CRISPR-Cas9 based screening to identify epigenetic candidates regulating virus-induced IFN-β production
To explore the epigenetic modifier landscape of influenza virus-induced IFN-β regulation of immune response, we designed a high-throughput screening strategy (Fig. 1a). First, we amplified the upstream 2000 bp promoter of human IFN-β while inserting the mCherry screening marker behind the in the promoter region, which was ligated into a pcDNA4/TO vector (pcDNA4/TO-IFN-β-promoter-mCherry) and transfected into A549. Cell lines stably expressing IFN-β-promoter-mCherry were obtained by zeocin screening. The lentiviral library generated from HEK293T cells was subsequently transfected with human epigenetic modifiers library and lentiviral helper plasmid. Overexpressing cell lines were infected with the lentivirus supernatants at a low MOI to ensure that the majority of cells received only one knockdown, and expanded in culture following puromycin selection. The cells expressing the mCherry red fluorescent signal, indicating activation of the IFN-β promoter, were isolated from the mixture by flow sorting after infection with H1N1. Additionally, uninfected H1N1 cells served as negative controls for clonal cell culture experiments (Fig. S1). The whole genome DNA was extracted for DNA sequencing. Using the MAGeCK algorithm, we compared the number of genes targeted by sgRNA in cells infected with or without H1N1, and identified the best candidate genes both positive regulators and negative regulators of IFN-β. We plotted volcanoes for positively and negatively selected genes and depicted the top 15 genes (Fig. 1b). Among these major targets, knockdown of HNRNPA1 and IGF2BP1 was previously reported to promote type I IFN (IFN-I) expression24, 25, and CTCF has been previously reported to interact with IFN-I26, 27, thereby confirming the validity of our screening system. Subsequent functional enrichment analysis of the above candidate genes (Fig. 1c) showed that the positive selection IFN-β was mainly enriched with chromatin organization, protein-containing complex assembly, protein-containing complex subunit organization, and RNA processing, while the negative selection was enriched mainly for nuclear lumen, membrane-enclosed lumen, organelle lumen, and intracellular organelle lumen. To determine the effects of candidate genes on influenza virus, we performed a secondary screen. We selected Five genes were selected from positive and negative selection, respectively, and the expression levels of these candidate genes were investigated in highly pathogenic influenza H5N1 (A/Turkey/582/2006). Transcriptomic data (GSE22319) are available in the GEO database (http://www.ncbi.nlm.nih.gov/geo)28. The transcript level of NEIL1 was significantly upregulated in cells infected with H5N1, whereas the transcript level of IGF2BP1 was downregulated (Fig. 1d). Subsequently, a significant increase in the transcript level of NEIL1 was observed in cells of A549 infected with H1N1 (Fig. 1e). In addition, mRNA (Fig. 1f) and protein expression (Fig. 1g) of NEIL1 gradually increased over time after infection with H1N1. All these data suggest that NEIL1 plays a potent role in IFN-β-mediated influenza virus replication.
