 3’UTR regions are relatively conserved (Fig 2C). To further confirm the presence of m6A modification in the ALV-J genome, we also investigated the pharmacological inhibition of m6A modification of ALV-J RNA using 3-deoxyadenosine (DAA), a known inhibitor of S-adenosylhomocysteine (SAH) hydrolase that catalyzes the reversible hydrolysis of SAH to adenosine and homocysteine. DAA causes the accumulation of SAH, which in turn increases the ratio of SAH-to-S-adenosyl methyl thionine (SAM), which is a substrate for m6A modification, and subsequently inhibits SAM-dependent methyltransferases. The DF-1 cells were treated with 10 μM of the m6A-modified inhibitor DAA and inoculated with HLJ09SH05. Compared with untreated DF-1 cells, DAA treatment of DF-1 cells did not affect the replication and release of HLJ09SH05 (Fig 2D), however, it reduced m6A modification levels in HLJ09SH05 RNA by approximately 73% (Fig 2E). These results indicate that the ALV-J genome exhibits the m6A modification, which is primarily enriched in the Env and 3′UTR regions.
(A and B) Distribution of m6A peaks in the ALV-J genome. The total RNA of DF-1 cells infected with HLJ09SH05 was extracted at 4 dpi and subjected to m6A-specific antibody immunoprecipitation, followed by high-throughput sequencing (MeRIP-seq). Burgundy red areas illustrate the distribution of m6A immunoprecipitation reads aligned to the ALV-J mRNAs, while the baseline signal from input samples is depicted as a continuous line. (B) The distribution area of m6A modification sites in the genomic RNA of HLJ09SH05 was demonstrated, and the presence of m6A motifs (RRACH) was searched for in the identified m6A peaks, verifying the predicted m6A sites. (C) Conservation analysis of 16 m6A motifs within the Env and 3’UTR regions. The Sequence Logos of 16 m6A motifs within the Env and 3’UTR regions were drawn by Web Logo software (http://weblogo.berkeley.edu/logo.cgi).All sequences of ALV-J strains were obtained from GenBank. (D) DAA treatment did not affect the replication and release of the virus. DF-1 cells were treated with DMSO as control or DAA (10 μM) for 4 h and transfected with the HLJ09SH05. The cell supernatants were collected at 4 dpi. (E) DAA treatment reduced the m6A modifications of ALV-J RNA. DF-1 cells were treated with DMSO as control or DAA (10 μM) for 4 h and transfected with the HLJ09SH05. ALV-J in the supernatants was collected at 4 dpi.
m6A-deficient ALV-J induces high IFN-β expression
To determine the role of viral m6A methylation in ALV-J-induced innate immunity, we first constructed a FTO-overexpressing DF-1 cell line (FTO-OE DF-1), which is an eraser protein that can remove m6A modification on mRNAs. The indirect immunofluorescent assay (IFA) results indicated that FTO-OE DF-1 cells expressed FTO and showed specific green fluorescence. However, the wt DF-1 cells displayed no fluorescence (Fig 3A). Similarly, western blotting results showed that FTO-OE DF-1 cells produced approximately 56-kDa band with the anti-FLAG monoclonal antibody (Fig 3B). These results showed that the FTO-OE DF-1 cell line was successfully constructed. To obtain an m6A-deficient virus, the FTO-OE DF-1 cells were infected with HLJ09SH05, and the cell supernatant was collected at 7 dpi. The Enzyme-linked immunosorbent assay (ELISA) of the m6A levels showed that the RNA of the virion harvested from FTO-OE DF-1 cells was demethylated, with approximately 90% decrease in the m6A modification levels compared with that of HLJ09SH05 RNA (Fig 3C). The m6A-deficient virus was named SH5-FTO-OE.
(A and B) Construction of FTO-OE DF-1 cell line. (A) IFA. The DF-1 cells and FTO-OE DF-1 cells were detected with the Flag monoclonal antibody and analyzed using a fluorescence microscope; the DF-1 cells were the reference. Scale bar: 400 μm. (B) Western blotting. The lysates of the DF-1 cells and FTO-overexpressing DF-1 cells were detected with the Flag monoclonal antibody. (C) Relative m6A levels in the virion RNA from ALV grown on FTO-OE DF-1 andwt DF-1 cells. We inoculated ALV-J into FTO-OE DF-1 cell lines to obtain viruses with m6A deficiency. The m6A levels for each virion RNA were quantified using an m6A methylation kit. (D) m6A-deficient and wt ALV-J induced IFN-β mRNA expression after infecting DF-1 cells. DF-1 cells were infected with m6A-deficient or wt ALV-J. At 3, 6, and 12 hpi, cells were collected for the analysis of IFN-β mRNA levels using RT-qPCR. (E) m6A-deficient and wt ALV-J induced IFN-β mRNA expression after infecting chicken primary macrophage cells. Chicken primary macrophage cells were infected with m6A-deficient or wt ALV-J. At 3, 6, and 12 hpi, cells were collected for the analysis of IFN-β mRNA levels using RT-qPCR. (F) The RNAs of m6A-deficient and wt ALV-J induced IFN-β mRNA expression after transfecting in DF-1 cells. DF-1 cells were transfected with 108 copies of the m6A-deficient or wt ALV-J RNA. Cells were collected at 3, 6, and 12 hpt to analyze IFN-β mRNA levels using RT-qPCR. (G) The RNAs of m6A-deficient and wt ALV-J induced IFN-β mRNA expression after transfecting in chicken primary macrophage cells. chicken primary macrophage cells were transfected with 108 copies of the m6A-deficient or wt ALV-J RNA. Cells were collected at 3, 6, and 12 hpt to analyze IFN-β mRNA levels using RT-qPCR.
