Kynurenine-3-monooxygenase (KMO) broadly inhibits viral infections via triggering NMDAR/Ca2+ influx and CaMKII/ IRF3-mediated IFN-β production.

Tryptophan (Trp) metabolism through the kynurenine pathway (KP) is well known to play a critical function in cancer, autoimmune and neurodegenerative diseases. However, its role in host-pathogen interactions has not been characterized yet. Herein, we identified that kynurenine-3-monooxygenase (KMO), a key rate-limiting enzyme in the KP, and quinolinic acid (QUIN), a key enzymatic product of KMO enzyme, exerted a novel antiviral function against a broad range of viruses. Mechanistically, QUIN induced the production of type I interferon (IFN-I) via activating the N-methyl-d-aspartate receptor (NMDAR) and Ca2+ influx to activate Calcium/calmodulin-dependent protein kinase II (CaMKII)/interferon regulatory factor 3 (IRF3). Importantly, QUIN treatment effectively inhibited viral infections and alleviated disease progression in mice. Furthermore, kmo-/- mice were vulnerable to pathogenic viral challenge with severe clinical symptoms. Collectively, our results demonstrated that KMO and its enzymatic product QUIN were potential therapeutics against emerging pathogenic viruses.

Introduction

Frequent outbreaks of emerging infectious diseases, including SARS-CoV-2, avian influenza H5N8, Zika virus (ZIKV), and Ebola virus (EBOV), have become a serious threat to global public health [1-6], and an urgent need in clinical practice is to explore the efficient and broad-spectrum antiviral agents against various viral infections. Increasing studies indicated the potential cross-talk between the immune responses to viral infections and the cellular metabolism in host cells [7,8]. In addition, viral infections may hijack the immune system and the metabolic system and thus cause metabolic disorders, including abnormal lipid metabolism, cardiovascular diseases, and neurological diseases [9-11]. As a result, in-depth investigation of the relationship between immune response and cellular metabolism may reveal novel targets to develop antiviral agents.

L-Tryptophan (L-Try) is a kind of essential amino acid for the human body, and its metabolites by the kynurenine pathway (KP) have critical functions in inflammation and immune homeostasis, thereby playing a key role in autoimmune diseases, tumorigenesis, and neurodegenerative diseases [12-15]. Recent studies also suggested that KP metabolites might play a regulatory role in host-pathogen interactions [16-18]. For example, the transcription level of indoleamine 2,3-dioxygenase 1 (IDO1), one metabolite of tryptophan, was significantly upregulated in response to influenza infection in mouse lungs and human primary macrophages [19,20]. Moreover, kynurenine biosynthesis in macrophages was increased in response to stimulation by the herpes simplex virus (HSV) as well as bacterial lipopolysaccharides [21]. In addition, another metabolite of KP-picolinic acid effectively induced the apoptosis of human immunodeficiency virus (HIV) or HSV-infected cells [22]. Thus, it is important to investigate further the host-pathogen interactions through the KP metabolites to explore the novel anti-infection targets.

Kynurenine-3-monooxygenase (KMO), a key rate-limiting enzyme during L-Try metabolism, belongs to the family of oxidoreductases. KMO can catalyze the conversion of L-kynurenine to 3-hydroxy-L-kynurenine and also regulate the balance between kynurenic acid (KA) and quinolinic acid (QUIN) [15]. So far, the role of KMO in innate immunity response to viral infections has not been reported yet. In the present study, we identified for the first time that KMO and its enzymatic product QUIN could act as a novel broad-spectrum antiviral factor against emerging pathogenic viruses.

Result

KMO is an interferon-dependent gene

To identify the potential genes with antiviral activity, we used a recombinant HSV-GFP-Luc reporter virus to screen the 288 ISGs induced by IFN-α and IFN-γ as we previously reported [23]. Besides the well-known antiviral genes including IRF1 [24], CH25H [25], IFITM2 [26], TAP1 [27], LY6E [28], we also identified some novel genes including KMO, SERPINA5, SLFN12, GPR146, and LIPG, which are uncharacterized with antiviral activity previously (Fig 1A). Our further analysis demonstrated that IFITM2, KMO, LIPG genes exerted the most obvious inhibition against viral infections in this study (Fig 1B).

Fig 1

The expression of KMO is interferon-inducible.

(A,B) 288 genes were screened by a luciferase-based analysis. Each dot represents one gene, and its effect on HSV-1 infection was normalized to that in pVAX-GFP transfected cells (negative control). Genes that inhibit HSV-1 infection by more than 50% were marked and shown in green. Genes that enhance HSV-1 infection by more than 2 fold were marked and shown in red. (C) Wild type bone marrow-derived macrophage (WT-J2-BMM) cells or interferon α receptor-deficient (Ifnar-/-)-J2-BMM cells were stimulated for 8 h with different TLR agonists, including LPS (1 μg/mL), R848 (100 nM), poly(I:C) (25 μg/mL), and then the expression level of kmo gene was measured by RT-qPCR. (D) Raw264.7 cells and J2-BMM cells were stimulated with IFN-α in different concentrations (500 U/mL, 1000 U/mL, and 2000 U /mL) for 8 h, and the expression level of the kmo gene was measured by RT-qPCR. (E) Raw264.7 cells were infected with indicated MOI of HSV-1 for 24 h or infected with various times (hours post-infection, hpi.) at 0.25 MOI of HSV-1. Then, the expression level of the kmo gene was measured by RT-qPCR. (F) J2-BMM cells and Ifnar-/—J2-BMM cells were stimulated with LPS (1 μg/mL), poly(I:C) (25 μg/mL), R848 (100 nM), IFN-α (2000 U/mL), or HSV-1 at MOI of 0.25 for 24 h, respectively, and then the expression level of KMO protein was measured by Western Blotting Analysis. GAPDH was used as intern control. The final data are presented as the mean ± SD of at least triplicate experiments. MOI: multiplicity of infection. *P < 0.05, **P < 0.01, ***P < 0.001.

Next, we sought to characterize the antiviral function of the KMO gene since it was previously well-described as a key rate-limiting enzyme in KP-mediated L-Try metabolism [29]. Our results showed that KMO expression was significantly upregulated in response to the stimulation of different toll-like receptors (TLRs) agonists, such as LPS, poly(I:C), R848 (Fig 1C). However, the up-regulation of KMO expression was abrogated in the IFN-I receptor (IFNAR) deficient (Ifnar-/-)-J2-BMMs (Fig 1C). In addition, in a dose-dependent manner, KMO expression was significantly induced by IFN-α stimulation in Raw264.7 and WT-J2-BMM cells (Fig 1D). We also found that KMO expression was significantly elevated by HSV-1 infection in both dose-dependent and time-dependent in Raw264.7 cells (Fig 1E). The Western Blotting analysis further confirmed that KMO protein was increased by the agonists mentioned above in J2-BMM cells while abolished in the (Ifna-/-)-J2-BMM cells (Fig 1F). Altogether, the KMO expression is IFN-dependent.

The antiviral activity of KMO

Next, we investigated the potential antiviral activity of KMO. Using the CH25H gene as a positive control [30], we demonstrated that KMO over-expression significantly suppressed the replication of HSV-1 by quantitative PCR analysis (Fig 2A), and this inhibition was further confirmed by Western Blotting analysis (Fig 2B) and plaque assay (Fig 2C). To further validate the antiviral function of KMO, the KMO expression was effectively inhibited using small interfering RNA (siRNA) (Fig 2D) and short hairpin RNA (shRNA) (Fig 2E), respectively. Results demonstrated that HSV-1 replication was significantly enhanced in KMO-knockdown cells compared to wild-type cells (Figs 2D and 3E). Furthermore, we generated the kmo cell line using the CRISPR/Cas9 system (Fig 2F) and found a significantly enhanced HSV-1 replication in kmo cells (Fig 2G) compared to wild-type (WT) cells. Moreover, the replenishment of the kmo gene could effectively rescue the antiviral activity in kmo cells (Fig 2H). Altogether, our studies with both loss-of-function and gain-of-function experiments validated the antiviral function of the kmo gene.

Fig 2

The antiviral activity of KMO through its enzymatic product QUIN.

