Microtubule disruption synergizes with STING signaling to show potent and broad-spectrum antiviral activity

The activation of stimulator of interferon genes (STING) signaling induces the production of type I interferons (IFNs), which play critical roles in protective innate immunity for the host to defend against viral infections. Therefore, achieving sustained or enhanced STING activation could become an antiviral immune strategy with potential broad-spectrum activities. Here, we discovered that various clinically used microtubule-destabilizing agents (MDAs) for the treatment of cancer showed a synergistic effect with the activation of STING signaling in innate immune response. The combination of a STING agonist cGAMP and a microtubule depolymerizer MMAE boosted the activation of STING innate immune response and showed broad-spectrum antiviral activity against multiple families of viruses. Mechanistically, MMAE not only disrupted the microtubule network, but also switched the cGAMP-mediated STING trafficking pattern and changed the distribution of Golgi apparatus and STING puncta. The combination of cGAMP and MMAE promoted the oligomerization of STING and downstream signaling cascades. Importantly, the cGAMP plus MMAE treatment increased STING-mediated production of IFNs and other antiviral cytokines to inhibit viral propagation in vitro and in vivo. This study revealed a novel role of the microtubule destabilizer in antiviral immune responses and provides a previously unexploited strategy based on STING-induced innate antiviral immunity.


Introduction
Over the course of human history, viruses have posed substantial challenges to human health.In the combat against viral infections, drugs targeting viral components such as viral proteases have been successfully developed and widely used in clinic.Meanwhile, antibodies have been developed to block interactions between viruses and host cells.However, the effectiveness of these strategies quickly dwindled due to mutations in viral genes and subsequent drug resistance.To deal with the continuous challenge of viral infection, more potent and safer antiviral drugs exploiting host immune system would offer potential advantages including broad-spectrum activities and a lower likelihood to develop drug resistance [1,2].
Innate immunity, which is the first line of host immune system against the invasion of both DNA and RNA viruses, is rapidly activated upon detection of viral components, including proteins, DNAs, or RNAs.Growing evidence shows that cGAS (cyclic GMP-AMP synthetase)-STING (stimulator of interferon genes) signaling pathway plays a crucial role in sensing and responding to invasions from DNA viruses and retroviruses [3].Upon binding to cytosolic DNAs, cGAS is activated and catalyzes the production of the second messenger, cGAMP [4].Subsequently, cGAMP binds and activates STING, which recruits and triggers the activation of Tank binding kinase (TBK1).TBK1 phosphorylates the transcriptional factor IRF3, resulting in its translocation into nucleus and inducing robust type I IFNs response and NF-kB immune response [5,6].The IFNs induce the expression of hundreds of interferon-stimulated genes (ISGs) in an autocrine and paracrine manner, and ISGs interfere with almost every step in the virus life cycle.Therefore, strategies to modulate STING signaling network and enhance STING-associated immune responses may have potential applications in antiviral therapies.Recently, STING agonists, both cyclic dinucleotides (CDN) and non-CDN agonists, have been developed [7,8] and demonstrated their therapeutic benefit by providing robust protection against different viruses [9][10][11][12][13][14], supporting STING as a novel antiviral target.
Studies have shown that the trafficking of STING is dynamically regulated by vesicle trafficking system [15,16].STING is an ER-localized transmembrane protein and senses CDN ligands, such as 2'3'-cGAMP [17], 3'3'-cGAMP, c-di-AMP, and c-di-GMP [18].Upon ligand binding, STING forms oligomers [5] and is transported from ER to ER-Golgi intermediate compartments (ERGIC) and to Golgi apparatus via trafficking pathway [19,20].STING activates the downstream kinase TBK1 in Golgi and STING vesicles then exit from trans-Golgi network (TGN) and continue their journey to autophagosomes, endosomes, and lysosomes, where STING is ultimately degraded and the signal is terminated [21,22].Therefore, perturbing STING trafficking may provide an alternative or additional approach in STING modulation.Mounting evidence indicate that microtubules, an essential component of the cytoskeleton, play a crucial role in intracellular trafficking.So, disruption of the microtubule network not only seriously affects the viral replication cycle, such as viral entry, intracellular transport, and cell-to-cell spread [23,24], but also affects the proper distribution and function

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of organelles and vesicles, including the trafficking of STING [25].Importantly, we found that a microtubule-destabilizing agent (MDA), podofilox, enhanced STING signaling with antitumor activity in our previous studies [26].We were intrigued to hypothesize that MDAs may have antiviral activity by increasing STING signaling and innate immunity.It was reported that MDAs, such as nocodazole, colchicine, and vinblastine, can inhibit viral infection in cells at micromolar concentrations [27,28], while the antiviral mechanism was not defined.Thus, it is of great interest to elucidate the relationship between the post-activation trafficking regulation of STING and microtubule network, and to seek potential therapeutic intervention strategies to combat viral infections.
In this study, we report that monomethyl auristatin E (MMAE), a potent MDA commonly used in antibody-drug conjugates (ADCs) [29], enhanced STING signaling and augmented IFN and NF-kB responses.Although MMAE has been successfully used in ADC for cancer treatment, the functions of MMAE in innate immunity and its antiviral effect have not been reported so far.Detailed mechanistic studies revealed that MMAE changed the pattern of STING trafficking, increased STING-containing puncta and promoted STING oligomerization, leading to enhanced IFN productions and IFN-dependent broad-spectrum antiviral immune responses.We demonstrated that the combination of cGAMP and MMAE showed potent antiviral activity in a STING-dependent manner both in vitro and in vivo.These results provided insights into the regulatory mechanisms of microtubule structures on cGAMP-mediated STING pathway and paved the way for its potential application in antiviral immunotherapy.