To determine the effect of m6A modification of ALV RNA on IFN-β induction during viral infection, SH5-FTO-OE and HLJ09SH05 were separately incubated with DF-1 cells. RT-qPCR results showed that SH5-FTO-OE induced approximately 2.3- and 2.85-fold higher IFN-β expression levels compared to that of HLJ09SH05 at 6 and 12 h post-inoculation (hpi) (Fig 3D). To validate the observations in the DF-1 cells, we also assessed IFN-β expression levels in chicken primary macrophages induced by SH5-FTO-OE and HLJ09SH05. The results also showed that SH5-FTO-OE induced 1.5–2.1-fold higher IFN-β mRNA levels compared to that of HLJ09SH05 at 3–12 hpi (Fig 3E). To eliminate the effects of ALV-J replication and viral protein synthesis on the induced IFN-β expression levels, we separately transfected the RNAs of SH5-FTO-OE and HLJ09SH05 into DF-1 cell and found that the viral RNA of SH5-FTO-OE also induced a significantly 1.9–2.7-fold higher IFN-β expression levels compared to that of the HLJ09SH05 RNA at 3–12 hpi (Fig 3F). Similar results were also observed in chicken primary macrophages (Fig 3G). Taken together, these results indicate that m6A-deficient ALV-J SH5-FTO-OE can induce significantly higher levels of IFN-β compared to that of m6A-sufficient HLJ09SH05.
m6A modification of ALV-J RNA regulates the recognition by MDA5
Previous research has demonstrated that MDA5 and TLR7 recognize the viral RNA of ALV when ALV infects host cells [18]. To investigate how ALV-J evades innate immune recognition through genomic m6A modification, we first selected three siRNAs for silenced TLR7. RT-qPCR results indicated that siTLR7–1 significantly reduced TLR7 expression levels (S1A Fig). Subsequently, SH5-FTO-OE and HLJ09SH05 were separately inoculated with siTLR7–1-transfected or siSC-transfected DF-1, and the IFN-β levels were determined using RT-qPCR. The results showed that SH5-FTO-OE and its RNA induced higher levels of IFN-β expression in siTLR7 DF-1 and wt DF-1 cells, compared to HLJ09SH05 (S1B Fig) and its RNA (S1C Fig). These results indicate that TLR7 does not play a role in recognizing the m6A-deficient viral genome of ALV-J. Next, we also investigated the potential role of MDA5 in sensing m6A-deficient ALV RNA. we first selected three siRNAs for silenced MDA5. RT-qPCR results indicated that three siMDA5s significantly reduced MDA5 expression levels (Fig 4A). Subsequently, SH5-FTO-OE and HLJ09SH05 were separately inoculated with siMDA5–1-transfected or siSC-transfected DF-1 cells, and the IFN-β levels were determined using RT-qPCR. The results indicated that SH5-FTO-OE triggered higher IFN-β levels compared to that of HLJ09SH05 in siSC-transfected DF-1 cells, as expected (Fig 4B). However, SH5-FTO-OE and HLJ09SH05 induced similar IFN-β expression levels in siMDA5–1-transfected DF-1 cells (Fig 4B). To further validate this result, the RNAs of both viruses were separately transfected into siMDA5–1-transfected or siSC-transfected cells, and the IFN-β expression levels were determined. Consistent with the results of virus infection, when MDA5 was knocked down, the RNA of m6A-deficient viruses could not induce high IFN-β expression levels (Fig 4C). Similarly, we inoculated siMDA5–1-transfected and siSC-transfected primary macrophages with HLJ09SH05 and SH5-FTO-OE or transfected them with the RNAs of HLJ09SH05 and SH5-FTO-OE to determine IFN-β expression levels using RT-qPCR. The results showed that there were no differences in the IFN-β expression levels induced by SH5-FTO-OE and its RNA compared to those induced by HLJ09SH05 and its RNA in siMDA5–1-transfected chicken primary macrophages (Figs 4D and 4E). In contrast, in siSC- transfected chicken primary macrophages, SH5-FTO-OE and its RNA induced IFN-β expression, which were 1.59- (Fig 4D) and 1.51-fold (Fig 4E) of those induced by HLJ09SH05 and its RNA, respectively. These results suggest that MDA5 is the cellular sensor for detecting m6A-deficient ALV-J RNA.
(A) Validation of the optimal siRNA targeting MDA5 using RT-qPCR. (B) The effect of MDA5 knockdown on m6A-deficient virus-induced IFN-β mRNA expression in DF-1 cells. The DF-1 cells were transfected with 2 μg siMDA5-1 or negative siRNA control siSc. for 24 h and were subsequently infected with m6A-deficient or wt ALV-J for 24 h. (C) The effect of MDA5 knockdown on the RNA of m6A -deficient virus-induced IFN-β mRNA levels in DF-1 cells. The DF-1 cells were transfected with 2 μg siMDA5-1 or negative siRNA control siSc. for 24 h and were subsequently transfected with 108 copies of m6A-deficient or wt ALV-J for 12 h. (D) The effect of MDA5 knockdown on m6A-deficient virus-induced IFN-β mRNA expression in chicken primary macrophages. The chicken primary macrophages were transfected with 2 μg siMDA5-1 or negative siRNA control siSc. for 24 h and were subsequently infected with m6A-deficient or wt ALV-J for 24 h. (E) The effect of MDA5 knockdown on the RNA of m6A -deficient virus-induced IFN-β mRNA levels in chicken primary macrophages. The chicken primary macrophages were transfected with 2 μg siMDA5-1 or negative siRNA control siSc. for 24 h and were subsequently transfected with 108 copies of m6A-deficient or wt ALV-J for 12 h. (F) The MDA5 mRNA level of DF-1 and MDA5-KO DF-1 cell lines. (G) Western Blotting. The DF-1 and MDA5-KO DF-1 cell lines were detected by MDA5 antibody. (H) Sequence analysis of DF-1 and MDA5-KO DF-1 cell lines. (I) Cell viability of DF-1 and MDA5-KO DF-1 cell lines. (J) Poly (I:C) stimulated IFN-β production in MDA5-KO and DF-1 cells. Similarly, 1 μg/mL of poly (I:C) was transfected into MDA5-KO DF-1 and DF-1 cells, and after 12 h, the mRNA levels of IFN-β were evaluated using RT-qPCR. (K) m6A-deficient and wt ALV-J induced IFN-β mRNA expression after infecting cells. DF-1 and MDA5-KO DF-1 cell lines were infected with m6A-deficient or wt ALV-J. At 12 hpi, cells were collected to analyze IFN-β mRNA levels using RT-qPCR. (L) The RNAs of m6A-deficient and wt ALV-J induced IFN-β mRNA expression after transfecting in cells. DF-1 and MDA5-KO DF-1 cell lines were transfected with 108 copies of the m6A-deficient or wt ALV-J RNA. At 12 hpt, cells were collected to analyze IFN-β mRNA levels using RT-qPCR.