(A) 293T cells were transfected with plasmids (1 μg/mL) expressing GFP, KMO, CH25H for 24 h respectively, followed by HSV-1 infection for 8 h at MOI of 0.25. Then the expression of KMO and HSV-1 UL27 were measured by RT-qPCR. (B) 293T cells were transfected with plasmids (1 μg/mL) expressing GFP, KMO, CH25H for 24 h respectively, followed by HSV-1 infection for 8 h at MOI of 0.25. Then the expression of KMO and HSV-1 ICP27 protein were measured by Western Blotting Analysis. (C) 293T cells were transfected with plasmids (1 μg/mL) expressing GFP, KMO, and CH25H for 24 h, respectively, followed by HSV-1 infection for 8 h at MOI of 0.25, the titer of HSV-1 in the supernatant was measured by plaque assay. (D) 293T cells were transfected with siRNA for 24 h, followed by HSV-1 infection for 8 h at MOI of 0.25, the expression of KMO and HSV-1 UL27 was measured by RT-qPCR. (E) 293T cells were infected with shRNA-expressing lentivirus for 24 h, followed by HSV-1 infection for 8 h at MOI of 0.25, the expression of KMO and HSV-1 UL27 was measured by RT-qPCR. (F) 293T kmo cell line was generated using the CRISPR/Cas9 system and confirmed DNA sequencing and Western Blotting Analysis. (G) 293T cells or 293T kmo cells were infected with HSV-1 for 8 h at MOI of 0.25. Then, the expression of KMO was measured by RT-qPCR, and the expression of HSV-1-Luc was measured by Luciferase analysis. (H) kmo293T cells were transfected with plasmids (2 μg/mL) expressing GFP, KMO, CH25H for 24 h, followed by HSV-1 infection for 8 h at MOI of 0.25. Then, the expression of HSV-1 UL27 was measured by RT-qPCR. (I) The residues Tyr 99 (M1), Tyr 194 (M2), and Glu 366 (M3) were critical to the enzymatic activity of KMO, and we generated the activity-dead KMO mutants (KMO-M1-3) by site-directed mutagenesis. (J) 293T cells were transfected with plasmids (1 μg/mL) expressing GFP, KMO, KMO-M1, KMO-M2, KMO-M3 for 24 h respectively, followed by HSV-1 infection for 8 h at MOI of 0.25. Then, the HSV-1-luciferase and UL27 gene expression were measured by Luciferase analysis and RT-qPCR, respectively. (K) Vero cells and 293T cells were pretreated with conditional medium from the indicated construct-transfected 293T cells for 24 h, and then the pretreated cells were infected with HSV-1 at MOI of 0.25 for 24 h. Then, the expression of HSV-1 UL27 was measured by RT-qPCR. (L) 293T cells were pretreated with conditional medium from the indicated construct-transfected 293T cells for 24 h, and the pretreated cells were infected with HSV-1 at MOI of 0.25 for 24 h. Then, the expression of HSV-1 ICP27 protein was measured by Western Blotting Analysis. (M) RAW264.7 cells were pretreated with the indicated concentration of QUIN for 8 h and then infected with HSV-1 at MOI of 0.25 for 8 h. The expression of HSV-1-luciferase was measured by Luciferase analysis. The final data are presented as the mean ± SD of at least triplicate experiments. MOI: multiplicity of infection. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3

The antiviral mechanisms of KMO and QUIN by activating IRF3 phosphorylation-mediated IFN-β production.

(A) RAW264.7 cells were transfected with plasmids (1 μg/mL) expressing GFP, KMO for 24 h, followed by HSV-1 infection at MOI of 0.25 for 8 h, and then the expression of KMO and IFN-β were measured by RT-qPCR. (B) RAW264.7 cells were transfected with siRNA for 24 h, followed by HSV-1 infection at MOI of 0.25 for 8 h, and the expression of KMO and IFN-β were measured by RT-qPCR. (C) RAW264.7 cells were infected with shRNA-expressing lentivirus for 24 h, followed by HSV-1 infection at MOI of 0.25 for 8 h, and the expression of KMO and IFN-β were measured by RT-qPCR. (D) RAW264.7 cells were infected with sgRNA-expressing lentivirus for 24 h, followed by HSV-1 infection at MOI of 0.25 for 8 h, and the expression of KMO and IFN-β were measured by RT-qPCR. (E) RAW264.7 cells were pretreated with the indicated concentration of KMO inhibitor for 8 h, followed by HSV-1 infection at MOI of 0.25 for 8 h, and the expression of IFN-β was measured by RT-qPCR. (F) RAW264.7 cells were pretreated with the indicated concentration of QUIN for 8 h, followed by HSV-1 infection at MOI of 0.25 for 8 h, and the expression of IFN-β was measured by RT-qPCR. (G) Raw 264.7 cells were transfected with plasmids expressing GFP or KMO (1 μg/mL) for 24 h respectively, followed by HSV-1 infection at MOI of 0.25 for 8 h. Then, the expression levels of indicated proteins were detected by Western Blotting Analysis. (H) Raw 264.7 cells were pretreated with the indicated concentration of QUIN or DMSO for 8 h and then infected with HSV-1 at MOI of 0.25 for another 8 h. Then, the expression level of indicated proteins was detected by Western Blotting Analysis. (I) Raw 264.7 cells were transfected with siNC or siKMO for 24 h respectively, followed by HSV-1 infection at MOI of 0.25 for 8 h. Then, the expression levels of indicated proteins were detected by Western Blotting Analysis. (J) Raw 264.7 cells were pretreated with the indicated concentration of KMO inhibitor (R061-8048) or DMSO for 8 h, and then infected with HSV-1 at MOI of 0.25 for 8 h, the expression levels of indicated proteins were detected by Western Blotting Analysis. (K) Raw 264.7 cells were pretreated with NMDAR inhibitor ((-)-MK 801 maleate (10 μM)) or DMSO for 1h, and then stained with Calbryte 520 AM and stimulated with QUIN (1 mM), PMA (8 ng/mL) and ionomycin (200 ng/mL), or DMASO respectively. Then, the samples were imaged by Leica microscopy with the original magnification of ×40. (L) Raw 264.7 cells were transfected with plasmids (1 μg/mL) expressing GFP or KMO for 24 h respectively, followed by HSV-1 infection at MOI of 0.25 for 8 h. Then, the expression levels of indicated proteins were detected by Western Blotting Analysis. Moreover, Raw 264.7 cells were transfected with siNC or siKMO for 24 h, followed by HSV-1 infection at MOI of 0.25 for 8 h. Then, the expression levels of indicated proteins were detected by Western Blotting Analysis. (M) Raw 264.7 cells were pretreated with the indicated concentration of QUIN or DMSO for 8 h and then infected with HSV-1 at MOI of 0.25 for 8 h. Then, the expression level of indicated proteins was detected by Western Blotting Analysis. (N) Raw 264.7 cells were pretreated with the indicated concentration of KMO inhibitor (R061-8048) or DMSO for 8 h and then infected with HSV-1 at MOI 0.25 for 8 h. Then, the expression level of indicated proteins was detected by Western Blotting Analysis. (O) Raw 264.7 cells were pretreated with NMDAR inhibitor ((-)-MK 801 maleate (10 μM)) or DMSO for 1 h, and then treated with QUIN(1mM) or DMSO for 8 h. Then, the expression level of indicated proteins was detected by Western Blotting Analysis. (P) Raw 264.7 cells were transfected with siNC or siCaMKII for 24 h, followed by QUIN(1mM) or DMSO treatment for 8 h, and then the expression level of indicated proteins were detected by Western Blotting Analysis. (Q) Raw 264.7 cells were pretreated with the indicated concentration of QUIN or DMSO for 8 h. Cell extracts were immunoprecipitated (IP) with anti-IRF3 or anti-CaMKII antibody. IRF3-conjugated proteins were detected with anti-CaMKII antibody, and CaMKII-conjugated proteins were detected with anti-IRF3 antibody by Western Blotting (top). The whole-cell extracts were also detected by Western Blotting using the indicated antibody (bottom). The final data are presented as the mean ± SD of at least triplicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

The antiviral effects of KMO through its enzymatic product QUIN

We also explored whether the antiviral function of KMO is dependent on its enzyme activity. Previous studies showed that the residues Tyr 99, Tyr 194, and Glu 366 were critical to the enzymatic activity of KMO [31,32], and we subsequently generated a series of kmo mutants (KMO-M1-3) by site-directed mutagenesis (Fig 2I). Results showed that the overexpression of these KMO enzyme-dead mutants had no significant inhibition effect on HSV-1 infection compared to that of wild-type KMO (Fig 2J), implying that the enzymatic activity of KMO is required for its antiviral function.

We subsequently assessed whether the enzymatic products in the supernatants of KMO-treated cell cultures exerted the viral inhibition effect. As expected, the supernatants from KMO-treated cells significantly inhibited HSV-1 infection in Vero cells and 293T cells (Fig 2K), and Western Blotting analysis further confirmed this observation (Fig 2L), suggesting that KMO exerted antiviral function by producing potential soluble antiviral factors. KMO is a well-known key enzyme in the KP to produce many kinds of metabolites [29-33], including quinolinic acid (QUIN), kynurenic acid, xanthic acid. Among them, we identified that QUIN had a significant antiviral effect in a dose-dependent manner (Fig 2M). Moreover, QUIN also effectively rescues the antiviral activity in kmo cells (S1 Fig). In addition, we investigated the cytotoxicity of QUIN, and the QUIN under 1 mM showed no obvious toxicity to different cell lines, including 293T, Vero, and Raw264.7 cells (S2 Fig).