MMAE specifically enhanced cGAMP-mediated STING signaling, including the phosphorylation signaling cascades and the innate immune response
THP1 Lucia ISG cells express the secreted luciferase reporter gene under the control of an IRF-inducible promoter containing five IFN-stimulated response elements (ISRE), allowing the monitoring of the IRF pathway.We found that a variety of MDAs increased cGAMPmediated luciferase signal in THP1 Lucia ISG cells through high-throughput screening (Fig 1A).To validate the findings, we investigated the effects of microtubule stabilizers (epothilone B, paclitaxel and docetaxel), DNA topoisomerase inhibitors (etoposide and topotecan), and other drugs that have microtubule-targeting activity.Indeed, only the microtubule destabilizers robustly enhanced the IRF-induced immune response in THP1 Lucia ISG cells induced by cGAMP (S1A and S1B Fig) .The results suggested that the potentiation effect of MDAs on the cGAMP-mediated signaling pathway may not be through mitotic arrest and its associated cell death.To gain insights into possible mechanisms of MDAs in innate immune responses, we selected MMAE as a representative member of MDAs for further investigations because of its relatively significant potentiation effect, well-known mode of action, and microtubule disruptive activity at lower doses [29].
We first examined the effect of MMAE alone on the cGAS-STING pathway in THP1-Lucia ISG cells.Compared with cGAMP treatment, MMAE could not induce activation of the ISRE luciferase reporter (Fig 1B), nor could it induce the phosphorylation of the STING signaling cascade on its own as shown by western blot (Fig 1C).However, multiple MDAs, including MMAE, increased the activation of the cGAMP-mediated STING signaling cascade further upon cGAMP co-treatment by analyzing the phosphorylation of STING, TBK1 and IRF3 (Figs 1D, S1C and S1D).Remarkably, the potentiation effect was observed within two hours after stimulation was initiated (Fig 1D ), suggesting that MMAE-mediated enhancement of STING signaling cascades is independent of mitotic arrest or cytotoxicity.We confirmed the immune potentiation effect of MMAE in RAW-Lucia ISG cells as well as in primary MEF cells (Fig 1E and 1F).Next, we investigated whether the effect of MMAE on cGAMP-mediated STING signaling was dependent on STING.THP1-Lucia ISG cells of wild type (WT) and STING-knockout (KO) were treated with cGAMP or cGAMP plus MMAE, and we found that STING KO To verify the physiological relevance of these observations, we evaluated the immuneenhancing effects of MMAE treatment in primary immune cells by using bone-marrowderived macrophages (BMDMs) from WT, Sting gt/gt , and Myd88 -/-mice.The Sting gt/gt and Myd88 -/-mice are specifically deficient in STING and MyD88 respectively.Similar to the results seen in THP1-Lucia ISG cells (S2B and S2C To determine if MMAE specifically enhanced the cGAMP-mediated STING pathway but not the RNA-sensing innate immune pathways or Toll-like receptor (TLR) signaling, THP1-Lucia ISG cells were stimulated with Sendai virus (SeV), polyinosinic-polycytidylic acid (Poly(I:C)), or lipopolysaccharide (LPS) with or without MMAE.We found that MMAE enhanced immune responses only after cGAMP treatment but not RNA-or TLR-sensing under the test conditions (S2D-S2G Fig) .These results suggested that the combination of MMAE and cGAMP selectively enhanced STING signaling.
We then tested the effect of MMAE on IFN-independent signaling of STING.Since STING could induce NF-κB signaling besides type I IFNs (Fig 2A ), we tested whether the STINGmediated NF-kB immune response was enhanced by MMAE.THP1-Lucia NF-kB cells were treated with cGAMP, LPS, or IL-1β in the absence or presence of MMAE for 24 h.We found that cGAMP and MMAE alone induced weak NF-kB reporter activity (Fig 2B).Of note, we observed that MMAE specifically enhanced cGAMP-mediated STING-dependent NF-kB reporter activity, but not the NF-kB signaling induced by LPS or IL-1β (Figs 2B, S2H and S2I).Moreover, we showed that the potentiation of NF-κB signaling induced by cGAMP and MMAE co-treatment was abolished when p50 or p65 were genetically ablated in THP1-Lucia NF-kB cells (Fig 2C and 2D), whereas the potentiation of ISRE reporter activity was not affected in p65-deficient THP1-Lucia ISRE cells (Fig 2E).To characterize the effect of MMAE on STING-induced autophagy in the absence of IFN, we overexpressed STING WT and S366A mutant in HEK 293T cells (without endogenous cGAS-STING expression) and THP1-Lucia ISG (STING KO) cells, respectively.We confirmed that MMAE enhanced cGAMP-mediated STING-IRF3 phosphorylation cascade and IFNβ production only in cells with WT STING expression, but not in cells with STING S366A mutant (Fig 2F -2H).This is consistent with the reports that the STING S366A mutant impaired the phosphorylation of STING and IRF3 and downstream signaling.Moreover, we showed that both the WT and S365A mutant of STING could activate autophagy in response to cGAMP.Of note, MMAE did not strongly enhance cGAMP-STING-mediated autophagy (Fig 2I and 2J).Together, our results suggested that MMAE robustly and specifically enhanced the cGAMP-STING-mediated type I IFNs and NF-kB signaling in tested cells.