To further confirm the results of the MDA5 knockdown, we constructed the MDA5-knockout (KO) DF-1 cell line with CRISPR/Cas9. RT-qPCR result showed that MDA5 was barely detectable in the MDA5-KO DF-1 cell line (Fig 4F). Western blotting results demonstrated that MDA5 was undetectable in MDA5-KO DF-1 cells when using the MDA5 monoclonal antibody prepared by Sino Biological, whereas MDA5 could be detected in DF-1 cells (Fig 4G). The sequence results showed that the 14 nucleotides at the N-terminus of the MDA5 open reading frame from the MDA5-KO DF-1 cell line were deleted (Fig 4H). The cell counting kit-8 (CCK8) assay also indicated that MDA5 KO did not affect cell viability (Fig 4I). Additionally, we transfected poly (I:C) into MDA5-KO and wt DF-1 cells and evaluated the IFN-β levels. These results showed that MDA5-KO DF-1 cells did not induce robust IFN-β expression in response to poly (I:C) stimulation (Fig 4J). These results demonstrated the successful construction of the MDA5-KO DF-1 cell line. Subsequently, SH5-FTO-OE and HLJ09SH05 were inoculated into MDA5-KO and wt DF-1 cells. Similar to HLJ09SH05, SH5-FTO-OE did not induce high IFN-β expression levels in MDA5-KO DF-1 cells (Fig 4K). However, it triggered a 1.5-fold increase in IFN-β expression levels in wt DF-1 cells. To further validate this result, the RNAs of both viruses were separately transfected into MDA5-KO and wt DF-1 cells. Consistent with the results of virus infection, the IFN-β expression levels of the RNAs from the two viruses in MDA5-KO DF-1 cells were no discernible difference (Fig 4L). However, the IFN-β expression induced by SH5-FTO-OE RNA in wt DF-1 cells was 2.4 times higher than those induced by HLJ09SH05 RNA (Fig 4L). These results suggest that MDA5 is a pivotal cellular sensor for detecting m6A-deficient ALV-J RNA.
m6A-deficient viral RNA enhances RNA recognition by and ubiquitination of MDA5
To further explore the mechanisms by which m6A inhibits the innate immune response to viral RNA, we directly compared the binding affinity of m6A-sufficient (HLJ09SH05) and m6A-deficient (SH5-FTO-OE) RNAs with MDA5. Briefly, cells expressing Flag-tagged MDA5 were lysed and pulled down with magnetic beads conjugated with Flag antibodies (Fig 5A). Then complexes were divided equally, and mixed with each of the virion RNAs, and the RNA in the complex was quantified using RT-qPCR. The amount of SH5-FTO-OE RNAs pulled down by MDA5 was approximately thrice that of HLJ09SH05 RNAs pulled down by MDA5 (Fig 5B). This data indicated that m6A deficiency enhanced the binding of ALV-J RNA to MDA5. After binding to RNA, MDA5 undergoes significant conformational changes, enabling ubiquitination of MDA5, which is crucial in activating its IFN-I signaling pathway [36]. To test whether m6A-deficient ALV-J RNA can promote MDA5 ubiquitination, the pFlag-MDA5 and pHA-Ub plasmids were co-transfected into DF-1 cells for 24 h, and SH5-FTO-OE and HLJ09SH05 RNAs were separately transfected into the cells. Western blotting result showed that SH5-FTO-OE RNA had a stronger ability to promote MDA5 ubiquitination compared to that of HLJ09SH05 RNA (Fig 5C). The above results demonstrate that m6A-deficient ALV-J RNA enhances its binding ability with MDA5 and promotes MDA5 ubiquitination.
(A and B) m6A-deficient virion RNA increased MDA5 binding. MDA5-conjugated magnetic beads were incubated with the RNAs of m6A-deficient and wt ALV-J. (A) One aliquot of beads was analyzed using western blotting. (B) RNA bound to magnetic beads was quantified using RT-qPCR. Results were normalized as the ratio between immunoprecipitated RNA from m6A-deficient and wt ALV-J. (C) The ubiquitination analysis of MDA5. The m6A-deficient ALV-J virion RNA enhanced MDA5 ubiquitination compared with the wt ALV-J RNA. Similarly, 1 μg each of pFlag-MDA5 and pHA-Ub was transfected into DF-1 cells after 24 h, and 108 RNA copies of m6A-deficient and wt ALV-J virions were transfected into DF-1 cells. Ubiquitination of MDA5 was analyzed using an anti-Flag blot. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001; ns: no significant difference.
ALV-J m6A-deficient RNA affects its replication and the IFN-β expression levels in vitro and in vivo
Owing to the m6A modification of RNA being reversible [37,38], to determine the impact of m6A modification in HLJ09SH05 genomic RNA on virus replication ability, we first rescued the wt ALV-J (rSH5) and m6A-deficient ALV-J virus (rSH5-D-m6A) by mutating all m6A modification sites in the Env and 3′UTR regions in DF-1 cells. The IFA results indicated that DF-1 cells inoculated with rSH5-D-m6A and rSH5 showed specific green fluorescence, uninfected cells did not show such fluorescence (Fig 6A). Western blotting results showed that rSH5-D-m6A and rSH5 produced a specific 27-kDa band with the 2E5 monoclonal antibody [39] (Fig 6B). Additionally, RNA was extracted from both viruses for m6A modification level analysis. ELISA result showed that the m6A modification levels in rSH5-D-m6A RNA were approximately 80% lower than those of rSH5 RNA (Fig 6C). These results indicate the successful rescue of the parental virus rSH5 and m6A-deficient virus rSH5-D-m6A.
