Manganese Mediates Its Antiviral Functions in a cGAS-STING Pathway Independent Manner

The innate immune system is the first line of host defense sensing viral infection. Manganese (Mn) has recently been found to be involved in the activation of the innate immune DNA-sensing cGAS-STING pathway and subsequent anti-DNA virus function. However, it is still unclear whether Mn2+ mediates host defense against RNA viruses. In this study, we demonstrate that Mn2+ exhibited antiviral effects against various animal and human viruses, including RNA viruses such as PRRSVs and VSV, as well as DNA viruses such as HSV1, in a dose-dependent manner. Moreover, cGAS and STING were both investigated in the Mn2+ mediated antiviral roles using the knockout cells made by the CRISPR-Cas9 approach. Unexpectedly, the results revealed that neither cGAS knockout nor STING knockout had any effect on Mn2+-mediated antiviral functions. Nevertheless, we verified that Mn2+ promoted the activation of the cGAS-STING signaling pathway. These findings suggest that Mn2+ has broad-spectrum antiviral activities in a cGAS-STING pathway independent manner. This study also provides significant insights into redundant mechanisms participating in the Mn2+ antiviral functions, and also indicates a new target for Mn2+ antiviral therapeutics.

The cGAS-STING pathway has been identified as the important DNA-sensing machinery in innate immunity against pathogens [6,7]. cGAS senses the presence of non-self and self-DNA, and utilizes substrates ATP and GTP to catalyze the production of the second messenger cyclic GMP-AMP (2 3 -cGAMP), which then activates the signaling adaptor protein STING [6,7]. Activated STING recruits the downstream TANK-binding kinase 1 (TBK1), and TBK1 is auto-phosphorylated [6,8]. Then, the transcription factor IRF3, which is recruited by STING and phosphorylated by TBK1, translocates to the nucleus and induces antiviral type I IFNs and IFN-stimulated genes (ISGs) [6,8]. Another transcription factor, NF-κB, is also activated by STING-TBK1 signaling, and drives proinflammatory gene expressions [6,9].
Manganese (Mn) is required as an enzymatic cofactor in many physiologic processes, such as protein and energy metabolism, immune function, development, reproduction, neuronal regulation, and antioxidant defenses [10][11][12]. Several canonical signaling pathways have been reported to be Mn-responsive, including the ataxia telangiectasia mutated 2 (ATM), p53, phosphatidylinositol 3 kinase (PI3K), insulin, and insulin-like growth factor-1 (IGF-1) pathways [13][14][15]. However, attention has been recently paid to Mn in the regulation of the cGAS-STING pathway, which exerts a potent host defense against DNA viruses [16]. Mn 2+ was shown to increase the sensitivity of cGAS to double-stranded DNA (dsDNA) and its enzymatic activity. It also facilitates STING activity by boosting cGAMP-STING binding affinity [16]. Further studies have revealed that Mn 2+ directly activates cGAS to induce a noncanonical catalytic synthesis of 2 3 -cGAMP, through similar overall conformation to dsDNA-activated cGAS [17,18].
Interestingly, recent publications have suggested the relevance of the cGAS-STING pathway in the process of RNA virus infections [19][20][21][22]. However, it is still largely unclear as to whether Mn 2+ plays a role in cGAS-STING pathway-mediated anti-RNA virus infections. Moreover, it has not been fully elucidated as to how Mn 2+ mediates its antiviral functions. In this study, we found that Mn 2+ exerts a broad antiviral function against various viruses, including some RNA and DNA viruses, which is independent of the cGAS-STING pathway.

Western Blot Analysis
Proteins were extracted in a radioimmunoprecipitation assay (RIPA) lysis buffer, mixed with a 4 × loading buffer in a 3:1 ratio, and boiled at 100 • C for 5-10 min. The protein samples were separated on 10% SDS-PAGE gels, and then transferred to PVDF membranes. After blocking with 5% skim milk solution at room temperature (RT) for 60 min, membranes were incubated with individual primary antibodies at 4 • C overnight. . Secondary antibody HRP-conjugated goat anti-mouse or rabbit IgG (TransGen Biotech, Beijing, China) was used to incubate the membranes for 60 min at RT. Signals were detected using enhanced chemiluminescence (ECL) substrate (Tanon, Shanghai, China), and images were visualized with an imaging system (Tanon, Shanghai, China).

Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted using TRIpure reagent (Aidlab, Beijing, China). The cDNA was synthesized using HiScript ® 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). The target gene expressions were examined using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) on a StepOne Plus real-time PCR system (Applied Biosystems, Foster City, CA, USA). The qPCR program was 95 • C for 30 s, followed by 40 cycles of 95 • C for 5 s and 60 • C for 1 min. β-actin served as an internal reference control. The relative mRNA levels were calculated using the 2 −∆∆CT method. For all the qPCR assays, an efficiency comprised between 90 and 110% was measured. The sequence of qPCR primers used in this study are listed in Table S1.

CRISPR gRNA Design and Preparation of Knockout (KO) Cells
The CRISPR gRNAs targeting porcine cGAS and monkey cGAS were designed using the web tool from Benchling (www.benchling.com (accessed on 4 January 2023)). For each gene, two gRNAs were chosen according to the predicted high scores, which are shown in Table S2. The recombinant pX458-gRNA plasmids were obtained when the annealed gRNA encoding DNA sequences were cloned into the Bbs I site of pX458-EGFP. Marc-145 cells or 3D4/21 cells were transfected with the corresponding recombinant pX458-gRNA plasmids using Lipofectamine 2000. At 24 h post transfection, the GFP positive cells were sorted by a FACS Aria SORP cell sorter (Becton Dickinson, Franklin lakes, NJ, USA) and cultured in 96-well plates by limiting dilution for monoclonal growth. The individual cell clones were screened by PCR using primers, as shown in Table S2. Briefly, the genomic PCR products were cloned into T vectors with a pClone007 versatile simple vector kit (TsingKe Biological Technology, Beijing, China). Base substitution, insertion, and deletion (ins/del) mutations were analyzed after the sequencing of inserted fragments, and cGAS -/monkey Marc-145 cells and cGAS -/porcine macrophages (3D4/21) were each acquired ( Figure S1). In addition, the STING -/-Marc-145 cells and STING -/-3D4/21 cells were previously obtained, and have been used in our lab [22,25].

Virus Tissue Culture Infectious Dose 50 (TCID50) Titrations
Marc-145 cells or 3D4/21 cells were seeded into 96-well plates, and then infected with 10-fold serial dilutions of various virus samples (Marc-145 cells for PRRSVs and 3D4/21 cells for VSV and HSV1). Next, the infected cell supernatants were replaced with fresh DMEM or RPMI 1640 containing 2% FBS, and the cells were monitored for the GFP fluorescence and cytopathic effects (CPE) characterized by cell clumping and shrinkage in Marc-145 cells or 3D4/21 cells after infections for 1-5 days. Finally, the viral titers were expressed as TCID50 and calculated using the method of Reed-Muench.

Dual-Luciferase Reporter Promoter Assay
The 293T cells were seeded in 96-well plates, followed by transfection the next day. Cells were co-transfected with reporter plasmids, ISRE-Firefly luc (Fluc) or IFNβ-Fluc (10 ng/well) and Renilla luciferase (Rluc) reporters (0.2 ng/well), plus the indicated porcine cGAS and STING plasmids or vector control (10-30 ng/well) using Lipofectamine 2000. The total DNA per well was normalized with control vectors to 50 ng. Twenty-four hours post transfection, the cells were treated with different concentrations of Mn 2+ for another 24 h. Then, the cells were harvested, and luciferase activities were detected with the TransDetect Double-Luciferase Reporter Assay Kit (Vazyme, Nanjing, Jiangsu, China). The fold changes were calculated relative to control samples after Fluc normalization by the corresponding Rluc.

Statistical Analysis
The results were analyzed using the software GraphPad Prism v.6.0 and expressed as the mean ± standard deviation (SD). Statistical analysis was conducted by one way ANOVA, followed by Tukey's post hoc test. The normality of the data distribution was assessed using the Shapiro-Wilk test. A p value of less than 0.05 was considered statistically significant.