MMAE boosted immune responses induced by distinct CDNs via the STING-IRF3 pathway directly
To further define the relevance of MMAE-enhanced cGAMP-STING pathway, we treated THP1-Lucia ISG cells with bacterial-derived CDNs, including 3'3'-cGAMP, c-di-AMP, and cdi-GMP.As expected, MMAE robustly enhanced the immune responses induced by bacterialderived CDNs in WT THP1 reporter cells, but this effect was completely abolished in

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Microtubule disruption enhances STING antiviral activity STING-KO cells (Fig 3A -3F).Consistently, the phosphorylation cascade of the STING signaling pathway induced by bacterial-derived CDNs was boosted upon MMAE treatment in WT (Fig 3G -3I) but not STING-KO THP1-Lucia ISG cells (Fig 3J -3L).These results suggested that MMAE had a ubiquitous potentiation effect on STING activation induced by distinct CDNs.
Since both cGAMP and human IFNβ could induce ISRE reporter activity, to further understand whether MMAE enhanced the cGAMP-mediated induction of ISGs through the canonical STING-IRF3 signaling pathway directly or the interferon-α/β receptor (IFNAR) signaling pathway in an indirect manner (S3A Fig) , we took both genetic and pharmacological approaches.We found that IFNβ-induced ISRE reporter activity was abolished by anti-IFNα/β receptor 2 (IFNAR2) antibody, whereas cGAMP-induced immune activity was not affected by IFNAR2 antibody (Fig 3M).By contrast, the ISRE immune response induced by cGAMP was significantly enhanced by MMAE, whereas IFNβ-induced immune activity was not promoted by MMAE (Fig 3M).Moreover, the enhancement of IFNβ production and IRF3 phosphorylation mediated by combination of cGAMP and MMAE were eliminated when IRF3 was genetically ablated (Fig 3N and 3O).These results indicated that MMAE enhanced cGAMP-induced

MMAE promoted the cGAMP-STING pathway by increasing puncta number and the extent of STING oligomerization
The CDNs trigger the translocation of STING from ER to perinuclear region, where it forms puncta-like structures and activates TBK1 and IRF3 to induce the production of IFNs and other immune-modulating molecules.To explore the dynamic regulatory mechanism of MMAE on cGAMP-mediated STING activation, we constructed HeLa cells stably expressing hSTING-GFP.There were no differences in the distribution pattern of hSTING-GFP between untreated cells and cells treated with MMAE alone (Fig 4A and 4B).Consistent with our previous results, cGAMP significantly induced STING translocation and perinuclear puncta formation [5] (Fig 4B ).By contrast, co-treatment of the cells with MMAE and cGAMP caused STING perinuclear puncta to disperse into numerous small vesicles throughout cytoplasm (Fig 4B).VcMMAE [30], which was employed as a negative control, failed to modulate cGAMP-mediated STING trafficking and translocation (Figs 4B and S4A).Moreover, brefeldin A, which is a specific inhibitor of protein trafficking by blocking the transport of membrane proteins from ER to Golgi apparatus [21] To confirm that STING signaling was maintained or even enhanced in the dispersed vesicles of STING, we extracted membrane fractions containing STING (supernatant, S1) from cell homogenate after centrifugation.We detected the oligomerization and activation of STING by native PAGE, confirming that MMAE induced more cGAMP-dependent STING oligomers than cGAMP alone or cGAMP plus VcMMAE (Fig 4C).Accordingly, phosphorylated STING signaling was significantly enhanced by MMAE (Fig 4C and 4D).To rule out the possibility that MMAE amplifies STING immune responses by promoting cGAMP entering into cells, we quantified intracellular cGAMP concentrations by liquid chromatography-tandem mass spectrometry.There were no significant differences in intracellular cGAMP levels after cGAMP treatment in the absence or presence of MMAE (Fig 4E).The results demonstrated that MMAE promoted STING oligomerization and vesicle dispersion which led to enhanced STING signaling.https://doi.org/10.1371/journal.ppat.1012048.g004S4D).Collectively, these data strongly suggested that MMAE promoted the cGAMP-mediated STING pathway by increasing puncta number and the extent of STING oligomerization.