(A) IFA assay. DF-1 cells are infected with wt ALV-J rSH5 and m6A-deficient virus rSH5-D-m6A for 72 h, and then detected with the 4A3 monoclonal antibody and analyzed using a fluorescence microscope, rSH5 as reference. Scale bar: 400 μm. (B) Western blotting. The wt ALV-J rSH5 and the m6A-deficient virus rSH5-D-m6A were detected with the 2E5 monoclonal antibody, rSH5 was the reference. (C) Relative m6A levels in the RNAs from rSH5-D-m6A and rSH5 virions. The m6A levels for each virion RNA were quantified using an m6A methylation kit. (D) The replication abilities of rSH5-D-m6A and rSH5 were detected using TCID50 in vitro. DF-1 cells were incubated with SH5-D-m6A and rSH5 at an MOI of 0.01 and harvested and quantified at 1, 2, 3, 4, 5, 6, and 7 dpi. (E) The replication abilities of rSH5-D-m6A and rSH5 were determined using TCID50 in vitro. MDA5-KO DF-1 cells were incubated with rSH5-D-m6A and rSH5 at an MOI of 0.01 and harvested and quantified at 1, 2, 3, 4, 5, 6, and 7 dpi. (F) rSH5-D-m6A and rSH5 induced IFN-β mRNA expression after infecting DF-1 cells. DF-1 cells were infected with rSH5-D-m6A and rSH5. At 12 and 24 hpi, cells were collected to analyze IFN-β mRNA levels using RT-qPCR. (G) rSH5-D-m6A and rSH5 induced IFN-β mRNA levels after infecting MDA5-KO DF-1 cells. MDA5-KO DF-1 cells were infected with rSH5-D-m6A and rSH5. At 12 and 24 hpi, cells were collected to analyze IFN-β mRNA levels using RT-qPCR. (H and I) 1-day-old SPF chickens were intraperitoneally inoculated with rSH5-D-m6A and rSH5 (n = 10, dose = 104 TCID50). (H) Viral loads in whole-blood samples were measured using RT-qPCR. (I) The IFN-β levels were also evaluated using a chicken IFN-β ELISA kit at different time points.
Subsequently, to assess the replication capacity of the rSH5 and the rSH5-D-m6A, DF-1 and MDA5-KO DF-1 cells were inoculated with rSH5 and rSH5-D-m6A, and cell samples were collected from 1 to 7 dpi. The 50% tissue infection dose (TCID50) results showed that the titer of the rSH5-D-m6A was reduced by approximately 1.8- to 6.4-fold at 2–7 dpi compared with that of the rSH5 in DF-1 cells (Fig 6D). However, in MDA5-KO DF-1 cells, there were no significant differences in the viral titer between both viruses from 1 to 7 dpi (Fig 6E). Furthermore, we examined IFN-β expression levels with rSH5 and rSH5-D-m6A in DF-1 and MDA5-KO DF-1 cells. The results showed that in DF-1 cells, rSH5-D-m6A induced a 2.4–2.6 fold increase in IFN-β expression levels compared to that of rSH5 (Fig 6F). In contrast, there were no significant differences in the IFN-β expression levels induced by both viruses in MDA5-KO DF-1 cells (Fig 6G). These results indicate that lack of m6A modification in the ALV-J genome RNA inhibits viral replication and induces a significantly higher level of IFN-β expression in DF-1 cells and further demonstrate that m6A modification of ALV-J RNA escapes recognition by MDA5.
To investigate the impact of m6A modification in the ALV-J genome on the replication of ALV-J and the induced expression levels of IFN-β in vivo, 104 TCID50 of rSH5 and rSH5-D-m6A were intraperitoneally injected into 1-day-old specific pathogen-free (SPF) chickens, respectively, and blood samples were collected between 1 and 7 dpi. RT-qPCR results showed that the viral loads of the rSH5 group were 2.2–3.8-fold higher than those of the rSH5-D-m6A group (Fig 6H). The ELISA result indicated that rSH5-D-m6A induced IFN-β expression in SPF chicken similar to that induced by the rSH5 group between 1 and 4 dpi. However, at 5–7 dpi, the IFN-β expression levels in the rSH5-D-m6A group were higher than those in the rSH5 and control groups (Fig 6I). These results also show that m6A-deficient ALV-J RNA inhibits viral replication and induces significantly higher IFN-β expression in vivo.
Discussion
Recent reports indicate that the m6A modification of RNA, as the most common mRNA modification, is significant in the life cycles of numerous viruses and the innate immune response to viral infection [29–31,40]. In this study, we found that ALV-J only induces a weak innate immune response, and its genome is enriched with m6A modifications in the Env and 3’ UTR regions. Further analysis revealed that the production of IFN-β is almost undetectable in the presence of m6A modifications in the ALV-J genome. More importantly, our result found that the m6A modification of ALV-J RNA primarily weakens MDA5 recognition of viral RNAs, diminishes the binding between MDA5 and viral RNA, and reduces MDA5 ubiquitination to inhibit IFN-β expression (Fig 7). These findings preliminarily elucidate the mechanisms by which m6A modifications enable ALV-J to evade host immune defenses and establish persistent infections.
ALV-J featuring m6A-modified RNA (blue dots) circumvents innate sensing in infected cells, effectively escaping immune detection. Conversely, ALV-J with m6A-deficient RNA, upon entering cells, enhances the interaction between MDA5 and viral RNA and MDA5 ubiquitination, then triggers the recruitment of the adaptor protein MAVS, which subsequently leads to IRF-7 phosphorylation ((indicated by the letter P). This process culminates in the formation of IRF-7 homodimers and/or heterodimers, their translocation into the nucleus, and ultimately, the expression of type I IFNs. This picture was drawn by Figdraw.