The antiviral mechanisms of KMO and QUIN by activating IRF3-mediated IFN-β production in HSV-1 infected cells

We next investigated the underlying mechanisms for the antiviral function of KMO and QUIN in HSV-1 infected cells. To clarify the possible roles of QUIN in antiviral immunity, we performed RNA-seq analyses for the HSV-1-infected RAW264.7 cells with or without QUIN treatment. Compared to the control group, QUIN treatment significantly altered the expression of 245 genes (161 upregulated and 84 downregulated) (S3A Fig). Gene ontology (GO) analyses showed that QUIN treatment upregulated the expression of multiple genes related to IFN-I production, cytokine production, cellular metabolic process (S3B Fig). KEGG analyses revealed enrichment in the Jak-STAT signaling pathway, Toll-like receptor signaling pathway, TNF signaling pathway (S3C Fig). After hierarchical clustering, we identified 19 genes involved in the antiviral signaling pathway by QUIN treatment (S3D Fig). These data suggested that QUIN and KMO might play the antiviral function by regulating IFN-I production and related signaling pathways and so on.

Consistent with these data, the over-expression of KMO significantly elicited the level of IFN-β expression in Raw 264.7 cells (Fig 3A). In contrast, siRNA or shRNA-mediated KMO knockdown and CRISPR/Cas9-mediated KMO knockout had significantly reduced the levels of IFN-β expression (Fig 3B–3D). Moreover, R061-8048, an inhibitor of KMO enzyme activity, could also inhibit the IFN-β production in a dose-dependent manner (Fig 3E). In addition, QUIN treatment significantly induced the IFN-β production in a dose-dependent manner (Fig 3F). These data suggested that induction of interferon production may contribute to the antiviral activity of KMO and QUIN.

We then studied the signaling pathway involved in KMO/QUIN-induced IFN-β production in HSV-infected cells and found that KMO over-expression effectively activated the phosphorylation of IRF3 at Ser396 (pSer396) (Fig 3G). Meanwhile, QUIN had a similar effect on the activation of IRF3 phosphorylation in a dose-dependent manner (Fig 3H). In contrast, KMO knockdown significantly decreased the phosphorylation of IRF3 in HSV-infected cells and consequently increased the expression of HSV-1 ICP27 protein (Fig 3I). Moreover, KMO inhibitor R061-8048 also suppressed the phosphorylation of IRF3 in response to HSV-1 infection (Fig 3J). Overall, these data indicated that KMO/QUIN promoted IFN-β production by enhancing IRF3 phosphorylation in HSV-infected cells.

Subsequently, we explored how KMO/QUIN modulated the IRF3 phosphorylation. QUIN was previously reported as an agonist of NMDAR to induce the Ca2+ influx [34,35]. In our study, we first validated the NMDAR expression in various cell lines (S4 Fig), and then we determined whether QUIN could elicit Ca2+ influx. Using Calbryte 520 to visually label the intracellular Ca2+, an immediate increase of the intracellular Ca2+ was observed after QUIN stimulation in Raw 264.7 cells, while this increase of Ca2+ influx was significantly suppressed in the samples treated with (-)-MK-801 (Fig 3K), which is an NMDAR inhibitor [36].

Considering that the increased Ca2+ influx can trigger IFN-I production through the Calcium/calmodulin-dependent protein kinase II (CaMKII) and IRF3 signaling pathway [37], we further determined whether KMO/QUIN modulated the phosphorylation of CaMKII, which is a major biochemical decoder of intracellular Ca2+ oscillations [38]. Results showed that KMO over-expression enhanced while KMO knockdown reduced the CaMKII phosphorylation (T286) in HSV-1 infected cells (Fig 3L). A similar observation of increased CaMKII phosphorylation (T286) was also observed with QUIN incubation (Fig 3M). In contrast, the QUIN-induced CaMKII phosphorylation was significantly inhibited in the presence of the KMO inhibitor (Ro 61–8048) (Fig 3N), (-)-MK-801(Fig 3O) or siCaMKII (Fig 3P). We have demonstrated a direct interaction between CaMKII and IRF3 in QUIN-stimulated cells in a dose-dependent manner (Fig 3Q). Taken together, KMO/QUIN induced the production of IFN-I via activating the NMDAR/Ca2+ influx to trigger the CaMKII/IRF3 signaling pathway.

Broadly antiviral activity of KMO and QUIN

As KMO had a novel function against viral infection by enhancing IFN-I induction, we wanted to determine the breadth of antiviral activity of KMO. Our results demonstrated that KMO could effectively inhibit the replication of DNA viruses, such as HSV-1 (a kind of enveloped DNA virus) and adenovirus (a kind of nonenveloped DNA virus) in a dose-dependent manner (Fig 4A and 4B). Moreover, we also found that KMO had a significant antiviral effect against RNA viruses, such as negative-strand RNA viruses, including vesicular stomatitis virus (VSV) and influenza virus (PR8) (Fig 4C and 4G), and positive-stranded RNA viruses, including Zika virus (ZIKV), dengue virus (DENV) and SARS-CoV-2 (Fig 4D and 4F). In addition, we found that KMO significantly enhanced IFN-β expression during infection with the viruses mentioned above (S5A Fig). Similarly, QUIN treatment also had dose-dependent inhibitory effects on these viruses by stimulating the production of IFN-β (Figs 4H–4N, and S5B). We found that QUIN also has broad antiviral activity in various cells, including A549 and BMM cells (S6 Fig). Thus, our data showed that KMO/QUIN exerted an unreported function to inhibit DNA viruses and RNA viruses broadly.

Fig 4

Broadly antiviral activities of KMO and QUIN.

(A) 293T cells were transfected with different concentrations of KMO-expressing plasmid for 24 h, followed by HSV-1 infection at MOI of 1 for 8 h. The expression level of HSV-1 UL27 was quantified by RT-qPCR. (B) 293T cells were transfected with different concentrations of KMO-expressing plasmid for 24 h, followed by Ad5 infection at MOI of 1 for 8 h. The expression level of Ad5 was quantified by RT-qPCR. (C) 293T cells were transfected with different concentrations of KMO-expressing plasmid for 24 h, followed by VSV infection at MOI of 1 for 8 h. The expression level of VSV was quantified by RT-qPCR. (D) 293T cells were transfected with different concentrations of KMO-expressing plasmid for 24 h, followed by ZIKV infection at MOI of 1 for 8 h. The expression level of ZIKV was quantified by RT-qPCR. (E) 293T cells were transfected with different concentrations of KMO-expressing plasmid for 24 h, followed by DENV4 infection at MOI of 1 for 8 h. The expression level of DENV4 was quantified by RT-qPCR. (F) CoCa-N-2 cells were transfected with different concentrations of KMO-expressing plasmid for 24 h, followed by replication-competent SARS-CoV-2 virus-like-particles (SARS-CoV-2 GFP/ΔN) infection at MOI of 1 for 8 h. The expression level of SARS-CoV-2 GFP/ΔN was quantified by RT-qPCR. (G) 293T cells were transfected with different concentrations of KMO-expressing plasmid for 24 h, and the expression level of PR8 was quantified by RT- qPCR. (H-N) Cells were pretreated with QUIN at different concentrations for 8 h, followed by viral infections for 8 h at MOI of 1, including HSV-1, VSV, ZIKV, DENV4, Ad5, SARS-CoV-2 GFP/ΔN (SARS-CoV-2) and PR8, and then the expression level of corresponding viruses was quantified by RT-qPCR. The expression level of mRNA was normalized to the expression of β-actin, and the data from at least triplicates were shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Previous studies have reported that IDO1 played a function in inhibiting viral replication [39,40]. To clarify that KMO and QUIN have antiviral activities independent of IDO1, we treated cells with 1-Methyl-D-tryptophan(1-MT), an inhibitor of IDO1. We found that 1-MT treatment weakened the antiviral effect by inhibiting IDO1 (S7A and S7B Fig). However, KMO and QUIN treatment could rescue the antiviral effect even after the IDO1 inhibition by 1-MT (S7C and S7D Fig), indicating that the antiviral effects of KMO and QUIN were independent on IDO1.

QUIN treatment protected mice from viral infections

Next, we sought to investigate the efficacy of QUIN as a potential antiviral agent against viral infections in vivo. Mice were infected with highly pathogenic HSV-1 McKrae strain, followed by daily treatment with QUIN for 7 days (Fig 5A). We also used the acyclovir (ACV) as a positive control, a clinical drug prescribed to control herpes infection. At 7 days post-infection (dpi.), the vehicle group’s progressive corneal scarring and visual impairment deteriorated significantly, while this symptom was greatly alleviated in the QUIN and the ACV groups (Fig 5B). More importantly, QUIN administration effectively protected mice and significantly improved their survival when challenged with the highly pathogenic HSV-1 McKrae strain, while all of the mice in the vehicle group succumbed to this lethal challenge within 7 dpi (Fig 5C). Consistent with this finding, mice treated with QUIN had significantly lower disease scores when compared to the vehicle group (Fig 5D).

Fig 5

QUIN treatment protected mice from viral infections.