MMAE altered the trafficking pattern of STING and delayed STING degradation
To further explore the mechanism for STING dispersal at an accurate spatial and temporal resolution, we performed live-cell time-lapse imaging and ER-Tracker live-cell staining using HeLa cells stably expressing hSTING-GFP (Figs 5A and S5A and S1 Movie).In line with the results in Fig 4B, we found that cGAMP induction caused strong activation of STING trafficking, moving from ER to perinuclear region within 60 minutes (Figs 5A and S5A and S1 Movie).Interestingly, MMAE treatment changed the cGAMP-mediated STING trafficking routes, resulting in STING vesicles to move aimlessly and scatter throughout the cytoplasm (S1 Movie and S5A Fig) .STING is a transmembrane protein on ER and its activities are dynamically regulated by vesicular trafficking.Previous studies have shown that proper distribution and movement of vesicles and organelles within the cytoplasm is highly dependent on the rapid assembly and disassembly of microtubules [31].We suspect that MMAE might disrupt cGAMP-mediated STING trafficking from ER to Golgi apparatus along microtubules by inhibiting tubulin polymerization.Imaging data revealed that MMAE, as an MDA, disrupted microtubule network and caused Golgi apparatus dispersal [29,32], whereas VcMMAE treatment at the same concentration had no such an effect (Fig 5B and 5C).Similarly, the disruption of microtubule network and STING dispersal phenotype were also confirmed by other MDAs (microtubule stabilizer paclitaxel is used as a negative control) (S5B Fig) .Notably, although MMAE changed distribution pattern of STING and Golgi in the MMAE plus cGAMP group, STING is still colocalized with Golgi (Fig 5B).Together, these data indicated that MDAs dispersed the Golgi apparatus and altered STING trafficking routes by disrupting the microtubule network.
Endogenous STING protein is quickly degraded by lysosome after stimulation by CDNs.Prevention of STING degradation could potentially lead to sustained STING signaling and immune responses, providing therapeutic advantages in antiviral treatment.To characterize the STING degradation, we employed BJ-5ta and THP1 cells, which are two cell lines with robust STINGdegrading signaling upon cGAMP stimulation.To investigate the effect of MMAE on STING degradation, we stimulated BJ-5ta and THP1 cells with a high concentration of cGAMP with or without MMAE and assessed STING degradation by measuring STING protein amounts.Consistent with previous studies, bafilomycin A1 (BafA1, inhibitor of lysosome function) or brefeldin A treatment potently blocked STING degradation.We found that the amounts of total STING were accumulated after MMAE plus cGAMP co-treatment compared to cGAMP treatment alone (Fig 5D and 5E), indicating that co-treatment delayed STING degradation.Furthermore, when protein synthesis is stopped by cycloheximide, MMAE significantly inhibited the degradation of STING over time in BJ-5ta and THP1 cells (Figs 5G, 5H and S5C).STING puncta were surrounded and engulfed by lysosome after cGAMP treatment.However, both lysosome and STING were dispersed with the addition of MMAE (Fig 5F ), which might cause delayed degradation of STING.Together, these data provide evidence that MMAE not only disrupted microtubule structure and altered STING trafficking, but also delayed STING degradation, leading to a stronger and more sustained cGAMP-STING signaling cascade.

MMAE significantly promoted cGAMP-mediated STING antiviral immunity in the cell
Activation of STING induces the production of IFNs and plays a critical role in controlling viral infections.Using herpes simplex virus 1 (HSV-1) and vesicular stomatitis virus (VSV) as an example of DNA and RNA virus, respectively, we examined the antiviral activity of MMAE and cGAMP combination.THP1 and L929 cells were infected with GFP-tagged herpes simplex virus 1 (HSV-1-GFP) or vesicular stomatitis virus (VSV-GFP) for 24 h.The mean fluorescence intensity (MFI) of viral GFP was used as an indicator of viral propagation.Both cGAMP https://doi.org/10.1371/journal.ppat.1012048.g005[33,34] and MMAE on its own inhibited viral propagation (Figs 6A, S6A and S6C), consistent with previous reports on antiviral activities of MDAs [27,35].Microtubule depolymerization not only disrupts virus trafficking in the endosome, but also affects the escape of virus particles from the endosome [36,37].Of note, MMAE in combination with cGAMP almost completely inhibited viral propagation in the cell (Figs 6A, S6A and S6C).When STING was genetically ablated, the cGAMP-mediated antiviral effect was abolished.By contrast, the antiviral effect of MMAE alone was not affected by STING-KO (S6B, S6D-S6F Fig) .The antiviral ability of MMAE plus cGAMP in STING-deficient cells became the same as that of MMAE alone, indicating that the enhanced antiviral effect of MMAE plus cGAMP is dependent on the STING signaling pathway (S6B and S6E Fig) .Immunoblotting analysis further confirmed that MMAE and cGAMP together significantly inhibited viral reproduction in the cells over time (Figs 6B and S7A).The viral infection still could be inhibited even the co-treatment was added after HSV-1-GFP virus infection or anti-IFNAR2 antibody pretreatment (Fig 6B).Meanwhile, the effect of treatment on cell viability could be ignored under the test conditions (Figs 6C and S7B).To explore the antiviral spectrum of MMAE plus cGAMP, we infected THP1 and L929 cells with pseudorabies virus (PRV-GFP, a DNA virus), vaccinia virus (VACV-GFP, a DNA virus), and enterovirus 71 (EV-A71, an RNA virus).We found that MMAE and cGAMP in combination provided cells with significant protection against all three tested viruses (S7C and S7D Fig) .We further confirmed that MMAE could enhance cGAMP-mediated STING signaling during infections by DNA or RNA viruses (Fig 6D and 6E).Together, these results suggested that the combination of cGAMP and MMAE had potent synergistic antiviral activity in a STING-dependent manner and could be used as a broad-spectrum antiviral strategy.
To see whether viral activity is modulated by MMAE-mediated microtubule destabilization, we constructed HeLa cells (STING deficient) stably expressing mCherry-TUBA1B.Imaging data revealed that MMAE at low doses dramatically disrupted microtubule network (Fig 6F).Importantly, in cells infected with HSV-1-GFP, HSV-1-GFP is localized along the microtubule, and MMAE significantly changed HSV-1-GFP invasion routes.HSV-1-GFP is trapped at certain positions inside the cells with MMAE treatment, regardless of STING expression level (Fig 6F and 6G).This is consistent with large number of reports on the role of microtubules in viral cycles.The intracellular transport of viral particles in the host cells, including particle trafficking at later stages of viral life cycles, is heavily dependent on the integrity of the microtubule network [38].Collectively, our findings reveal a novel role of microtubule destabilizer MMAE, and its combination with cGAMP provides a promising therapeutic approach for future antiviral therapy.