Viruses need to evade the host’s innate immune response for infection establishment and persistence. It is reported that HIV infection in cells cannot effectively activate the innate immune response, causing the establishment of persistent infection [41]. Another retrovirus, Simian immunodeficiency virus, is also a poor inducer of innate immunity in pig-tailed macaques, African green monkeys, and mandrills, causing long-term infection [42,43]. In addition, our study proved that ALV-J, as a simple retrovirus, was not effective in inducing the innate immune response after infecting DF-1 cells, chicken primary macrophages, and SPF chickens. These data suggest that retroviruses are poor inducers of innate immune responses, preventing the host from clearing the virus and leading to persistent infection.
Viruses usually evade host immune surveillance through protein and nucleic acid modifications [44–46]. Among these, m6A modification of nucleic acids affects mRNA metabolism, translation, and splicing, influencing virus replication, and also plays a critical role in the innate immune response [47,48]. Studies have reported that m6A modifications occur in the genomic RNAs of viruses such as HCV, ZIKV, HIV, HSV-1, and HBV [49–55]. Ji et al. recently predicted the m6A modification of ALV-J RNA using SRAMP software. However, this result was not further validated [56]. In this study, we systemically confirmed the presence of m6A modifications in ALV-J RNA using MeRIP-seq and m6A methylase drug inhibition experiments, further identifying its primary localization in the Env and 3′UTR regions. Furthermore, we rescued the virus by mutating all m6A modification sites in the Env and 3′UTR regions, resulting in an 80% decrease in genomic m6A modification levels of ALV-J. Additionally, we performed m6A sequencing of the genomes of ALV-A and ALV-B, discovering that m6A is also mainly concentrated in the Env and 3’ UTR regions (S2 Fig). Notably, previous study has reported that the m6A modifications in the genomic RNA of human immunodeficiency virus (HIV) are also enriched in the Env and 3’ UTR regions [57], suggesting that m6A modifications in the genomes of retroviruses are focused in these areas. This is similar to the pattern observed in flavivirus genomes, where m6A modifications are primarily concentrated in the last viral gene.
The binding of the protein encoded by Env to its specific receptor on the target cell is the first and critical step in determining viral infection [2,54,58,59]. Env is the most variable gene in ALV-J [7,60,61], however, a comparative analysis of its nucleotide sequences from multiple strains of ALV-J revealed that 92.3% (12 out of 13) of the m6A motifs were highly conserved. Further analysis indicates that the relatively conserved E element within the 3’UTR region is another m6A enrichment site, which is closely associated with tumorigenicity [62,63]. This suggests that the ALV-J genome m6A modification sites are relatively conserved. Studies on human respiratory syncytial virus also revealed that despite the G gene having the highest genetic variability, the m6A modification sites within it are highly conserved [64]. This suggests that m6A modification sites in viral genes may provide an evolutionary advantage for viral infection, replication, and transmission, causing their selective retention.
To explore the potential role of m6A modification in ALV-J-induced weak innate immunity, in this study, we obtained m6A-deficient viruses through the FTO-OE DF-1 cell line or by mutating the m6A modification sites of ALV-J genome. These two viruses and their RNAs induced higher IFN-β expression levels in the different cells compared to that of the wt virus and its RNA. These results further demonstrate that m6A modification is crucial in inhibiting the IFN-β production after ALV-J infects the host. Similar phenomena have also been observed in viruses such as HIV, HCV, and hMPV [65–68]. Recent studies suggest that epitranscriptomic modifications of RNA contribute to viral infection by assisting RNA to escape recognition by PRRs. For example, HIV-1 RNA uses 2’-O-methylation to escape MDA5 recognition, inducing lower IFN-I levels [69], m6A modifications assist VSV, RSV, hMPV, and HIV RNAs in evading RIG-I recognition, escaping the innate immune response [67,68,70]. However, chickens lack RIG-I, prompting the investigation of how the m6A modification of ALV-J genomic RNA aids RNA in evading PRRs recognition. Our results confirmed that m6A modification can weaken the recognition and binding of MDA5 to ALV-J genomic RNA and its ubiquitination. These data emphasize that chicken MDA5 can compensate for the function of RIG-I and also suggest that m6A modification in RNA is a molecular marker of host innate immunity, which can be used by RLR pattern recognition receptors to distinguish between self and non-self RNAs.
m6A modifications not only promote viral replication by evading innate immunity but also affect mRNA metabolism, translation, and splicing. For example, the m6A modifications on the mRNA of the highly mutated G protein of the RSV enhance the stability of the mRNA, promoting its translation into more G protein. This leads to increased incorporation of G protein into viral particles, resulting in the production of more infectious viral particles [64]. Additionally, the increased soluble G protein can influence leukocyte migration, thereby regulating the host immune response to RSV [71]. In this study, during the early stage of infection, there is no difference in IFN-β induction between rSH5 and rSH5-D-m6A, but the level of gp85 mRNA is higher in rSH5. It is speculated that the m6A modification in the mRNA of the Env gene may enhance its stability, promote the translation of Env protein and viral assembly, and increase the production of infectious viral particles. Moreover, studies show intracellular Env inhibits IFN-β production, exerting immunosuppression [72], which could explain the reduced IFN-β at 7 dpi, but further verification is needed.
In conclusion, our study demonstrated the pivotal role of m6A modifications within the Env and 3’UTR regions of ALV-J genomic RNA in dampening the host’s innate immune defenses. Specifically, these modifications curtail the capacity of MDA5 to identify viral RNA, thereby hindering a robust immune reaction. Our findings not only elucidated the molecular mechanism by which m6A modification of ALV-J genomic RNA caused the virus evasion of innate immune responses but also provided insights into further elucidating the infection and pathogenic mechanisms of ALV.