NEIL1 negatively regulates IFN-β and its downstream related gene production caused by RNA virus
We next focused on investigating the effect of NEIL1 on virus-induced IFN-β production. Firstly, the IFN-β mRNA level was investigated after IAV H1N1-inoculated and compared with overexpressing and interfering with NEIL1. Validation of the NEIL1 RNA interference by RT-PCR detection of endogenous NEIL1 transcripts levels (Fig. S2). Overexpression of NEIL1 significantly inhibited H1N1-induced IFN-β production, whereas interference with NEIL1 increased IFN-β expression (Fig. 2a). Moreover, ELISA assay discloses that the secretion of IFN-β from the supernatants of H1N1-infected A549 cells significantly inhibited after overexpression NEIL1, instead, silencing of NEIL1 significantly elevated the secretion in a dose-dependent manner (Fig. 2b). To investigate the effect of NEIL1 in activation of IFN-β in HEK293T cells, we co-transfected HEK293T cells with NEIL1-Flag, IFN-β-Luc and pRL-TK reporter plasmids, and then infected with H1N1. After 12 h of infection, cell lysates were prepared to assess the luciferase activity driven by IFN-β promoter. Meanwhile, IFN-β-Luc and pRL-TK reporter plasmids were transfected after interfering with NEIL1 in HEK293T, and then infected with H1N1 to detect luciferase activity. The results shown that H1N1-mediated IFN-β promoter activity was inhibited in the presence of NEIL1, whereas interfering away NEIL1 significantly increased H1N1-induced IFN-β promoter activity in a concentration-dependent manner (Fig. 2c). To verify whether the regulation of IFN-β levels by NEIL1 could affect the expression of ISGs, we examined the expression of IFN-stimulating factors (ISG) in A549 cells stimulated by H1N1 using qPCR after NEIL1 overexpression or knockdown. The results showed that NEIL1 overexpression suppressed mRNA expression levels of ISGs (MX1, OAS1, and CCL5) induced by H1N1 (Fig. 2d). On the contrary, NEIL1 knockdown promoted ISGs (MX1, IRF7, OAS1, and CCL5) mRNA expression levels induced by H1N1 compared with controls (Fig. 2d). We next sought to determine the effects of NEIL1 on RNA virus-induced IFN-β production. Further, when the production of IFN-β was induced by treating cells with double-strand RNA (poly (I:C)), mimicking the infection of RNA viruses29, overexpression of NEIL1 significantly inhibited the activation of IFN-β promoter in reported assays. In contrast, the poly (I:C)-induced in IFN-β promoter activity was upregulated after interference with NEIL1 (Fig. 2e). Consistent with the above results, we observed a significant decrease in IFN-β promoter activity in the overexpressed NEIL1-treated group relative to the control group post-infection with vesicular stomatitis virus (VSV)△51. The IFN-β promoter activity was strengthened after interference with NEIL1 and infection of VSV△51 (Fig. 2f). The above results demonstrated that NEIL1 negatively regulated RNA virus-mediated IFN-β production, which further affects ISG expression.
NEIL1 suppressed the transcriptional activation of IFN-β
RIG-I-like receptors recognize cytosolic RNA and then activate MAVS, which physically interact with TBK1 to phosphorylate and activate IRF3 or IRF7, resulting in IFN-I production30, 31. We know overexpression of these signal pathway plasmids activate IFN-I production. To elucidate the underlying mechanisms by which NEIL1 reduced the IFN-β production, we tested the effects of NEIL1 on the activation of IFN-β promoter by transfected plasmids related to IFN activation signaling pathway, including proximal regulators (MAVS, TBK1) and key transcriptional factors (p65, IRF3, and IRF7). Overexpression of NEIL1 suppressed IFN-β promoter-reporter activation by MAVS, TBK1, p65, IRF3-5D, and IRF7 in a concentration-dependent manner (Fig. 3a-3e). To test whether NEIL1 uses additional mechanisms to target the IFN-I signaling pathway, we assayed phosphorylated TBK1 (p-TBK1), phosphorylated IRF3 (p-IRF3), and phosphorylated p65 (p-p65) in A549 cells interfering with NEIL1 after H1N1 infection. In contrast to the lower IFN-β expression (Fig. 2a-c), western blotting data indicated that knockdown of NEIL1 did not obviously promote PR8-induced phosphorylation of TBK1, p65, and IRF3 (Fig. 3f). Collectively, our data suggested that NEIL1 inhibited IFN-β transcriptional activation without impacting TBK1-IRF3 activation and that the action target is located in the nucleus. The NEIL1 might function downstream of IRF3/IRF7 and directly acts on the IFN-β locus to impact IFN production.