MMAE promoted the antiviral effect of cGAMP in vivo in a STINGdependent manner
The cGAS-STING pathway plays pivotal roles in controlling HSV-1 propagation in mice.To investigate the antiviral effect of MMAE in vivo, we treated HSV-1-GFP infected WT and Sting gt/gt mice with cGAMP, MMAE, or in combination.We found that HSV-1-GFP infected mice treated with cGAMP or MMAE alone showed higher survival rates compared with the control group, while the mice group treated with MMAE and cGAMP together showed the highest survival rate (Fig 7A).It's worth noting that HSV-1-GFP infected mice experienced dramatic body weight loss before death, treatment with cGAMP or MMAE alone alleviated this weight loss symptom, and the cGAMP and MMAE combination largely prevented body weight loss and body condition score decrease caused by viral infection (Figs 7B, 7C and S8A-S8C).Consistently, viral titers and the expressions of viral genes were reduced in drug treated mice, especially in the MMAE plus cGAMP group (Figs 7D-7F and S8D-S8F).Crucially,   7P and S8H).Consistent with this data, we found that Sting gt/gt mice were more vulnerable to HSV-1 infection, resulting in death even with a much lower virus titer than WT mice.In addition, MMAE alone did not prevent the death of Sting gt/gt mice caused by HSV-1 infection (Fig 7I).It did not alleviate body weight loss and body condition score worsening in these mice, and viral titers or the expressions of viral genes in brain and spleen were not significantly changed, indicating a slight or undetectable antiviral effect of MMAE in Sting gt/gt mice (Figs 7J-7N, S8I and S8J).
We then tested whether the combination strategy of MMAE and cGAMP could be employed to treat infection in vivo after virus replication had been well established.We infected mice with HSV-1-GFP first and treated the mice with MMAE, cGAMP, or in combination 16 hours later after the viral infection.We further confirmed that the combination of cGAMP and MMAE substantially decreased viral titers and the expressions of viral genes as compared with monotherapy (S8K-S8P Fig) .These results suggested that the MMAE and cGAMP co-treatment strategy still could be used after the viral infection.Together, we demonstrated that MMAE promoted cGAMP-mediated host antiviral immunity in a STING-dependent manner and protected mice form HSV-1 infection in vivo.These data suggest that the combination of MMAE and cGAMP has great potential in antiviral immunotherapy.