Materials and methods
Ethics statement
All animal experiments were approved by the Committee on the Ethics of Animal Experiments of Harbin Veterinary Research Institute (HVRI), Chinese Academy of Agricultural Sciences (CAAS) (240130–01-GR). Specific pathogen-free (SPF) chickens were purchased from the Experimental Animal Centre of the HVRI and housed in negative-pressure isolators with adequate food and light. All animal procedures were performed according to the international standards for animal welfare.
Cells, viruses, and plasmids
HEK293T and DF-1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (L110KJ, BasalMedia, China) supplemented with 10% (vol/vol) fetal bovine serum (FBS) (FDN500, Excell Bio, China) in a humidified incubator with 5% CO2 at 37 °C. DF-1 cells were cultured in DMEM containing 10% FBS. Chicken primary macrophages were prepared from the bone marrow of 3-week-old SPF chickens according to a previously described method [73] and cultured in an RPMI-1640 medium (C11875500BT, Gibco, USA) supplemented with 5% FBS, 5% chicken serum (S9080-500ML, Solarbio, China), 10% tryptose phosphate broth (T8782-500G, Sigma, USA), 1% sodium pyruvate (11360–070, Gibco, USA), and 0.1% β-mercaptoethanol (21985023, Gibco, USA). DF-1 and chicken primary macrophages cells were cultured in a humidified incubator with 5% CO2 at 38.5 °C. The epidemic strain HLJ09SH05 was identified and maintained at the Harbin Veterinary Research Institute, CAAS (Harbin, China).
The pLVX-IRES-Puro, psPAX2, pMD2.G, CRISP/Cas9, pHA-Ub, pFlag-MDA5, pCAGGS, and pBluescript II KS(+) plasmids were maintained in our laboratory. FTO (GenBank accession number: HM050377.1) was amplified from the cDNA of DF-1 cells through RT-PCR and was inserted into the pLVX-IRES-Puro plasmid with a Flag tag at the C-terminus to construct the FTO-OE DF-1 cell line. This plasmid was named pLVX-FTO-Flag.
To construct a pBlueSH5 infectious clone, the full-length cDNA of HLJ09SH05 was inserted into the pBluescript II KS(+) plasmid as described in the previous study [39,62]. Based on MeRIP-seq data, 16 potential m6A sites were found in the Env and 3’UTR regions of the ALV-J genome, which were mutated using synonymous mutations. The resultant plasmid was named pBlueSH5-D-m6A. All plasmid sequences were confirmed through DNA sequencing. The primer sequences for all oligonucleotides used in this study are available upon request.
MeRIP-seq
First, the total RNAs from DF-1 cells infected with HLJ09SH05 were extracted, and the mRNA with poly-A tails within the total RNAs were enriched using Oligo-dT beads. Subsequently, the enriched mRNAs were randomly broken into approximately 100 bp segments by adding divalent cations. The randomly fragmented RNAs were divided into two parts: one part was used to construct a traditional transcriptome sequencing library as a control, while the other part was added to magnetic beads coated with m6A antibodies to enrich for m6A-modified mRNA fragments. Using the enriched mRNA as a template, we synthesized cDNA with random primers and ligated it using splint adapters. Then, we performed PCR amplification on the adapter-ligated products to construct the m6A-seq library. After constructing the library, we used agarose gel electrophoresis to detect RNA contamination and degradation and simultaneously employed Ubit 3.0 to accurately measure the RNA concentration. Once both sequencing libraries were successfully constructed and passed quality control, we subjected them to high-throughput sequencing. The raw sequencing data obtained were then filtered to produce high-quality sequencing data (clean data). Subsequently, we aligned the clean data to the reference genome of the project species to obtain comprehensive transcriptome information, and performed gene expression quantification as well as GO and KEGG Pathway analyses. Additionally, we conducted whole-genome de novo peak calling on the alignment results to investigate the binding preferences of proteins on RNA. Finally, we performed motif analysis on these binding sites to further reveal their functions and characteristics.
Generation of m6A-deficient ALV-J from DF-1 cells treated with DAA
DF-1 cells were pre-treated with 10 μM DAA or DMSO for 4 h before inoculation with 0.01 MOI HLJ09SH05. Subsequently, the cells were supplemented with DMEM containing 10 μM DAA and 2% FBS and subjected to continuous culturing for 4 days. The cell supernatants were collected for virus purification, and the RNA m6A modification was analyzed using an m6A RNA methylation assay kit.
Colorimetric quantification of viral m6A methylation
According to the manufacturer’s instructions, the m6A modification levels of virion RNA were quantified using an m6A RNA methylation assay kit (ab185912, Abcam, UK). Briefly, the same amount of sample RNA, negative control, and positive control were separately added into the wells and incubated at 37 °C for 90 min, followed by three washes with 1× wash buffer. Subsequently, 50 µL of diluted capture antibody (1:1000) was added to each well and incubated at room temperature for 60 min, followed by three washes with 150 µL of 1× wash buffer. Next, 50 µL of diluted detection antibody (1:2000) was added to each well, followed by incubation at room temperature for 30 min, and each well was washed four times with 150 µL of 1× wash buffer. Furthermore, 50 µL of diluted enhancer solution (1:3000) was added to each well and incubated at room temperature for 30 min, followed by five washes with 150 µL of 1× wash buffer. Similarly, 100 µL of substrate solution was added to each well, followed by incubation at room temperature in the dark for 5 min, and 100 µL of stop solution was added to each well to terminate the enzyme reaction. Finally, the absorbance was measured and recorded at a wavelength of 450 nm.
Generation of FTO-OE DF-1 cell line
To obtain a DF-1 cell line stably expressing FTO, we first co-transfected the recombinant plasmid pLVX-FTO-Flag along with the helper plasmids psPAX2 and pMD2.G into HEK293T cells. 48 hours post-transfection (hpt), we collected the cell supernatants and centrifuged them at 5,000×g for 10 min at 4°C. The centrifuged supernatants were then filtered through a 0.45-μM filter and subsequently inoculated into DF-1 cells. After 96 h, the inoculated cells underwent three consecutive rounds of selection using 1.5 μg/mL puromycin to select stably expressing cells. Once the stably expressing cell line was established, we utilized western blotting and IFA to detect the expression of FTO.