(A) Schedule for evaluating the therapeutic efficacy of QUIN in mice. Briefly, C57BL/6 mice were corneally inoculated with 105 PFU HSV-1 McKrae strain and treated daily with QUIN for 7 days. The vehicle group (negative control) was received intraperitoneal (IP) injection of DMSO and PBS. QUIN group was received an IP injection of QUIN (2.5 mg/kg). The ACV group (positive control) received an IP injection of ACV (5 mg/kg). The eyes of mice were washed at 2 dpi and 4 dpi to perform the plaque assay. The disease symptoms of experimental mice were monitored until 15 dpi. (B) The representative picture of progressive corneal scarring, visual impairment of experimental mice in different groups at 7 dpi. (C) Survival curve of experimental mice in different groups over time post-infection (n = 8). (D) Statistical analysis of disease scores of experimental mice in different groups over time (n = 5). Score 0, healthy; Score 4, being severe disease. (E) The HSV-1 titer in the eye washing fluid at 2 dpi and 4 dpi were measured by plaque assay (n = 5 per group). (F) The concentration of IFN-β in serum, brain, and spleen of experimental mice at 5 dpi was measured by ELISA assay (n = 5 per group). (G) The frequency of HSV-1 antigen-specific IFN-γ-secreting cells was determined by the enzyme-linked immunosorbent spot (ELISPOT) assays at 5 dpi. Data represents spot-forming cells (SFC) per million cells. The final data are presented as the mean ± SD of triplicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

In addition, the viral copies in the eye washing fluid from QUIN-treated mice were significantly reduced than that of mock-treated mice (Fig 5E). We also quantitatively measured the concentration of IFN-β in serum, brain, and spleen of these experimental mice and found a significant increase of IFN-β secretion in the QUIN-treated mice than that of mock-treated mice (Fig 5F). Furthermore, there was an increased frequency of HSV-1 antigen-specific IFN-γ-secreting cells, determined by the enzyme-linked immunosorbent spot (ELISPOT) assays, in the QUIN group compared to that of the vehicle group (Fig 5G), implying that QUIN therapy also modulated the adaptive immune responses, especially the antigen-specific cellular immune responses. Collectively, these data indicated that QUIN treatment effectively protected mice from viral infection.

kmo mice are more susceptible to viral infections

Finally, to determine whether KMO played a physiological role in host defense against viral infections, we generated the kmo mice by CRISPR/Cas9-based strategy (S8 Fig) and then compared whether there was an increased susceptibility to viral infections in kmo mice as compared to the age-matched wild-type mice (kmo+/+). After challenging with sub-lethal doses of highly pathogenic HSV-1 McKrae strain (Fig 6A), kmo mice developed a more severe disease progression and clinical symptoms, leading to a significantly lower survival rate than that of kmo mice (Fig 6B). Furthermore, plaque assay indicated that the viral copies in the eye washing fluid from kmo mice were significantly higher than kmo mice (Fig 6C). Our data also showed that peritoneal and bone marrow-derived macrophages from the kmo mice were more susceptible to HSV-1 infection than that of kmo mice (Fig 6D). Consistent with the above in vitro data (Fig 3D), the level of IFN-β expression in serum, brain, and spleen of kmo mice was significantly lower than that of kmo mice (Fig 6E and 6F). In addition, we further showed that there was an obvious dysfunction of adaptive T cell immune responses in kmo mice, characteristic with the decreased frequency of the polyfunctional CD4+ T cells and CD8+ T cells secreting interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and interleukin-2 (IL-2) cytokines (Fig 6G).

Fig 6

kmo mice are more susceptible to viral infections.

(A) Schedule for evaluating the susceptibility to viral infections in kmo mice. Briefly, both kmo C57BL/6 mice and kmo C57BL/6 mice were corneally inoculated with 3×104 PFU HSV-1 McKrae strain, disease symptoms of experimental mice were monitored until 15 dpi. (B) Survival curve and disease score of experimental mice in different groups over time post-infection (n = 8 per group). (C) The HSV-1 titer in the eye washing fluid at 3 dpi and 5 dpi was measured by plaque assay (n = 5 per group). (D) Bone Marrow-derived and Peritoneal macrophages were isolated from kmo or kmo mice and then infected with HSV-1-Luc at MOI of 0.25 for 8 h. Then, the luciferase expression was measured by Luciferase Assay kit (n = 5 per group). (E) The concentration of IFN-β in serum, brain, and spleen of experimental mice at 7 dpi was measured by ELISA assay (n = 5 per group). (F) The expression of IFN-β in the brain and spleen of kmo or kmo mice were measured by RT-qPCR (n = 5 per group). (G) The frequency of polyfunctional CD4+ and CD8+ T cell populations in kmo or kmomice was assessed by detecting the secretion of TNF-α, IFN-γ, and IL-2 cytokines response to HSV-1 peptide pool stimulation. (H) The pathological changes of experimental mice’s liver, spleen, and trigeminal ganglia (TG) were observed by hematoxylin-eosin (H&E) staining at 7dpi. The scale bars: 100 μm. (I) The numbers of white blood cell (WBC), lymphocyte (Lym), monocyte (Mon), granulocyte (Gran) in HSV-1 infected kmo mice and kmo mice were detected by complete blood count analysis at 7 dpi (n = 5 per group). The final data are presented as the mean ± SD of at least triplicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

We also performed immunohistochemistry and hematoxylin-eosin (H&E) staining to examine the pathological changes in the tissues of these experimental mice. Compared with kmo mice, the McKrae HSV-1 infected kmo mice had the increased cytoplasmic eosinophilia (black arrow) and focal infiltration of inflammatory cells (red arrow) in the liver lobules, and the increased neutrophil infiltration in the red pulp of the spleen (black arrow) (Fig 6H). Importantly, an obvious pathological injury was observed in the trigeminal ganglia in kmo mice, characteristic with the increased neuron necrosis (black arrow), decreased neuronal soma count, enhanced inflammatory cell infiltration (red arrow), and widened the gap between neuronal soma (yellow arrow) (Fig 6H). This status of immune imbalance in kmo mice was also confirmed by complete blood count analysis, and the results indicated that the numbers of white blood cell (WBC), lymphocyte (Lym), monocyte (Mon), granulocyte (Gran) were significantly decreased in kmo mice than those of kmo mice (Fig 6I).

We next performed RNA-seq analyses for the HSV-1-infected BMM cells from kmo and kmo mice. Compared to the kmo group, the kmo group significantly altered the expression of 2929 genes (975 upregulated and 1954 downregulated) (S9A Fig). Gene ontology (GO) analyses showed that the kmo group upregulated the expression of multiple genes related to IFN-β and IFN-γ production, immune system process, defense response (S9B Fig). KEGG analyses revealed enrichment in the Jak-STAT signaling pathway, Toll-like receptor signaling pathway, TNF signaling pathway (S9C Fig). After hierarchical clustering, we identified 25 genes involved in the antiviral signaling pathway (S9D Fig). These data suggested that kmo knockout might reduce the antiviral function by regulating IFN-I production and related signaling pathways.

To further clarify whether QUIN has a rescue effect on virus-infected kmo mice, kmo and kmo mice were challenged with HSV-1 McKrae strain, followed by daily treatment with different concentrations of QUIN for 7 days (S10A Fig). QUIN administration effectively protected the mice and reduced disease scores in a dose-dependent manner (S10B and S10C Fig). The viral copies in the eye washing fluid from QUIN-treated (2.5 mg/kg) mice were significantly reduced than that of mock-treated mice. Of note, in the kmo group, the viral copies were significantly reduced in the QUIN-treated group at a lower dose of 1 mg/kg (S10D Fig). In addition, IFN-β secretion was significantly increased in the serum of QUIN-treated mice (S10E Fig).

Overall, these findings indicated that KMO is a key antiviral factor physiologically involved in modulating antiviral immunity.

Discussion

Independent of its known regulatory role on tryptophan metabolism, we herein reported that KMO and its enzymatic product QUIN exerted a novel broad-spectrum antiviral function against numerous emerging pathogenic viruses, thorough triggering the NMDAR/Ca2+ influx and CaMKII/ IRF3-mediated IFN-β production. Previously, KMO had been shown to associate with pathological conditions like tumorigenesis [41-43] and neurodegenerative diseases [44-48]. However, our study reported its new function in host immunity against viral infections. Importantly, in the animal infection model, QUIN treatment effectively inhibited viral infections and alleviated disease progression in vivo. Moreover, the kmo gene is a physiologically key antiviral factor because kmo mice were more vulnerable to pathogenic viral challenge than kmo mice (Fig 7). As a result, our findings revealed the critical role of tryptophan metabolism in antiviral immunity, which highlighted the importance of further understanding the profound intricacy between innate immunity and cellular metabolism.

Fig 7

A schematic illustrating KMO and its enzymatic product QUIN exerted a novel broadly antiviral function.

QUIN triggered the NMDAR/Ca2+ influx and CaMKII/IRF3-mediated IFN-β production.