Mn 2+ Exerted Antiviral Functions against PRRSV, VSV and HSV-1
To investigate the effect of Mn 2+ on the different viruses, the Marc-145 cells and 3D4/21 cells were pretreated with various concentrations of Mn 2+ , and then the Marc-145 cells were infected with two PRRSV strains, whereas the 3D4/21 cells were infected with VSV and HSV-1, as shown in Figure 1A

Mn 2+ Triggered Antiviral Activity against PRRSVs Was cGAS-STING Independent
Mn 2+ has been found to promote the sensitivity of the cGAS-STING pathway for double-stranded DNA [16]. Thus, we first explored whether Mn 2+ affects PRRSV replications depending on cGAS and STING. The cGAS -/-Marc-145 cells ( Figure S1A

Mn 2+ Triggered Antiviral Activity against PRRSVs Was cGAS-STING Independent
Mn 2+ has been found to promote the sensitivity of the cGAS-STING pathway for double-stranded DNA [16]. Thus, we first explored whether Mn 2+ affects PRRSV replications depending on cGAS and STING. The cGAS -/-Marc-145 cells ( Figure S1A) and STING -/-Marc-145 cells were both utilized for PRRSV infections in the presence of Mn 2+ (200 and 500 μM), and the anti-PRRSV activity by Mn 2+ was examined. Western blotting and TCID50 assay showed that PRRSV XJ17-5 replication was upregulated in the cGAS -/-Marc-145 cells relative to those in normal Marc-145 cells. However, Mn 2+ -triggered anti-PRRSV XJ17-5 activity did not appear altered in cGAS -/-Marc-145 cells at both 24 h and 48 h post infection (Figure 2A,B). Similar results were obtained with the infections of PRRSV JXA1-  Then, the effect of Mn 2+ on PRRSV replication was examined in the STING -/-Marc-145 cells. Western blotting and TCID50 assay showed that the PRRSV XJ17-5 replication was upregulated in the STING -/-Marc-145 cells relative to normal Marc-145 cells. However, Mn 2+ (200 and 500 μM) triggered anti-PRRSV XJ17-5 activity, which was retained at both 24 h and 48 h post infection ( Figure 3A,B). Similar results were obtained with the PRRSV JXA1-R infections ( Figure 3C,D). Together, the results indicated that, although the cGAS-STING pathway plays a role in the anti-PRRSV innate immunity, it is not required for Mn 2+ -triggered anti-PRRSV activity. Then, the effect of Mn 2+ on PRRSV replication was examined in the STING -/-Marc-145 cells. Western blotting and TCID50 assay showed that the PRRSV XJ17-5 replication was upregulated in the STING -/-Marc-145 cells relative to normal Marc-145 cells. However, Mn 2+ (200 and 500 µM) triggered anti-PRRSV XJ17-5 activity, which was retained at both 24 h and 48 h post infection ( Figure 3A,B). Similar results were obtained with the PRRSV JXA1-R infections ( Figure 3C,D). Together, the results indicated that, although the cGAS-STING pathway plays a role in the anti-PRRSV innate immunity, it is not required for Mn 2+ -triggered anti-PRRSV activity.

Mn 2+ Triggered Antiviral Activity against VSV and HSV-1 Was Independent of cGAS-STING
To detect whether the antiviral functions of Mn 2+ against VSV and HSV-1 in 3D4/21 cells were dependent on cGAS and STING, the cGAS -/-3D4/21 cells ( Figure S1B) and STING -/-3D4/21 cells were both examined after virus infections. The results showed that the replications of VSV and HSV-1 were both upregulated in cGAS -/-3D4/21 cells, compared with those in normal 3D4/21 cells. However, the antiviral functions of Mn 2+ (100 and 200 μM) were not attenuated in cGAS -/-3D4/21 cells, as evidenced by Western blotting and TCID50 assay ( Figure 4A-D). The VSV and HSV-1 replications were also upregulated in STING -/-3D4/21 cells compared with those in normal 3D4/21 cells, but the Mn 2+ (100 and 200 μM)-triggered antiviral activities were not diminished in STING -/-3D4/21 cells ( Figure 5A-D). Altogether, the results clearly suggest that the cGAS-STING pathway is