Virus production and purification
The wt and FTO-OE DF-1 cells were infected with ALV-J at an MOI of 0.01, and the cell culture supernatants were harvested. First, the cell culture supernatants were clarified through centrifugation at 1,500 × g for 5 min to remove cellular debris, and this supernatant virus was passed through a 0.45-μm syringe filter. Furthermore, the virus was concentrated through a 25% (wt/vol) sucrose cushion via centrifugation at 28,000 × g for 2 h at 4 °C in a type SW32Ti rotor (Beckman, Brea, CA). Finally, the pellet was resuspended in DMEM.
RNA transfection of cells
For RNA transfection, 5×105 DF-1 cells, MDA5-KO DF-1celLs, and chicken primary macrophage cells were then transfected with 108 copies ALV-J RNA using TransIT mRNA transfection kits (MIR 2225, Mirus, USA) according to the manufacturer protocol. At 12 hpt, cells were harvested for measuring IFN-β and GAPDH mRNA by RT-qPCR.
RT-PCR
According to the manufacturer’s instructions, RNA was extracted from cells and virion RNA from purified virus using the RNAiso Plus reagent (9108, TaKaRa, Japan). The RNA was reverse-transcribed into cDNA using a Reverse Transcription Kit (R223-01, Vazyme, China). The quantity of each cDNA was determined through RT-qPCR using the THUNDERBIRD SYBR qPCR Mix Kit (QPS-201, TOYOBO, Osaka, Japan) and analyzed with the QuantStudio 5 system (Applied Biosystems, USA). The specific primers for IFN-β, ZAP, Env, ISG12–1, Mx-1, and IFITM3 were designed according to references [74] and synthesized by Invitrogen (Shanghai, China). The relative mRNA levels of these genes were normalized to chicken GAPDH mRNA levels in each sample.
RNA interference
Three siRNAs specifically targeting chicken TLR7 (siTLR7–1: 5′- GGUGAUGACAGAAUUGGUUdTdT-3′; siTLR7–2: 5′-CCAGAACUCAAGAUACUAAdTdT-3′; siTLR7–3: 5′-CCACCCAACUUAUCUUCAAdTdT-3′), chicken MDA5 (siMDA5–1: 5′- GGUAUCAAGUUAUUGGCUUdTdT-3′; siMDA5–2: 5′-GCAGAACACUUGAAGAAAUdTdT-3′; siMDA5–3: 5′-CCGCCAGAAGAGUAUUUAAdTdT-3′) and a scramble negative control siRNA (5′-UUCUCCGAACGUGUCACGUdTdT-3′) were synthesized by Seven Biotech (Beijing, China). According to the manufacturer’s instructions, the siRNA transfections were performed in DF-1 cells using X-tremeGENE siRNA Transfection Reagent (04476115001, Roche, USA). After 24 h of transfection, cells were harvested to determine the knockdown efficiency using RT-qPCR. In addition, after 24 h of siRNA transfection, DF-1 cells were transfected with viral RNA or inoculated with ALV-J for subsequent analyses.
Generation of MDA5-KO DF-1 cell line
CRISPR/Cas9 was used to knock out the MDA5 gene in DF-1 cells. The sgRNA (5′-CGGATGGTTCACTGAATTCC-3′) was designed using E-CRISPR (http://www.e-crisp.org/E-CRISP/designcrispr.html). The DNA fragments containing the U6 promoter, target gRNA, and the gRNA scaffold were fused using overlap PCR and inserted into the pMD-18T vector (6011, TaKaRa, Japan). Subsequently, the gRNA-carrying pMD-18T and pMJ920 plasmids were transfected into DF-1 cells using the TransIT-X2 delivery system (MIR 6000, Mirus, USA) according to the manufacturer’s instructions. Cells with green fluorescence were sorted into a 96-well plate using flow cytometry, and monoclonal cells were identified through sequence analysis, RT-qPCR, and poly (I:C) stimulation.
CCK8 assay
To detect MDA5-KO DF1 cell proliferation rates using the CCK8 kit, the detection principle of this kit is that WST-8 in the CCK8 reagent, with the assistance of the electron carrier (1-Methoxy PMS), can be reduced by dehydrogenases within cells to produce a highly water-soluble yellow formazan dye, which is directly proportional to the number of viable cells. The simplified procedure is as follows: First, 100μL of DF-1 cell and MDA5-KO DF1 cell suspensions are separately inoculated into a 96-well plate and incubated for 24 h in a 38.5°C culture incubator. Then, 10μL of CCK8 solution is added to each well and incubated for another 2 hours. Finally, the absorbance value of each well at a wavelength of 450 nm were measured by using a microplate reader, and the cell proliferation rate based on the absorbance values.
Determination of IFN-β using ELISA
The IFN-β levels in the serum of ALV-J- or mock-infected chicken were determined using a chicken IFN-β ELISA kit (SEA222Ga, Cloud-Clone, China), following the manufacturer’s instructions.
Protein-RNA pull down
After transfecting pFlag-MDA5 into HEK293T cells for 48 h, the cells were washed thrice with PBST and lysed with NP40 buffer containing 1 mM phenylmethanesulfonyl fluoride protease inhibitors on ice for 30 min; the cell lysate was centrifuged at 12,000 × g for 10 min at 4 °C. Subsequently, 80 μL of the cell lysate was taken and mixed with 20 μL of 5×SDS loading buffer, followed by denaturation at 100 °C for 10 min as input control. The remaining cell lysate was incubated with 20 μL of Anti-FLAG(R) M2 beads (M8823-5ML, Sigma-Aldrich, USA) for 1 h at room temperature on a 4D rotator. The cell lysate bound to the Anti-FLAG(R) M2 magnetic beads was divided into four 1.5 mL EP tubes, with two tubes receiving 108 copies of HLJ09SH05 RNA and SH5-FTO-OE RNA, followed by incubation for 1 h at room temperature, while the other two tubes served as negative controls. The samples were washed five times with 0.5 mL of NP40 lysis buffer, each time for 10 min, and the supernatants were discarded. One negative control was mixed with 80 μL of PBS and 20 μL of 5×SDS loading buffer and denatured at 100 °C for 10 min. The other three samples were mixed with 200 μL of RNAiso plus for RNA extraction and reverse transcription, followed by RT-qPCR to detect RNA levels.