The tryptophan metabolism, mainly processed by the kynurenine pathway (KP), is an essential physiological process to maintain our body’s health, and the key rate-limiting enzymes in the KP include IDO1, IDO2, TDO, and KMO [49]. Recently, KP has also been reported to play a critical role in modulating immune homeostasis [15], and the KP metabolites might be associated with immune disorders [50-52]. For example, IDO1 could inhibit the replication of human cytomegalovirus, HSV-1, and retroviruses through the increased consumption of L-Try [53-55]. However, the role of KMO in modulating innate immunity against viral infections has not been investigated. Our study examined the serum of kmo and kmo mice. We found that kynurenine accumulated because of the absence of kmo (S11 Fig), which might have a negative feedback inhibition on IDO1. Still, our further research showed that the KMO and QUIN could independently inhibit the virus when IDO1 was inhibited. In addition, IDO-mediated tryptophan consumption was not involved in the IFN-α/β-mediated antiviral effects [40], but the antiviral effects of KMO and QUIN in our study were associated with IFN-β production. Therefore, the antiviral effects of KMO and IDO1 may have a synergistic relationship through different antiviral pathways.

Our study found that the clinical symptoms in pathogenic HSV-1 infected kmo mice were more severe than those in kmo mice, and this observation was consistent with a lower IFN-γ secretion in kmo mice than in kmo mice. One study showed that KMO deficiency increased the frequency of Foxp3+ regulatory T cells and the levels of anti-inflammatory cytokines, including transforming growth factor-β and interleukin-10 [56]. On the contrary, another study showed that KMO deficiency reduced the mortality in encephalomyocarditis virus-infected mice [57]. Therefore, KMO might play dual roles in regulating immune responses through various mechanisms, thus exerting beneficial and harmful effects on various diseases. Further studies are consequently needed to clarify how KMO precisely modulates inflammation, innate immunity, and adaptive immunity, especially in patients with autoimmune diseases and chronically infectious diseases.

Tryptophan metabolism through the KP is often preferentially directed to produce QUIN catalyzed by KMO [50]. Our studies indicated that the antiviral activity of KMO was mediated through its enzymatic product QUIN. We have provided evidence that QUIN activates the N-methyl-d-aspartate receptor (NMDAR) and Ca2+ influx, which trigger the phosphorylation of CaMKII and IRF3, leading to IFN-I production. The previous study also found that Ca2+ and its major downstream effector, CaMKII, are important for immune cells’ functions, and CaMKII could significantly enhance the production of IFN-α/β in macrophages [37,58]. However, other studies demonstrated that NMDAR antagonists such as MK-801 inhibited Japanese encephalitis virus (JEV)-mediated neuropathogenesis and VSV infection [59,60]. Therefore, modulation of NMDAR in different cells or tissues might be a double-edged sword.

Of note, besides the enhanced IFN-β-mediated innate immunity, our data showed that QUIN treatment also promoted the IFN-γ-mediated cytolytic T lymphocytes (CTL) against HSV-1 antigen-specific stimulation. Stimulating antigen-specific CTL is essential for eradicating virus-infected host cells [61] and inhibiting the activation of viral latency [62], which is important for controlling chronically viral infections, such as HIV-1, HSV-1. Especially, IFN-γ could effectively suppress the reactivation from HSV-1 latency in sensory neurons [63,64]. Therefore, the enhancement of HSV-1-specific CTL responses by QUIN treatment contributed to suppressing herpes disease progression and providing long-term protection in our study.

The structural skeleton of QUIN and its derivatives were previously reported as an important pharmacophore and explored to develop anti-infectious agents, including anti-malaria [65,66], anti-tuberculosis [67], and anti-tumor [68]. Our studies showed that QUIN had broad antiviral activities against a broad range of DNA and RNA viruses, including HSV-1, adenovirus, VSV, influenza virus (PR8), ZIKV, DENV, and SARS-CoV-2. We further demonstrated that administration of QUIN significantly inhibited viral infections and alleviated disease progression in the mouse model of pathogenic HSV-1 infection. To be noted, QUIN is a neuroactive metabolite and can induce oxidative neurotoxicity [69-71]. A high level of QUIN was reported to associate with Alzheimer’s disease, anxiety, depression, epilepsy, and Huntington’s disease [72]. Therefore, its potential neurotoxicity should be addressed when developing the QUIN as an antiviral drug candidate. Since QUIN cannot penetrate the blood-brain barrier [73], and thus QUIN was injected intraperitoneally to exert antiviral effects in our in vivo experiment to avoid the potential side effect. Nevertheless, the safety of the long-term administration of QUIN should be further monitored in the future.

Some studies reported that the concentration of the QUIN was increased because of KP activation in response to some pathogen infections (such as HIV-1, HSV-1, influenza A virus) [74-80]. Importantly, recent studies also reported that KP metabolites, including kynurenate, kynurenine, 8-methoxykynurenate, were enriched in COVID-19 patients, and the decrease in tryptophan and the increase in kynurenine were correlated to the COVID-19 disease severity [81-83]. Given that we have encountered numerous challenges with emerging pathogenic viruses in the past decades years, including SARS, MARS, EBOLA, ZIKV, and SARS-COV-2, further studies on immune regulation of metabolic genes such as KMO may provide insights to develop novel broad antiviral agents like QUIN to treat diseases associated with many unpredictable, viral pathogens.

In summary, it is of great interest to further investigate the possibility of applying KMO and QUIN as antiviral drug candidates in future studies.

Materials and methods

Ethics statement

The animal study was approved by the Institutional Review Boards and Animal Care and Use Committees of Sun Yat-Sen University (Approval No. SYSU-IACUC-2021-000048).

Cell lines

293T cells (from the embryonic kidney of a female human fetus), Vero cells (from the kidney of a female normal adult African green monkey), A549 cells (from human alveolar adenocarcinoma basal epithelial cells), Hela cells (from human cervical cancer tissue)and RAW 264.7 cells (from macrophage of a male adult mouse) were cultured in complete Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Gibco), at 37°C in an atmosphere of 5% of CO2. THP-1 cells (from the peripheral blood of a boy with acute monocytic leukemia) were cultured in conditioned RPMI 1640 medium containing 10% fetal bovine serum (FBS, Gibco), 1% penicillin/streptomycin (Gibco) at 37°C in an atmosphere of 5% of CO2. The above cells were stocked in our laboratory.

293T kmo cells were constructed in our lab. In brief, 293 T cells were infected with sgRNA-expressing lentivirus with polybrene and then added with puromycin. The single clone of the kmo cell line was selected and validated by Western Blotting analysis and DNA sequencing.

Wild-type bone-marrow-derived macrophage cells (WT-J2-BMM) and interferon-α receptor-deficient cells (Ifnar-/—J2-BMM) were gifted by Genhong Cheng (UCLA, USA) and cultured in conditioned RPMI 1640 medium containing 10% fetal bovine serum (FBS, Gibco), 1% penicillin/streptomycin (Gibco), 10 mM HEPES (pH 7.8), and 1% M-CSF (Gibco).

The kmo bone marrow-derived and peritoneal macrophages were isolated from kmo mice as previously described [84]. Briefly, bone marrow-derived macrophages were isolated from femurs and tibias of kmo mice. Peritoneal macrophages isolation is performed by injecting 4 mL PBS 1× through the peritoneal wall into the peritoneal, gently massaging the mouse abdomen, slowly recovering as much PBS 1× as possible. Cells were cultured in 10 mL conditioned medium (DMEM medium (Corning) with 10% fetal bovine serum, 1% L glutamine, 1% penicillin-streptomycin (Gibco), 100 ng/mL M-CSF (Biolegend) at 37°C in an atmosphere containing 5% CO2 in an incubator. On day 3, most cells were adherent to the dish. Discard the medium from the dish, and then the dish was supplemented with 10 mL of conditioned medium for further growth until day 6. On day 7, the cells were used to perform the corresponding experiments.

Viruses

VSV-GFP (VSV) was gifted by Dr. Tian Lan (VectorBuilder, Guangzhou, China). DENV4 and ZIKV were kindly gifted by Dr. Zhongyu Liu (Sun Yat-sen University, Guangzhou, China). Influenza virus A/PR8 was gifted by Prof. Yuelong Shu (Sun Yat-sen University, Guangzhou, China). Replication-competent SARS-CoV-2 virus-like-particles (SARS-CoV-2 GFP/ΔN) and Caco-2 cells expressing N protein (Caco-2- N) were gifted by Prof. Qiang Ding (Center for Infectious Disease Research, School of Medicine, Tsinghua University, Beijing, China). Ad5-GFP and HSV-GFP-Luc were stored in our lab. These viruses were used in cell studies.

The pathogenic HSV-1 McKrae strain (McKrae) was gifted by Prof. Jumin Zhou (Kunming Institute of Zoology, Chinese Academy of Sciences. Kunming, China). McKrae was used in an animal study.

Mice

WT C57BL/6 mice were purchased from the Laboratory Animal Resource Center of Sun Yat-sen University and bred in the SPF animal facility of Sun Yat-sen University in individually ventilated cages.

kmo-deficient C57BL/6 mice (kmo mice) were constructed by Cyagen Biosciences and bred in the SPF animal facility of the Laboratory Animal Resource Center of Sun Yat-sen University in individually ventilated cages. Briefly, using CRISPR/Cas9 technology, we obtained the kmo-knockout mice by applying high-throughput electro-transformation of sgRNA to fertilized eggs. The heterozygous F1 mice were obtained after being bred with wild-type C57BL/6 mice, and then the homozygous kmo F2 mice after being bred with heterozygous F1 mice. The tail tip was cut, and the tissue DNA was extracted to perform a PCR assay to identify the homozygous kmo mice (S8 Fig). In addition, the PCR products were also be sequenced for further confirmation.