The functional domain of NEIL1 is crucial for the suppression of IFN-β transcription
In addition to histone posttranslational modifications and regulation of the stages of the transcription cycle, DNA methylation can also play a role in controlling IFN-β transcription13. 5-mC is associated with the repression of regulatory DNA and endogenous retro-element repression32. The NEIL glycosylases can excise oxidized cytosines by the BER machinery and may activate demethylation and reactivation of epigenetically silenced genes22. We were surprised to observe that NEIL1 decreased IFN-β production, so we next questioned whether the enzymatic activity of NEIL1 was critical for IFN-β production. To explore the effect of NEIL1 on the function of IFN-β, we designed a truncated plasmid according to the functional domain of NEIL133. A recent study generated NEIL1 with a site-directed mutation in the catalytic N-terminal proline or glutamic acid residues (P2T and E3Q) is inactive33, 34. We deployed three different glycosylase activity dead mutants (P2T, E3Q and double mutant P2T + E3Q) (Fig. 4a). HEK293T cells vector, wt NEIL1 and the NEIL1 mutants (sit mutants or functional truncation mutants) were overexpressed in HEK293T cells to analyze their suppressive function using an IFN-β luciferase assay stimulated with IRF3-5D or IRF7. Functional truncation33, 35 mutants △CBD (Catalytic/DNA binding domain), △ZF (Zincless Finger), and △DPID (Disordered Protein Interaction Domain) could reverse the inhibition of IFN-β by wt NEIL1 to levels comparable to that found in the control vector (Fig. 4b, c). In contrast, glycosylase-dead mutants of NEIL1 (P2T, E3Q, and P2T + E3Q) inhibited IFN-β luciferase activity induced by IRF3-5D (Fig. 4d) and IRF7 (Fig. 4f) in a manner similar to that of full-length wt NEIL1. All those suggest that the all functional domain of NEIL1 is critical for IFN-β suppression but the enzymatic activity of NEIL1 is dispensable.
Furthermore, to examine the methylation level on the IFN-β promoter, we studied, by bisulfite genomic PCR (BSP), the methylation levels of 4 CpG sites located around 350 bp upstream of the translation starting site of the IFN-β promoter. The BSP results showed that the GC site (-345) away from the IFN-β transcription start site (TSS) was unmethylated in untreated cells after infection with the influenza virus, and NEIL1 overexpression significantly increased the methylation level of the IFN-β promoter (Fig. 4f and g). However, neighboring the GC site of the TSS (-55 and − 12), NEIL1 treatment slightly reduced (by 10%) IFN-β methylation. Interestingly, sequencing at the specific site (site − 22 of the IFN-β TSS)13 revealed that the methylation level of this particular GC site was conserved (90%) with or without NEIL1 treatment (Fig. 4f and g). The above results demonstrate that the functional domain of NEIL1 plays a crucial role in the regulation of IFN-β and NEIL1 may affect the methylation status of IFN-β at different sites to regulate its expression.
NEIL1 has a positive effect on IAV replication
Based on the above findings, we verified that IAV positively regulated host NEIL1(Fig. 1) and NEIL1 inhibited the expression of IFN-β (Fig. 2–3). To validate the role of NEIL1 in IAV replication, the NEIL1-flag plasmid was transfected into A549 cells following IAV infection. QPCR assay data shown that NEIL1 overexpression increased the influenza virus matrix (M) gene relative expression and gene copy numbers (Fig. 5a, Fig. S3a). Also, the culture supernatant was harvested at 24 h post-infection. For titration of infectious virus by using plaque assays, the results showed that overexpression of NEIL1 led to a 2.3-fold increase in viral titers at 24 h post-infection (Fig. 5b). Western blotting showed that high-dose overexpression of NEIL1 promoted nucleoprotein (NP) protein of PR8 expression compared with control (Fig. 5c). As expected, NP was increased in NEIL1-expressing A549 cells by indirect immunofluorescence microscopy using a mAb against IAV NP (Fig. 5d, Fig. S3b). Next, we examined the effect of NEIL1 downregulation on virus replication by using NEIL1 knockdown. The data revealed that NEIL1 knockdown led to a significant decrease in M gene relative expression and gene copy numbers using qPCR assays (Fig. 5e, Fig. S3c). Meanwhile, the viral titers in two siNEIL1 of A549 cells dropped 2.6- and 5-fold compared to that control A549 cells respectively (Fig. 5f). We also measured the expression levels of viral proteins in A549 NEIL1 knockdown and control cells. Viral NP protein expression was lower in A549 cells transfected with siNEIL1 and in a dose-dependent manner (Fig. 5g, Fig. S3d). Our findings suggest that cellular NEIL1 is essential for positive regulation of IAV infection.