Ubiquitination assay
To investigate whether m6A-deficient ALV-J RNA can increase the ubiquitination modification of MDA5, 1 μg of pFlag-MDA5 and pHA-Ub or pCAGGS plasmids were transfected into DF-1 cells for 24 h, followed by the transfection of 108 RNA copies. After 12 h of RNA transfection, the cells were washed thrice with PBS and lysed with 500 μL of NP40 lysis buffer on ice for 30 min. The cell lysate was centrifuged at 12,000 × g for 10 min at 4 °C, and 80 μL of the cell lysate was mixed with 20 μL of 5×SDS loading buffer and denatured at 100 °C for 10 min as an input control. The remaining sample was incubated with 20 μL of Anti-FLAG(R) M2 magnetic bead on a 4D rotator for 8 h at 4 °C. After washing the cells thrice with precooled PBST, 80 μL of PBS and 20 μL of 5×SDS loading buffer were mixed and denatured at 100 °C for 10 min. The samples were detected and analyzed using western blotting.
Rescue of recombinant viruses
The plasmids pBlueSH5 and pBlueSH5-D-m6A were transfected into DF-1 cells using the TransIT-X2 Dynamic Delivery System (MIR6000, Mirus Bio LLC, USA). After 7 days, the culture supernatant containing the viral stock was harvested and blindly passed to the next generation of DF-1 cells. The rescued viruses were named rSH5 and rSH5-D-m6A, respectively. Subsequently, the titers of the rescued recombinant viruses were determined using the Reed and Muench method [75].
IFA
First, after inoculating DF-1 cells with 200 TCID50 of rSH5 and rSH5-D-m6A for 72 h, the cells were fixed with cold solute ethanol at room temperature for 15 min. Subsequently, the cells were washed thrice with PBST and incubated with 4A3 (mouse anti-gp85 antibody, diluted at 1:200 ratio) at 37 °C for 1 h [39]. Next, after washing the cells thrice with PBST, they were incubated with a 1:200 dilution of fluorescein isothiocyanate-conjugated secondary antibody (FITC-conjugated goat anti-mouse IgG) at 37 °C for 1 h. Finally, DF-1 cells were observed using a fluorescence microscope (TU-80, Nikon, Tokyo, Japan). Normal DF-1 cells were used as a negative control.
Experimental infections
To detect the innate immune response induced by the HLJ09SH05 infection in SPF chickens, 20 1-day-old SPF chickens were divided into two groups, with 10 chickens in each group, and were maintained in different negative pressure isolators. One group was infected with 104 TCID50 of HLJ09SH05 via intraperitoneal injection; the other group received intraperitoneal PBS injection. Blood samples were collected on 1, 2, 3, 4, 5, 6, and 7 dpi to assess viremia and IFN-β levels using RT-qPCR and ELISA, respectively.
To investigate whether the m6A modification of the ALV-J genome affects the replication ability of the ALV-J and the induction of innate immunity, 104 TCID50 of rSH5 and rSH5-D-m6A were injected intraperitoneally into 1-day-old SPF chickens. Blood samples were collected between 1 and 7 dpi to assess viremia and IFN-β levels.
Statistical analysis
GraphPad Prism software (version 7.03, GraphPad Software, San Diego, CA, USA) was used for the statistical analysis. The two-way ANOVA was used to assess differences between groups. Statistical significance was established at P < 0.05. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.
Supporting information
S1 Fig. TLR7 is not the primary RNA sensor for recognizing m6A-defective ALV-J RNAs.
(A) Validation of the optimal siRNA targeting TLR7 using RT-qPCR. (B) The impact of TLR7 gene knockdown on the expression of IFN-β mRNA in chicken primary macrophages induced by m6A-defective viruses. Chicken primary macrophages were transfected with 2 μg of siTLR7–1 or a negative siRNA control (siSc). Subsequently, the cells were infected with m6A-defective or wt ALV-J for 24 h. (C) The effect of TLR7 knockdown on the levels of IFN-β mRNA in chicken primary macrophages induced by m6A-defective viruses. Chicken primary macrophages were transfected with 2 μg of siTLR7–1 or a negative siRNA control (siSc), followed by transfection with 108 copies of m6A-defective or wt ALV-J for 12 h.
https://doi.org/10.1371/journal.ppat.1013064.s001
(TIF)
S2 Fig. Distribution of m6A peaks in the ALV-A and ALV-B genome.
The total RNA of DF-1 cells infected with RAV-1 and RAV-2 was separately extracted at 4 dpi and subjected to m6A-specific antibody immunoprecipitation, followed by high-throughput sequencing (MeRIP-seq). Purple areas illustrate the distribution of m6A immunoprecipitation reads aligned to the ALV-A mRNAs, while the baseline signal from input samples is depicted as a continuous line. Yellow areas demonstrate the distribution of m6A immunoprecipitation reads aligned to the ALV-B mRNAs, while the baseline signal from input samples is depicted as a continuous line.
https://doi.org/10.1371/journal.ppat.1013064.s002
(TIF)
S1 Data. Microsoft Excel workbook provided source data matrices and associated statistical computations used to generate the graphical representations in Figures.
https://doi.org/10.1371/journal.ppat.1013064.s003
(XLSX)
Acknowledgments
We thank Personal Biotechnology Co., Ltd for providing m6A Sequencing service (Shanghai, China).
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