Plasmid constructs

pVAX-KMO, pVAX-CH25H, pVAX-GFP: The full-length human KMO and human CH25H were cloned into the pVAX vector. pVAX-Green fluorescent protein (GFP) plasmid was stored in our laboratory used as a mock transfection in our study. KMO hydroxylase activity-dead mutant (KMO-M) was generated by site-directed mutagenesis kit (TransGen Biotech) from pVAX-KMO construct as described above. The primers were listed in the S1 Table.

Lentivirus production

Small guide RNA (sgRNA)

sgRNAs targeting kmo gene were designed by using a CRISPR design tool (http://zlab.bio/guide-design-resources)), and three highest-scoring oligonucleotides were selected and cloned into pLentiCRISPRv2 (Addgene #52961) respectively. The recombinant lentivirus was generated by co-transfecting 293T cells with a cocktail of lentiCRISPRv2, packaging plasmid pMD2.G (Addgene #12259) and psPAX2 (Addgene #12260) using Lipofectamine 2000 transfection reagent (Invitrogen). The primers were listed in the S1 Table.

Short hairpin RNA (shRNA)

shRNAs targeting KMO mRNA were designed using an shRNA design tool (http://sirna.wi.mit.edu/), and the top-scoring targets were selected in this study. The forward and reverse oligonucleotides were synthesized and then annealed and cloned into the pLKO.1 vector. Lentiviral vectors were generated by co-transfecting 293T cells with a cocktail of pLKO.1 shRNA plasmid, packaging plasmid psPAX2, envelope plasmid pMD2.G using Lipofectamine 2000 transfection reagent (Invitrogen). The primers were listed in the S1 Table.

siRNA and cell transfection

According to the manufacturer’s manuals, the cultured cells with 80% confluence were transfected with different plasmids using Lipofectamine 2000 Transfection Reagent. The medium was replaced with DMEM containing 5% FBS and 1% penicillin/streptomycin after 5 h transfection, and then the cells were incubated for 24 h or 48 h.

The siRNA oligonucleotide duplexes targeting KMO were synthesized by Sangon (Shanghai, China). The negative-control siRNAs were purchased from Sangon. According to the manufacturer’s protocol, the cells were transfected with 100 nM of the indicated siRNAs for 48–72 h by using Lipofectamine RNAiMax transfection reagent (Invitrogen). The knockdown efficacy of the target genes was detected by quantitative real-time PCR (RT-qPCR) or Western Blotting Analysis. The sequences of all siRNAs were listed in S1 Table, and all primers for RT-qPCR were listed in the S2 Table.

Western Blotting analysis

The Western Blotting assay was performed as previously described [27]. Reagents and antibodies are listed in S4 Table.

RNA-seq library preparation, sequencing, and data processing

RNA-seq experiments were performed as previously described [85]. In brief, RAW264.7 cells were incubated with or without QUIN at a dose of 1mM for 8 hours. Then the cells were infected with HSV-1 for another 8 hours. Bone marrow-derived macrophages from the kmo mice and kmo mice were infected with HSV-1 for 8 hours. According to the manufacturer’s instructions, these samples were collected for total RNA extraction and then used to generate RNA-seq libraries with a TruSeq PE Cluster Kit v4-cBot-HS (Illumina, USA). The prepared libraries were sequenced on an Illumina platform by Sangon Biotech (Shanghai, China). Genes with P values < 0.05 and fold change > 1.5 were differentially expressed. Gene Ontology (GO) analysis was performed by using the GO knowledgebase (https://geneontology.org/), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed by using the KEGG database (hyyp://www.kegg.jp). The volcano plot was drawn by the Volcano mapping tool in SangerBox, and the GraphPad Prism software drew the heatmap.

Co-immunoprecipitation

The cells were harvested using NP40 lysis buffer containing protease inhibitors (Beyotime, China). After incubation on ice for 30min, the lysates were centrifuged at 13,000 rpm for 20 min at 4°C. Subsequently, a proportion of the cell lysate was performed for analysis as input, and another proportion was subjected to precipitation with appropriate antibodies overnight at 4°C. The next day, the beads were washed three times with wash buffer (50 mM Tris pH 7.4, 500 mM NaCl, 0.1% (v/v) NP-40, 1 mM EDTA). The precipitated proteins were eluted from beads or gel by the heating sample in SDS loading buffer at 100°C for 10 min. The precipitates were subjected to Western Blotting Analysis.

Viral plaque assay

The Vero cells were seeded in a 12-well plate at a density of 5 × 105 for 12 h, and the virus samples in 10-fold serial dilution were added. After 2 h of incubation at 37°C in 5% CO2, the supernatant was aspirated, and the cells were washed with PBS. Then, each well added a medium containing 1% FBS, 1% low-melting-point agarose, and 1% penicillin-streptomycin. After 5 days of incubation, the cells were fixed with paraformaldehyde for 1 h and stained with 2% crystal violet for another 1 h. Plaques were visualized and enumerated, and the virus titer was calculated as plaque-forming units per mL (PFU/mL).

Luciferase assay

The Steady-Glo Renilla Luciferase detection system and Dual-Luciferase Reporter Assay were employed according to the manufacturer’s instructions (Promega, Madison, WI, USA).

Ca2+ imaging

Calbryte 520 dye is a cell-permeable calcium indicator. Cells were loaded with this dye for 25 min in the dark at room temperature and washed 3 times with PBS. Next, cells were treated with appropriate stimulation. Calbryte 520 fluorescence imaging was recorded under a Leica microscope (Leica, Wetzlar, Germany).

Cell viability assay

1×104 cells were inoculated in each well of 96-well-plate, and a series of concentrations of QUIN were added. After incubating for the required time, 10 μl CCK8 solution was added into each well, followed by 1 h in the dark at 37°C. The OD at 450 nm was then detected by a microplate reader (Biotek, Synergy HTX, USA).

In vivo therapy experiment

C57BL/6 mice were bred in the SPF animal facility of the Laboratory Animal Resource Center of Sun Yat-sen University, and this study was approved by the Institutional Review Boards and Animal Care and Use Committees of Sun Yat-Sen University (Approval No. SYSU-IACUC-2021-000048). 6 to 8-week-old mice were anesthetized, and corneal epithelial debridement was performed using a 30-gauge needle, followed by the inoculation of 105 PFU HSV-1 (McKrae). Intraperitoneal injections of ACV (5 mg/kg), QUIN (2.5 mg/kg), or vehicle alone in 2% DMSO were administered daily for one week, and the ocular swabs, disease scores, and corneal images (Carl Zeiss stereoscope) were acquired during the experiment. The corneal surface was washed with PBS (20 μl/eye) at various times post-infection, and then the virus titer of these samples was quantified by plaque assay.

Challenge experiment in kmo-deficient mice

kmo-deficient C57BL/6 mice (kmo mice) were constructed and identified as shown in S8 Fig and infected with 3×104 PFU HSV-1 (McKrae) by corneal inoculation as described above. The disease symptoms of experimental mice were monitored, and samples were collected as above to perform the corresponding detection.

QUIN rescue experiment

Both kmo+/+ and kmo-/- C57BL/6 mice were infected by HSV-1 (McKrae) as described above, and then intraperitoneal injections of QUIN (0, 0.5, 1, 2.5 mg/kg) were daily administered for one week. The disease symptoms of experimental mice were monitored, and samples were collected to perform the corresponding detection.

ELISA assay

The samples were collected and analyzed with the Mouse IFN Beta ELISA Kit (Solarbio Life Science, Beijing, China) according to the manufacturer’s instructions.

ELISPOT

Enzyme-linked immunosorbent spot (ELISPOT) assays were performed as previously described [86]. Briefly, 96-well plates (Millipore, Immobilon-P membrane) were coated with anti-IFN-γ monoclonal antibody (BD Pharmingen) overnight at 4°Cand then blocked with 10% fetal bovine serum for 2 h at 37°C. Freshly isolated splenocytes were added at 4 × 105 cells/well, and the HSV-1 peptides (Genscript, Nanjing, China, listed in S3 Table were immediately added at a final concentration of 2 mg/mL. The cells were incubated for 24 h at 37°C, and the expression of IFN-γ was then detected using biotinylated polyclonal anti-mouse IFN-γ (BD Pharmingen) and NBT/BCIP reagent (Pierce). Finally, the numbers of spots were quantified using an ELISPOT reader (Bioreader4000, BIOSYS, Germany). The data was reported as spot-forming cells (SFC) per million cells.