Discussion
In this study, by using MMAE as an example we show that MDAs could act synergistically with cGAMP to defend viral infections by modulating STING signaling and increasing STING-mediated immune response.We showed here that the combination of MMAE with cGAMP displayed potent and broad-spectrum antiviral activity in the cell and provided protection against HSV-1 infection in a mouse model.
Recent studies have demonstrated that STING agonists evoke potent innate immune responses via the induction of type I IFNs and IFN-stimulated genes.cGAMP, the second messenger of the cGAS-STING pathway, is critical for this host innate immune responses against viral infections.Accordingly, some viruses could degrade 2'3'-cGAMP using specific enzymes to restrict cGAMP-STING signaling [39,40], while some could cleave or repress cGAS to decrease cGAMP production [41][42][43][44].Therefore, the strategies to enhance cGAMP (or cGAMP analog)-induced STING innate immune response may have potentials in antiviral treatment.In this study, we found that the combination of MMAE and cGAMP might be able to fulfill this purpose.We showed that MMAE enhanced cGAMP-mediated antiviral immune response by promoting the oligomerization of STING and increasing STING puncta.The imaging data clearly showed that MMAE treatment disrupted the microtubule network, dispersed the Golgi apparatus, and completely disturbed the normal transport routes of intracellular STING vesicles (Fig 8).Notably, MMAE delayed trafficking-mediated STING degradation by altering lysosome localization patterns.These results suggested that MMAE could lead to sustained and reinforced STING-dependent immune response, culminating in the production and secretion of antiviral cytokines.Moreover, our results suggested that score was measured and calculated as in previous research with minor modifications [52] (normal = 0).(D-H and L-P) Six days after virus infection, three C57BL/6 mice (WT and Sting gt/gt ) were randomly selected for subsequent experiments.(D and L) The viral titers in mouse brains were measured by qRT-PCR assay (n = 3 biological replicates).(E, F, M, and N) Expressions of viral genes in brains were measured by immunoblotting and qPCR analysis (n = 3).(G and O) Expressions of IFNβ and ISGs in brains were analyzed by qPCR analysis (n = 3).(H and P) IFNβ production in brains were qualified by ELISA assay.https://doi.org/10.1371/journal.ppat.1012048.g007

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MMAE directly regulated cGAMP-STING signaling, but not the secondary interferon-α/β receptor (IFNAR) signaling pathway.When cGAMP-mediated STING pathway was modulated by MMAE, the enhanced signaling pathway and immune response via STING signaling are dominant.Thus, the effect of IFNAR signaling could be ignored in this condition.Indeed, MMAE directly promoted the phosphorylation of the TBK1-STING-IRF3 cascade, and the enhancement could be detected after short time drug exposure (less than 2 hours).Additionally, the enhancement effect could only be detected with microtubule destabilizers, but not microtubule stabilizers.These data suggested the effect of MMAE on STING signaling was not caused by antimitotic and cytotoxic effects of MDAs.MMAE augmented cGAMP-mediated activation of the TBK1-STING-IRF3 signal axis in a cGAS-independent manner, which is different from the regulation mechanism of other microtubule stabilizers and destabilizers by promoting the activation of the cGAS-STING pathway through micronucleus formation and mitochondrial DNA release [45,46].
Studies have shown that microtubules are required for efficient intracellular mature virus (IMV) formation and are essential for intracellular enveloped virus (IEV) assembly [38,47].In particular, the completion of infection cycles of HSV-1, PRV, and VACV is strictly dependent on microtubule movement [27,48].Accordingly, it's reasonable to hypothesize that chemicals disrupting microtubule networks could suppress viral infection.For example, nocodazole treatment reduced the number of virus particles by three fold in the cell [27].Moreover, selective depolymerization of microtubules by nocodazole or colchicine effectively prevents the migration of murine polyomavirus (double stranded DNA virus) toward nucleolus and the exit of progeny virus in cells [35].These findings are supported by a recent study demonstrating that podofilox, an antiviral drug used typically for the treatment of warts, destroys microtubules and prohibits virus entry following initial binding [49].Intriguingly, these microtubule destabilizers, including nocodazole, colchicine, and podofilox, were among the compounds that enhanced STING signaling in our high throughput screening assay.Therefore, we systematically compared different microtubule destabilizers and chose MMAE for further study because of its potent antiviral activity and less pronounced cytotoxic effects.While the results suggest that other clinically available MDAs could be investigated for their potential antiviral activities, it would be required to conduct side-by-side experiments to compare different MDAs in future studies.
We revealed that MMAE alone could inhibit viral infection, however the inhibition was independent of STING-mediated activation of IFN pathway and was through viral transport by disrupting the microtubule network (Fig 6G).Remarkably, the combination of MMAE and cGAMP strongly increased antiviral activity.We found that MMAE enhanced cGAMP-STING signaling cascade at low doses (nanomolar level in cellular assay) and exerted excellent broad-spectrum antiviral activities.The combination of MMAE and cGAMP enhanced the production of IFNs in vitro and in vivo.IFNs are essential components of the immune response against infections, but prolonged exposure to IFNs can lead to suppression of the immune response or hyper-activation [50,51].Thus, the treatment with MMAE and cGAMP need to be evaluated and controlled carefully to avoid side effects.Moreover, out data showed that MMAE could enhance the NF-kB immune signaling induced by cGAMP-STING pathway.The exact mechanism of STING-mediated NF-kB signaling still need to be revealed, and further studies are required to investigate the mechanism for how MMAE and cGAMP regulates NF-kB signaling.In addition, MMAE showed synergistic effect with not only cGAMP, but also other STING agonists [26], suggesting that the combination of MMAE with other available STING agonists could also have antiviral activities, expanding possible therapeutic options.Collectively, these results support the notion that MMAE, a potent MDA with proven clinical safety at tested concentrations, might have potential to be used as antiviral drug in combination with STING agonists.Given that MMAE is widely used in the treatment of tumor, the combination of MMAE and STING agonists may also have potential in cancer therapy.
Certain HSV-1 strains sensed by the cellular cGAS-STING pathway could induce strong type I IFNs for the host to curtain viral replication [52,53].Hence, we chose the mouse infection model with HSV-1 to study the antiviral activity of MMAE combined with cGAMP in vivo and to verify the effect of MMAE on the innate immune response to DNA virus.Although MMAE alone did not induce IFN response, it generated comparable antiviral effect to that of cGAMP in WT mice by disrupting microtubule networks.In addition, we showed that MMAE augmented cGAMP-mediated STING signaling and thus mediated the production of IFNs and the induction of ISGs in vivo, which is the major antiviral effector against HSV-1 infection.Ablation of STING in mice resulted in impaired IFN response and more vulnerable defense against HSV-1 infection.This led to the abolition of the antiviral effect of cGAMP with or without MMAE in Sting gt/gt mice.These data suggested that STING-mediated IFNs induced by MMAE and cGAMP co-treatment play an essential role in this antiviral response.Further studies are required to characterize which immune cells are activated by the enhanced STING immune signaling and what is the precise role of related IFN expression during this process.Understanding this modulatory network may provide useful information for antiviral treatment.
Our work revealed a new role for microtubules in the regulation of STING-induced immune signaling and antiviral responses.Our results suggested that the combination of MMAE and cGAMP may be a potential strategy in antiviral treatment.The findings open a new possibility to fight against viral outbreaks by combining STING agonists, MDAs or even MDA-conjugated antibodies.