Mn 2+ Triggered Antiviral Activity against VSV and HSV-1 Was Independent of cGAS-STING
To detect whether the antiviral functions of Mn 2+ against VSV and HSV-1 in 3D4/21 cells were dependent on cGAS and STING, the cGAS -/-3D4/21 cells ( Figure S1B) and STING -/-3D4/21 cells were both examined after virus infections. The results showed that the replications of VSV and HSV-1 were both upregulated in cGAS -/-3D4/21 cells, compared with those in normal 3D4/21 cells. However, the antiviral functions of Mn 2+ (100 and 200 µM) were not attenuated in cGAS -/-3D4/21 cells, as evidenced by Western blotting and TCID50 assay ( Figure 4A-D). The VSV and HSV-1 replications were also upregulated in STING -/-3D4/21 cells compared with those in normal 3D4/21 cells, but the Mn 2+ (100 and 200 µM)-triggered antiviral activities were not diminished in STING -/-3D4/21 cells (Figure 5A-D). Altogether, the results clearly suggest that the cGAS-STING pathway is not essential for Mn 2+ triggered antiviral functions, despite the important role that the cGAS-STING pathway plays in innate antiviral immunity. not essential for Mn 2+ triggered antiviral functions, despite the important role that the cGAS-STING pathway plays in innate antiviral immunity.

Mn 2+ Treatment Promoted cGAS-STING Signaling Activity
To further validate whether Mn 2+ can activate the cGAS-STING pathway, the effect of Mn 2+ on the cGAS-STING signaling pathway was investigated in both 293T and 3D4/21 cells. In 293T cells, the co-transfection of porcine cGAS and STING activated downstream IFNβ and ISRE promoter activities, as well as IFNβ and ISG60 gene transcriptions ( Figure  6A,B), whereas the Mn 2+ (100 and 200 μM) treatments increased the two promoter activi-

Mn 2+ Treatment Promoted cGAS-STING Signaling Activity
To further validate whether Mn 2+ can activate the cGAS-STING pathway, the effect of Mn 2+ on the cGAS-STING signaling pathway was investigated in both 293T and 3D4/21 cells. In 293T cells, the co-transfection of porcine cGAS and STING activated downstream IFNβ and ISRE promoter activities, as well as IFNβ and ISG60 gene transcriptions ( Figure 6A,B), whereas the Mn 2+ (100 and 200 µM) treatments increased the two promoter activities and IFNβ and ISG60 transcriptions in dose-dependent manners ( Figure 6A,B). In the 3D4/21 cells, both cGAS agonist polydA:dT and STING agonist 2 3 -cGAMP activated downstream IFNβ and ISG15 gene transcriptions, and the phosphorylations of TBK and IRF3 in Western blotting ( Figure 6C,D). Similarly, Mn 2+ (100 and 200 µM) further promoted the IFNβ and ISG15 transcriptions, and the phosphorylations of TBK and IRF3 in dose-dependent manners ( Figure 6C,D). The results suggested that the cGAS-STING activation may be one of the redundant mechanisms of Mn 2+ -triggered antiviral functions in cells.

Discussion
The element Mn is critical for almost all forms of life [10][11][12]. Cytosolic Mn 2+ has been reported to be involved in the dsDNA-sensing activity of cGAS, and protects against DNA viruses [16]. Recent studies have shown that Mn 2+ could directly activate cGAS, which is independent of dsDNA [17,18]. In addition, the overlapping mechanisms between the antiviral innate immunity developed against RNA and DNA viruses have been reviewed