Intracellular Cytokine Staining (ICS)

ICS was processed according to our previous method [87]. Briefly, 1×106 freshly isolated mouse splenocytes were stimulated with HSV-1 peptides (Genscript, Nanjing, China, listed in S3 Table at a final concentration of 2 mg/mL for 2 h at 37°C. The splenocytes were then incubated with brefeldin A (BD Pharmingen) for 16 h at 37°C. After incubation, the cells were collected and stained with anti-CD3-FITC, anti-CD4-BB700, and anti-CD8-PE-Cy7 monoclonal antibodies (BD Biosciences) for 1 h. Then the resuspended cells were permeabilized in FACS Perm/wash buffer for 20 min before staining with anti-IFN-γ-Alexa Fluor 647, anti-TNF-α-PE, and anti-IL-2-BV605 (BD Pharmingen). Samples were tested using the flow cytometer (CytoFLEX S, Beckman, America) instrument with CytExpert software (version 2.4).

The complete blood counts

Peripheral blood and plasma samples of experimental mice were collected following standard protocols, and the complete blood count was conducted with Mindray veterinary automatic blood cell analyzer BC-2800Vet.

Hematoxylin and eosin (H&E) staining

The different tissues of experimental mice were immersed completely in 4% paraformaldehyde, gradually dehydrated, embedded in paraffin, and cut into sections. H&E staining was performed according to standard protocol by Wuhan Service Biotechnology CO., LTD.

LC-MS/MS analysis for Trp-Metabolites

The LC-MS/MS analysis of mouse serum (100 μl) was performed by Shanghai Applied Protein Technology, Shanghai, China.

Statistical analysis

Statistical analyses were performed using GraphPad Prism software version 8 (GraphPad Software, Inc.). Statistical significance was calculated using Student’s two-tailed unpaired t-test or ANOVA with Holm-Sidak’s multiple comparisons test. *P < 0.05; **P< 0.01; ***P < 0.001.

QUIN has a rescue effect on 293T kmo cell lines.

293T kmo cell lines were pretreated with QUIN for 8 h and then infected with HSV-1 at MOI of 0.25 for 8 h. Then, the expressions of HSV-1 and IFN-β were measured by RT-qPCR. The expression level of mRNA was normalized to the expression of β-actin, and the data from at least triplicates were shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

(TIF)

Click here for additional data file.

QUIN showed no obvious toxicity to different cell lines.

The different concentrations of QUIN were added into 293T, Vero, and Raw264.7 cells for 72 hours, and then the cell viability was detected with CCK8 assay. The final data are presented as the mean ± SD of at least triplicate experiments.

(TIF)

Click here for additional data file.

RNA-Seq analysis to reveal the modulation of host antiviral signal pathway during viral infections by QUIN treatment.

(A) Representative of the volcano plots to identify the differential gene expression (DGE) between QUIN-treated cells and non-QUIN-treated cells. Adjust P-value < 0.05, fold change |FC| > 1.5. Red dots represent those up-regulated genes. Green dots represent those down-regulated genes. Black dots represent those non-changed genes (Non-DEG). (B) The gene ontology (GO) annotation analysis for the related DEGs involved in QUIN treatment. (C) The enrichment analysis of Kyoto encyclopedia of genes and genomes (KEGG) of the related signaling pathways by QUIN treatment (P-value < 0.05). (D) Heatmap of the 19 selected genes involved in antiviral signaling pathway by QUIN treatment (P-value < 0.05).

(TIF)

Click here for additional data file.

The expression of NMDAR in different cells.

The Western Blotting analysis confirmed NMDAR protein expression in 293T, 293T kmo, Hela, A549, BMM, THP-1, Raw 264.7 cell lines. Mouse brain protein lysate as the positive control.

(TIF)

Click here for additional data file.

KMO and QUIN significantly enhanced IFN-β expression during viral infections.

(A) Cells were transfected with different concentrations of KMO-expressing plasmid for 24 h, followed by viral infections for 8 h at MOI of 1, including HSV-1, VSV, ZIKV, DENV4, Ad5, SARS-CoV-2 pseudovirus, and PR8, and then the expression of IFN-β was quantified by RT-qPCR. (B) Cells were pretreated with QUIN at different concentrations for 8 h, followed by viral infections for 8 h at MOI of 1, including HSV-1, VSV, ZIKV, DENV4, Ad5, SARS-CoV-2 pseudovirus, and PR8, and then the expression of IFN-β was quantified by RT-qPCR. The expression level of mRNA was normalized to the expression of β-actin, and the data from at least triplicates were shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

(TIF)

Click here for additional data file.

QUIN inhibits viral replication in different cell lines.

A549 cell lines and BMM cell lines were pretreated with QUIN (1mM) for 8 h, followed by infected with different viruses at MOI of 1 for 8 h. The expression of viruses was measured by RT-qPCR. The expression level of mRNA was normalized to the expression of β-actin, and the data from at least triplicates were shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

(TIF)

Click here for additional data file.

KMO and QUIN exerted the antiviral effect independent on IDO1.

(A,B) 293T cell lines were pretreated with IDO1 inhibitor 1-MT (10 μM) for 4 h, followed by HSV-1 infection at MOI of 1 for 8 h. (C,D) Cells were pretreated with 1-MT, and then treated with KMO over-expression or QUIN followed by HSV-1 infection. The expression of HSV-1 and IDO1 were measured by RT-qPCR. The expression level of mRNA was normalized to the expression of β-actin, and the data from at least triplicates were shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

(TIF)

Click here for additional data file.

Construction and identification of kmo mice in this study.

(A) kmo knockout strategy. The position shown by the scissors was the two sites of kmo exons targeted by designed CRISPR/Cas9. The red line represents the knocked-out fragment. F1/R1 primer pair and F1/R2 primer pair were the positions of the two primers to identify whether the knockout was successful. (B) The kmo mice and wild-type mice were identified by F1/R1 primers. Theoretically, the amplified fragment of kmo mice should be 574 bp (base pair) in length, while that of wild-type mice is too long to be amplified. (C) The kmo mice and wild-type mice were identified by F1/R2 primers. Theoretically, the amplified fragment of wild-type mice should be 514 bp (base pair) in length, while that of kmo mice cannot be amplified because of the lack of R2 sequence. (D) The kmo mice and wild-type mice were identified by Western Blotting (top) and RT-qPCR (bottom). The expression level of mRNA was normalized to the expression of β-actin, and the data from at least triplicates were shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

(TIF)

Click here for additional data file.

RNA-Seq data to analyze the transcriptome difference of primary macrophages between kmo mice and kmo mice.

(A) Representative of the volcano plots to identify the differential gene expression (DGE) between bone marrow-derived macrophages from wild kmo mice and kmo mice. Adjust P-value < 0.05, fold change |FC| > 1.5. Red dots represent those up-regulated genes. Green dots represent those down-regulated genes. Black dots represent those non-changed genes (Non-DEG). (B) The gene ontology (GO) annotation analysis for the upregulated DEGs in bone marrow-derived macrophages from wild kmo mice compared to kmo mice. (C) The enrichment analysis of Kyoto encyclopedia of genes and genomes (KEGG) of the upregulated signaling pathways in bone marrow-derived macrophages from wild kmo mice compared to kmo mice (P-value < 0.05). (D) Heatmap of the selected genes involved in antiviral signaling pathway (P-value < 0.05).

(TIF)

Click here for additional data file.

QUIN has a rescue effect in virus-infected kmo mice.

(A) Schedule for evaluating the rescue effect of QUIN on antiviral infections in kmo mice. Briefly, both kmo C57BL/6 mice and kmo C57BL/6 mice were corneally inoculated with 1×105 PFU HSV-1 McKrae strain, disease symptoms of experimental mice were monitored until 7 dpi. (B,C) Survival curve and disease score of experimental mice in different groups over time post-infection (n = 5 per group). (D) The HSV-1 titer in the eye washing fluid at 6 dpi was measured by plaque assay (n = 3 per group). (E) The concentration of IFN-β in the serum of experimental mice at 7 dpi was measured by ELISA assay (n = 3 per group). The final data are presented as the mean ± SD of triplicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

(TIF)

Click here for additional data file.

The kynurenine, a metabolic substrate of KMO, was accumulated in kmo mice.

The blood of virus-infected and non-virus-infected kmo mice and kmo mice were collected by orbital bleeding, and the plasma was separated to determine tryptophan metabolites including Trytophan (A), N-formyl-kynurenine (B), Kynurenine (C). The final data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

(TIF)

Click here for additional data file.

The sequences of siRNA, sgRNA, shRNA used in this study.

(DOCX)

Click here for additional data file.

Primers for RT-qPCR used in this study.

(DOCX)

Click here for additional data file.

The sequence of HSV-1 peptide used in this study.

(DOCX)

Click here for additional data file.

Key Resources used in this study.

(DOCX)

Click here for additional data file.