Measurement of cell viability
The cell viability was represented by the total ATP detected with the CellTiter-Glo Luminescent Cell Viability Assay (G7570, Promega) according to instructions.Briefly, THP1-Lucia ISG cells were infected with VSV-GFP (MOI = 0.1) or HSV-1-GFP (MOI = 1), and then cultured with cGAMP (0.5 μM) and/or MMAE (0.25 μM) for 24 h.Cell suspension was mixed with the same volume of CellTiter-Glo Substrate (50 μl), and added to each well of a 96-well white plate.The luminescence was measured by a Cytatio Cell Imaging Reader (BioTek), and the fold change of luminescence was normalized to DMSO-treated cells.

LC-MS/MS quantification of cGAMP
THP1-Lucia ISG cells were treated with cGAMP (0.5 μM) for 4 h with or without MMAE (0.5 μM).Cells were lysed in 500 μl-1 ml lysis buffer containing 80% of analytical pure methanol and 2% of pre-chilled acetic acid, and subjected to freeze-thaw cycles with liquid nitrogen.The supernatant was evaporated to dryness, and the extracted cGAMP was reconstituted in 60 μl of water, followed by centrifugation at 16,000 g for 15 min at 4˚C.The supernatant was collected for LC-MS/MS analysis.The LC-MS/MS analysis was performed on an ACQUITY UPLC I-Class (Waters, USA) coupled to an AB Sciex 6500 Triple Quad mass spectrometer (AB Sciex, USA) with the electrospray ionization (ESI) source.The purchased cGAMP compound (InvivoGen) was used as the standard.Optimized ion transitions (m/z: 675.1-524.1,675.1-506.1)were used for quantification.

Western blot analysis
Activation of the STING pathway was assessed by western blot to analyze phosphorylation status of STING, TBK1, and IRF3 using commercially available antibodies.Cells were lysed in lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, 1% Sodium Deoxycholate, 1 mM Na 3 VO 4 , 25 mM β-glycerol-phosphate) supplemented with 0.1 mM PMSF (Beyotime Biotechnology) and 0.5 mg/ml Leupeptin (Solarbio) on ice for 30 min.Lysates were centrifuged at 13,000 rpm for 15 min at 4˚C, and the soluble fraction was transferred to a new tube.Protein concentration was determined by the absorbance of 280 nm on NanoDrop One (Thermo Fisher Scientific), and the protein sample was boiled with SDS loading buffer at 95˚C for 5 min.Proteins were separated on 12% SDS-PAGE gels, immunoblotted onto nitrocellulose membranes, and subsequently incubated with different primary antibodies overnight.After incubation with HRP-labelled secondary antibodies (Huaxingbio) for 1 h at room temperature, the proteins were detected using ECL substrates (Mei5 Biotechnology).
normalized to DMSO-treated cells.(N and O) THP1-Lucia ISG (WT and IRF3 KO) cells were treated with cGAMP and/or indicated doses of MMAE for 12 h (N) or 6 h (O).IFNβ production was measured by ELISA analysis (N).Expression of IRF3 and the activation of STING pathway was analyzed by immunoblotting (N and O).https://doi.org/10.1371/journal.ppat.1012048.g003Wethen assessed the number of STING puncta formed upon treatment with cGAMP with or without MMAE or VcMMAE.MMAE, but not VcMMAE, significantly increased the number of STING puncta induced by cGAMP treatment (Fig4F).The positive effect of MMAE and the negative effect of VcMMAE were confirmed by the luciferase reporter assay with THP1/ RAW-Lucia ISG cells (Figs 4G and S4C).Similarly, MMAE, but not VcMMAE, significantly promoted cGAMP-mediated production of secreted IFNβ and ISGs expression(Figs 4H, 4I and