Discussion
The element Mn is critical for almost all forms of life [10][11][12]. Cytosolic Mn 2+ has been reported to be involved in the dsDNA-sensing activity of cGAS, and protects against DNA viruses [16]. Recent studies have shown that Mn 2+ could directly activate cGAS, which is independent of dsDNA [17,18]. In addition, the overlapping mechanisms between the antiviral innate immunity developed against RNA and DNA viruses have been reviewed previously [26]. Many RNA viruses of families Flaviviridae, Coronaviridae, and Arteriviridae have been found to be associated with the cGAS-STING pathway [19][20][21][22]27]. Likewise, in our study, we demonstrated that Mn 2+ exerts antiviral functions against a DNA virus (HSV-1) and some RNA viruses (PRRSV XJ17-5, PRRSV JXA1-R, and VSV) in a dose-dependent manner. Mn 2+ exhibited a broad antiviral activity, which is similar to that mediated by the cGAS-STING pathway. At the first glance, it is logical to deduce that Mn 2+ exerts antiviral activity by acting on the cGAS-STING signaling pathway. However, further investigation revealed that Mn 2+ -triggered antiviral activity is cGAS-STING independent, suggesting that there is another cell mechanism which mediates the Mn 2+ antiviral functions.
Previously, Mn 2+ has been found to participate in the phosphorylation of p53 [15,28]. p53 is a tumor suppressor gene, and functions most commonly in cell cycle arrest, differentiation, and apoptosis [29]. Moreover, p53 has been reported to be involved the regulation of antiviral functions [30][31][32]. For example, p53 overexpression represses HIV-1 long terminal repeat (LTR) transcriptional elongation by preventing the phosphorylation of serine 2 of the pol II C-terminal domain (CTD), resulting in the inhibition of HIV-1 transcription and replication [30]. Influenza virus infection promotes the activation of the p53 pathway leading to apoptosis, and suppression of p53 activity contributes to influenza virus infection [31]. Similarly, depletion of p53 was shown to promote porcine epidemic diarrhea virus (PEDV) infection susceptibility [32]. p53 has robust antiviral immunity by activation of the IFN pathway and the induction of several antiviral proteins [33,34]. Miciak et al. have identified p53 as contributing to the perpetuation of IFN signaling through ISG-dependent positive feedback loops [35]. Hao et al. discovered that p53 facilitates IFN signaling and secretion, and activates ISREs and ISG expression during viral infection [32].
In addition, a recent report found that Mn 2+ alone activates the phosphorylation of TBK1 with ATM involved, and enhances DNA-or RNA-mediated innate immune responses [36]. TBK1 is the downstream mediator of multiple DNA sensors, such as Ku70, IFI16, cGAS, and DDX41 [37]. Similarly, it is also involved in the signaling pathway of RNA sensors, such as RIG-I and MDA5 [38,39]. Here, our study demonstrates that Mn 2+ has antiviral functions against RNA viruses (PRRSVs, VSV) and a DNA virus (HSV-1) in cGAS -/and STING -/cells. Mn 2+ may initiate ATM-TBK1 signaling to exert its broad-spectrum antiviral functions [36]. Together, we speculate that Mn 2+ may induce the activation of p53 and/or ATM-TBK1 to establish a powerfully antiviral state through cGAS-STING independent signaling pathways.
Nevertheless, we observed that the cGAS-STING signaling is indeed activated by Mn 2+ treatment. This observation is consistent with the previous discovery that Mn 2+ suppressed virus replicates by sensitizing both cGAS and STING [16], and suggests that cGAS-STING is one of the cell machineries that mediates Mn 2+ antiviral functions. In our study, although Mn 2+ -mediated antiviral functions via a cGAS-STING independent pathway, it was also identified as promoting cGAS-STING signaling activity. In which case, what is the relationship between cGAS-STING signaling and other cell machinery triggered by Mn 2+ ? These cell machineries must be redundant, and together participate in Mn 2+ -triggered antiviral functions. However, the exact molecular mechanisms underlying Mn 2+ -mediated antiviral functions merit further exploration for a complete elucidation.
In summary, we demonstrated that Mn 2+ inhibits PRRSV XJ17-5, PRRSV JXA1-R, VSV, and HSV-1 replications in a cGAS-STING independent manner. Our results reveal that Mn 2+ may harbor multiple cellular mechanisms to exert its broad-spectrum antiviral activity, and suggest that Mn 2+ has the potential to be used not only as an antiviral therapeutic, but also as the immune adjuvant in some animal vaccines.