NEIL1 interacts with the NP of IAV
Considering that NEIL1 has a promotion effect on IAV proliferation, we determined whether NEIL1 was directly associated with IAV replication. First, we examined if NEIL1 physically interacts with the viral proteins of IAV. We identified NP as a viral protein potentially interacting with NEIL1 using immunoprecipitation combined with mass spectrometry screening (Fig. S4a). To prove our hypothesis, we first checked whether IAV NP protein could interact with NEIL1 in transient-transfection experiments. We performed co-immunoprecipitation (co-IP) experiments in HEK293T cells transfected with epitope-tagged NP and NEIL1. As indicated in Fig. S4b, epitope-tagged NP and NEIL1 were co-immunoprecipitated in HEK293T cells. To further explore the interaction between NEIL1 and NP during IAV infection, PR8 were incubated with HEK293T cells transfected with or without NEIL1, and then subjected to co-IP of the cell lysates postinfection 24 hours.
We found that IAV NP efficiently interacted with NEIL1 during virus replication (Fig. 6a). Consistent with this observation, our immunofluorescence staining showed that NEIL1 co-localized with NP in cell nuclei (Fig. 6b, Fig. S4c). The NP protein, composed of 498 amino acids, is a major component of viral ribonucleoprotein complex (vRNP)36. It contains an RNA-binding region at its N terminus (residues 1 to 181)37 and two domains, responsible for NP-NP dimer formation at residues 189 to 358 and 371 to 46538. In an attempt to map the region of IAV NP responsible for interaction with NEIL1, we created three truncate variants and investigated them in co-immunoprecipitation experiments (Fig. S4d). Deletion of the RNA-binding region at N terminus (amino acids 1-181) and NP-NP self-interaction at C terminus (amino acids 371–465) completely abolished the binding of NP to NEIL1, whereas NEIL1 with middle domains of NP-NP self-interaction had a binding affinity similar to that of full-length NP (Fig. 6c). This suggests that the N- and C-termini of NP are regions essential for interaction with NEIL1.
NP is highly conserved among each type of influenza virus (A and B) and has received significant attention as a good target for universal influenza vaccine39. Next, we evaluated the interactions of NEIL1 with more influenza strains NP. The co-IP results showed that NEIL1 interacted with NP proteins of H5N1, H7N9, and avian H9N2 strains (Fig. 6d-e). However, for the NP proteins of influenza B viruses, no interaction with NEIL1 was found (Fig. 6f). The vRNP complex of AIV is composed of RNA, NP, and the RNA-dependent RNA polymerase (RdRP: PB2, PB1, and PA)40, thus, we wish to address the specificity of the interaction between NEIL1 and other vRNP subunits. HEK293T cells expressing Flag-tagged NEIL1 were infected with or without H1N1, and we found that NEIL1 only co-immunoprecipitated with NP, but there was no interaction between NEIL1 with PB2, PB1, and PA (Fig. S4e). Together, our results indicated that NEIL1 interacts with IAV NP in transiently transfected mammalian cells.
NEIL1 contributes to vRNP nuclear trafficking and efficient influenza viral replication
As previously described NEIL1 is expressed in the nucleus41 and was identified to localize with NP in the nucleus and promote IAV replication by the above assay. In addition to being a structural RNA-binding protein, NP is a key adapter between viral and host cellular processes, as it is essential for nuclear or cytoplasmic trafficking of vRNP 42–44.