7 Dec 2021

Dear Dr. Sun,

Thank you very much for submitting your manuscript "Kynurenine-3-monooxygenase (KMO) broadly inhibits viral infection via triggering NMDAR/Ca2+ influx and CaMKII/ IRF3-mediated IFN-β production" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Jun Zhao, Ph.D.,

Guest Editor

PLOS Pathogens

Adolfo García-Sastre

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: Exploring the novel antiviral agents is a public health priority against frequent outbreaks of emerging infectious diseases. In this manuscript, Zhao and colleagues reported that KMO and its enzymatic product QUIN exerted a novel broad-spectrum antiviral function in vitro and in vivo via triggering NMDAR/Ca2+ influx and CaMKII/ IRF3-mediated IFN-�  production. Previously, KMO was recognized as a key rate-limiting enzyme in the Tryptophan metabolism, but its role in the innate immunity in response to viral infections has not been clarified. Thus, this work addresses an important topic to help determine the possibility of applying KMO as a novel target of antiviral drugs. The authors' intriguing data is therefore of considerable interest. There are still some issues to be addressed by the authors that would improve the manuscript.

Reviewer #2: This study showed that the mid-stream enzyme from the kynurenine pathway of the tryptophan metabolism, KMO, plays a role in modulating anti-viral activity. They showed that KMO and quinolinic acid (QA) can exert antiviral activity involving NMDA-induced calcium flux and IRF-mediated IFN-beta activity. They further showed that KMO expression and QA were responsible for inhibiting a broad spectrum of viruses that may confer a protective and susceptibility role in the host against viral infection.

A major strength of the study is that the authors used various approaches to examine the role of KMO and QA as novel antiviral targets. For this, I appreciate and acknowledge the efforts of this study. Another interesting result from this study that was understated in this study is the result of KMO and QA having a direct impact on the immune activity in driving antiviral activity.

A major concern I have is to what extent does IDO-1 involved in this study co-commitment with KMO activity? This is something that needs to be teased out since IDO-1 also has antiviral activity. For example, O Adams et al (J Virol, 2004 - https://dx.doi.org/10.1128/JVI.78.5.2632-2636.2004) showed that IDO-1 can exert antiviral effect via interferon activity. We know that viral infection can trigger IDO-1, which is likely to be the case in this study. Did the authors look at IDO-1 expression to make sure their result is due to KMO and not IDO-1? It is also possible that inhibition of KMO leads to accumulation of kynurenine which can have a negative feedback inhibition on IDO-1, thereby suppressing antiviral activity as well. Hence this is important missing info to look into.

In short, if the authors can prove that the antiviral effect is due to KMO and not IDO. This will be a novel role in the field of tryptophan research.

Reviewer #3: This study examined the mechanism of action of kynurenine-3-monooxygenase (KMO)-mediated antiviral effect. It was shown that the enzymatic product of KMO, quinolinic acid (QUIN), activates N-methyl-d-aspartate receptor (NMDAR) and Ca2+ influx, which subsequently activates Calcium/calmodulin29 dependent protein kinase II (CaMKII) / Interferon regulatory factor 3 (IRF3). Over-expression of KMO led to the inhibition of viral replication, while knockout or mutate KMO promote viral replication. Similarly, QUIN treatment led to the inhibition of viral replication of multiple viruses in cell culture. QUIN was also shown to have in vivo antiviral efficacy in HSV-1 infection mouse model.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: 1. The author analyzed the difference in transcriptomics of cell lines after virus infections with or without QUIN incubation and found the changes in the interferon pathway. It is recommended to analyze further the difference in transcriptomics of primary cells between KMO-/- mice and wild KMO+/+ mice after viral infections.

2. Does QUIN have rescue effects on KMO-/- cell lines or KMO-/- mice infected with the virus?

3. Fig S5 provided some vital information regarding the immune dysfunction status in kmo-/- mice compared to wild mice, and thus this figure should be moved to the Text.

4. Line 269-271 and Line 285-288: repeated descriptions

5. Line 288-290: The cited literature seems not related to the topic what authors meant

6. phrases such as in vitro, in vivo, and KMO (or kmo) is sometimes in italics and sometimes not in italics

Reviewer #2: 1) Demonstrate that all antiviral activities is independent of IDO-1 expression.

2) If IDO-1 expression confound with the finding, does pharmacological inhibition of IDO-1 (e.g. 1-MT) still confer antiviral activity by KMO?

Reviewer #3: 1. The antiviral effect shown in Fig. 4 is very moderate, and in most case less than 1 log viral reduction was observed. It is also not shown whether the antiviral effect is cell type dependent. For example, does QUIN inhibit influenza virus replication in epithelial cells?

2. NMDA receptor is found in neurons. However, not all the viruses studied in this ms including HSV-1, VSV, PR8, ZIKA, Dengue, and SARS-CoV-2 are neurotropic viruses. Therefore there is a lack of physiological relevance for the use of NMDA activator as antivirals. Are the cell lines used for the antiviral assay all express NMDAR? If yes, what are the evidences?

3. The claim of repurposing quinolinic acid as a broad-spectrum antiviral is misleading. Quinolinic acid (QUIN) is a neuroactive metabolite, and can induce oxidative neurotoxicity. High quinolinic acid levels have been associated with Alzheimer's disease, anxiety, depression, epilepsy, and Huntington's disease. Therefore, a section should be added to state the well-known side effects of quinolinic acid so the readers are not biased.

Ref:Davis I & Liu A (2015) What is the tryptophan kynurenine pathway and why is it important to neurotherapeutics? Expert Rev Neurother 15, 719–721.

4. In the in vivo study, QUIN is dosed by IP injection, and it has been shown that QUIN could not pass the BBB, therefore, how could it elicit the antiviral effect? A relatively low dose of 2.5mg/kg of QUIN was used, is this sufficient to achieve the in vivo drug concentration that is needed to suppress viral infection?

5. NMDAR antagonists such as MK-801 have been shown to inhibit Japanese encephalitis virus (JEV)-mediated neuropathogenesis (ref below). In addition, NMDAR antagonists were also reported as VSV and influenza antivirals. Therefore, modulation of NMDAR might be a double-edge sword.

Ref: Journal of Neuroinflammation volume 15, Article number: 238 (2018)

Bioorg Med Chem. 2012 Jan 15;20(2):942-8.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: The language needs professional editing, here are a few examples.

Line 21: replace "through kynurenine pathway" with "through the kynurenine pathway"

Line 43: replace "mechanism" with "mechanisms"

Line 44: replace "in vivo" with italics "in vivo"

Line 83:replace "infection" with "infections"

Line 118: replace "overexpression" with "the overexpression"

Line 271: remove the space after "kmo+/+ mice"

Line 202, 204, 225, 319: replace "viral infection" with "viral infections"

Reviewer #2: The overall narrative of the introduction and discussion that links KMO to cancer and neurodegeneration seems slightly out of context with what the study is trying to demonstrate. Hence, the direct focus of the role of KP in immunology and virology should be discussed more thoroughly. Some earlier work on KP (especially IDO-1) and antiviral is missing such as O Adams et al (as mentioned above), Gaeling L et al (2016) - https://doi.org/10.1111/febs.13966, Fernandez-Pol (2001) - https://pubmed.ncbi.nlm.nih.gov/11911246/. The authors may also want to consider linking some of the KP discoveries performed in COVID-19. See this paper: https://doi.org/10.1016/j.cels.2020.10.003.

Another question that is worth discussing in the paper is that if the host uses KMO-mediated QA to suppress viral activity, is there a long-term consequence where increased QA production chronically can promotes neurodegeneration?

Reviewer #3: (No Response)

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Chunfu ZHENG

Reviewer #2: Yes: Chai K Lim

Reviewer #3: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at .

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here on PLOS Biology: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

27 Jan 2022

Submitted filename: Response to reviewers.docx

Click here for additional data file.

14 Feb 2022

Dear Dr. Sun,

We are pleased to inform you that your manuscript 'Kynurenine-3-monooxygenase (KMO) broadly inhibits viral infections via triggering NMDAR/Ca2+ influx and CaMKII/ IRF3-mediated IFN-β production' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Jun Zhao, Ph.D.,

Guest Editor

PLOS Pathogens

Adolfo García-Sastre

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: They have well addressed all my concerns, now it is acceptable.

Reviewer #2: This is a revised manuscript. Overall, the authors had addressed the questions raised by the reviewers. I'm happy with all the revision made. Hence, my recommendation is accepting the manuscript. Thanks for the great effort and excellent work!

Reviewer #3: Comments from the previous round of review were largely addressed. I therefore recommend acceptance.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: They have well addressed all my concerns, now it is acceptable.

Reviewer #2: No issue.

Reviewer #3: (No Response)

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: They have well addressed all my concerns, now it is acceptable.

Reviewer #2: No issue

Reviewer #3: (No Response)

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Chunfu ZHENG

Reviewer #2: Yes: Chai K Lim

Reviewer #3: Yes: Jun Wang

24 Feb 2022

Dear Dr. Sun,

We are delighted to inform you that your manuscript, "Kynurenine-3-monooxygenase (KMO) broadly inhibits viral infections via triggering NMDAR/Ca2+ influx and CaMKII/ IRF3-mediated IFN-β production," has been formally accepted for publication in PLOS Pathogens.

We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Pearls, Reviews, Opinions, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064