Fig 4 .
Fig 4. MMAE boosted the cGAMP-mediated STING pathway by increasing STING-containing membrane puncta numbers and the extent of STING oligomerization.(A) Chemical structure of Monomethyl auristatin E (MMAE).(B) HeLa cells stably expressing human STING-GFP were stimulated with cGAMP (8 μM) and/or MMAE (1 μM), or VcMMAE (1 μM) for 2 h in the absence or presence of brefeldin A (BFA, 1 μM).Fluorescent images of cells were acquired on a Zeiss LSM980 Airyscan2 Confocal microscope using a 63× (NA 1.45) objective and processed in Zen Blue 3.1 software.STING (green), nuclei were stained with Hoechst (blue).Scale bars, 10 μm.(C-E) THP1-Lucia ISG cells were stimulated with cGAMP with or without MMAE (0.5 μM) or VcMMAE (0.5 μM) for 4 h (C and E).STING oligomerization was analyzed by native PAGE, and indicated proteins were detected by immunoblotting.The results are representative of three independent biological replicates (C).The activation of STING pathway was analyzed by immunoblotting (D).cGAMP quantification by LC-MS/MS in THP1-Lucia ISG cell lysates (E).(F) HeLa cells are stimulated similarly as in B. The STING (green) puncta are shown as 3D projections of Z-stack images.Scale bar, 5 μm.The STING puncta volume was quantitated by Imaris software (version 9.7) (n = 20).(G) THP1-Lucia ISG and RAW-Lucia ISG cells were treated with cGAMP with or without MMAE (1 μM), or VcMMAE (1 μM) for 24 h.Fold change of luminescence was normalized to DMSO-treated cells.(H and I) THP1-Lucia ISG cells were stimulated by cGAMP with or without MMAE (1 μM), or VcMMAE (1 μM) for 12 h (H), or 6 h (I).IFNβ induction was measured by ELISA and qPCR analysis.

Fig 5 .
Fig 5. MMAE changed STING trafficking routes and promoted cGAMP-mediated STING activity.(A-C and F) HeLa cells stably expressing human STING-GFP were stimulated with cGAMP (8 μM) and/or MMAE (1 μM), or VcMMAE (1 μM) for 2 h.(A and F) Live cells were stained by ER-Tracker Blue-White DPX and LysoTracker Deep Red.(B and C) Cells were fixed, permeabilized, and stained for GM130 (a Golgi protein, red) or tubulin (red).Nuclei were stained with DAPI (blue).All structured illumination microscope (3D-SIM) images are z-stack images.Scale bars, 10 μm.3D-SIM images was acquired and processed using the Highly Intelligent and Sensitive SIM (HIS-SIM), and Wiener deconvolution was used in reconstructed images.Dashed white boxes in each main image indicate enlarged areas of interest shown below.Co-localization was quantified using Pearson's correlation coefficient (r), shown on the right of each row of images (n = 50).(D and E) BJ-5ta cells were stimulated with cGAMP (8 μM) with or without MMAE (pre-treatment for 30 min), bafilomycin A1 (BafA1, 100 nM), or brefeldin A (BFA, 1 μM) for 2 h.Total STING protein was quantified by image J software (n = 3 biological replicates).(G and H) STING stability was analyzed by immunoblotting in the absence or presence of cycloheximide (CHX, 50 μg/ml).BJ-5ta cells were treated and analyzed as in (D and E).

Fig 7 .
Fig 7. MMAE relied on the host STING pathway to enhance cGAMP antiviral immune response.(A-H and I-P) WT and Sting gt/ gt C57BL/6 mice (n = 10) were treated with PBS, cGAMP (30 μg/mice), MMAE (0.5 mg/kg), or cGAMP along with MMAE by intraperitoneal injection (i.p.) for 2 h.Then, the mice were infected intravenously with HSV-1-GFP at 2 × 10 8 pfu per WT mouse or at 1 × 10 7 pfu per Sting gt/gt mouse.(A and I) Survival curves of virus-infected mice after treatments were analyzed using the log-rank (Mantel-Cox) test.(B, C and J, K) Body weight and body condition score of mice were observed and recorded daily.Body condition

Fig 8 .
Fig 8.A graphic model for MMAE enhanced cGAMP-mediated antiviral immunity.MMAE changed cGAMPmediated STING trafficking routes from ER to Golgi apparatus by disrupting the microtubule network, and delayed the trafficking-mediated STING degradation.MMAE dispersed the cGAMP-mediated STING perinuclear puncta into large number of tiny vesicles throughout the cytoplasm.The accumulated STING vesicles further amplified the cGAMP-mediated TBK1-STING-IRF3 signaling cascade, and promoted the production of IFNs and ISGs expression.MMAE alone restricted viral replication and infection by destroying microtubule networks, while MMAE combined with cGAMP exerted potent and broad-spectrum antiviral activity in vitro and in vivo in a STING-dependent manner.https://doi.org/10.1371/journal.ppat.1012048.g008