Hence, to test whether NEIL1 inhibits vRNP formation, NEIL1-expressing A549 cells were infected with H1N1 and then nuclei and cytoplasm were fractionated at early time points after infection. At 90 min post-infection, levels of H1N1 vRNP-containing proteins in cytoplasmic and nuclear fractionation of mock and NEIL1-expressing cells indicated that the efficiency of viral vRNP nuclear import was slightly higher in NEIL1-expressing cells in A549 (Fig. 7a top panel). At 6 h post-infection, the levels of H1N1 vRNP protein in the cytoplasm of mock and NEIL1-expressing cells were equal. However, vRNP proteins (especially NP and PB2) were significantly more expressed in the nuclei of NEIL1-expressing cells than in controls (Fig. 7a bottom panel). Subsequently, we investigated the distribution and expression of NP protein in the cytoplasm and nucleus of Hela cells 12 h post-infection using a high-throughput imaging system. The fluorescence intensity of NP in the nucleus was higher than that in the cytoplasm after infection with H1N1, and the fluorescence intensity of NP in the nucleus of NEIL1-expressing Hela was noticeably higher than that in the empty vector group (Fig. 7b). These data further illustrate that NEIL1 promotes vRNP entry into the nucleus.
In addition, NP interacted with components of the polymerase protein complex (PB1 and PB2) but not PA42. From the front results, we verified that NEIL1 did not physically interact with PB2 and PB1 (Fig. S4e). This made us speculate whether NEIL1 could mediate the interaction between NP and PB2, in turn, affect the stability of the RNA complexes. First, we found addition of NEIL1 resulted in reinforcing interaction of NP with PB2 and PB1 by immunoprecipitation of overexpression of NEIL1 and RNP complexes (Fig. 7c). To investigate whether this effect of NEIL1 is consistent during IAV infection, HEK293T cells were transfected with constructs for the expression of Myc-tagged NP and HA-tagged-vector or NEIL1, followed infecting with H1N1 (PR8). At 24 h post-infection, cell lysates were immunoprecipitated with an anti-Myc mAb. The results revealed that NEIL1 significantly promoted the interaction of NP with PB2 in the presence of IAV infection (Fig. 7d). The above results confirm that NEIL1 contributes to the entry of vRNP into the nucleus, which in turn improves the stability of NP with PB2.
The transcription and replication of the lAV genome take place in the nucleus of virus-infected cells45, 46, catalyzed by the vRNP 47. We next tested whether vRNP replication activity was affected by NEIL1 using qPCR assays to measure viral mRNA, vRNA, and cRNA species at early time points after infection in the presence or absence of cycloheximide (CHX, a translation elongation inhibitor). The inhibitory effect of CHX on protein synthesis results in attenuation and/or inhibition of lAV genome replication activity that is dependent on newly synthesized NP monomers48. In this case, the vRNA level represents the amount of virus initially inoculated. After infection with AIV, the expression levels of mRNA, cRNA and vRNA of the NP gene of AIV were significantly up-regulated in cells overexpressing NEIL1 (Fig. 7e), in which the up-regulation levels of cRNA and vRNA were more obvious relative to mRNA, while cRNA was the template for synthesizing RNA of the zygotic virus, and newly synthesized vRNA served as the genome of the zygotic virus. Meanwhile, the knockdown of NEIL1 was able to inhibit vRNA replication of AIV (Fig. 7f). In CHX-untreated cells, viral cRNA, vRNA, and mRNA levels were insensitive to NEIL1 for the first 6 h post-infection, but cRNA, vRNA subsequently increased at 9h (Fig. 7g). Meanwhile, cRNA, vRNA and mRNA accumulation was significantly reduced after 6 h in NEIL1 knockdown cells (Fig. 7h). Collectively, these data indicate that NEIL1 positively affects lAVs mainly at the level of viral genome replication and transcription.