Compared with terrestrial mammals, bats have a longer lifespan and greater capacity to co-exist with a variety of viruses. In addition to cytosolic DNA generated by these viral infections, the metabolic demands of flight cause DNA damage and the release of self-DNA into the cytoplasm. However, whether bats have an altered DNA sensing/defense system to balance high cytosolic DNA levels remains an open question. We demonstrate that bats have a dampened interferon response due to the replacement of the highly conserved serine residue (S358) in STING, an essential adaptor protein in multiple DNA sensing pathways. Reversing this mutation by introducing S358 restored STING functionality, resulting in interferon activation and virus inhibition. Combined with previous reports on bat-specific changes of other DNA sensors such as TLR9, IFI16, and AIM2, our findings shed light on bat adaptation to flight, their long lifespan, and their unique capacity to serve as a virus reservoir.

Main Text

Zhang et al., 2013 Zhang G.J.

Cowled C.

Shi Z.L.

Huang Z.Y.

Bishop-Lilly K.A.

Fang X.D.

Wynne J.W.

Xiong Z.Q.

Baker M.L.

Zhao W.

et al. Comparative analysis of bat genomes provides insight into the evolution of flight and immunity. Ahn et al., 2016 Ahn M.

Cui J.

Irving A.T.

Wang L.F. Unique loss of the PYHIN gene family in bats amongst mammals: implications for inflammasome sensing. Zhou et al., 2011 Zhou P.

Cowled C.

Todd S.

Crameri G.

Virtue E.R.

Marsh G.A.

Klein R.

Shi Z.

Wang L.-F.

Baker M.L. Type III IFNs in pteropid bats: differential expression patterns provide evidence for distinct roles in antiviral immunity. Zhou et al., 2016 Zhou P.

Tachedjian M.

Wynne J.W.

Boyd V.

Cui J.

Smith I.

Cowled C.

Ng J.H.J.

Mok L.

Michalski W.P.

et al. Contraction of the type I IFN locus and unusual constitutive expression of IFN-alpha in bats. Escalera-Zamudio et al., 2015 Escalera-Zamudio M.

Zepeda-Mendoza M.L.

Loza-Rubio E.

Rojas-Anaya E.

Mendez-Ojeda M.L.

Arias C.F.

Greenwood A.D. The evolution of bat nucleic acid-sensing Toll-like receptors. There are limited studies on bat DNA sensors despite the belief that bat cells are likely to be more at risk of cytosolic DNA exposure. A recent comparative genomics study showed that the most positively selected genes of bats seemed to be concentrated in the DNA damage checkpoint pathway and innate immunity (). One of these genes encodes the inflammasome sensor NLRP3. More strikingly, the entire PYHIN gene family, including AIM2 and IFI16, is lost in all bat genomes sequenced so far, implying a dampened DNA-triggered inflammasome response (). Bats have been shown to have a contracted type I IFN locus and different expression patterns of type III IFNs compared with those in human and mouse (). Also, TLR9 seems to be under greater positive selection in bats compared with other mammals (). Taken together, these findings suggest that bats may have evolved to adopt a DNA sensing and IFN response mechanism in adaptation to flight, which is sufficiently different from terrestrial mammals.

Ge et al., 2013 Ge X.Y.

Li J.L.

Yang X.L.

Chmura A.A.

Zhu G.

Epstein J.H.

Mazet J.K.

Hu B.

Zhang W.

Peng C.

et al. Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. Figure 2 Dampened IFN Activation and Virus Inhibition by Bat STING Show full caption (A) Splenocytes of Rhinolophus sinicus bats and mice (n = 3 cells from 3 animals each) were transfected with cGAMP (1 μg/mL) or poly I:C (1 μg/mL), or infected with SeV (100 hemagglutinin units/mL). Six hours later, the induction of IFNβ and IRF7 genes was determined by qPCR. Primers can be found in Table S2 (B) Transcriptome next-generation sequencing of splenocyte RNAs. The differentially expressed genes (DEGs) were analyzed by RSEM at FDR (false discovery rate) < 0.05. The ISG in the DEG sets of mice and Rs bat are listed. Fold change is indicated in color from 0 to 110. (C) Restoration of STING function by introducing S358 in bat STING. HEK293T cells were co-transfected with STING, cGAS, IFNβ promoter firefly luciferase, and renilla luciferase plasmids. Luciferase activity was determined 24 hr post-transfection. The blots showing protein levels can be found in Figure S2 (D) cGAMP treatment of HEK293T stably expressing various STING. Cells stably expressing the indicated proteins were transfected with IFNβ promoter firefly luciferase and renilla luciferase plasmids. Six hours later, cells were permeabilized in digitonin buffer with or without 1 μg/mL cGAMP. Luciferase activity was determined 16 hr after treatments. (E) PaKiT03 cells were transfected with indicated STING plasmids followed by infection with HSV-luciferase at MOI = 0.1 at 24 hr post-transfection. At 24 hr post-infection, HSV replication was determined by luciferase activity. Data from (A), (C), (D), and (E) are presented as the means ± SD, n = 3, ∗∗p < 0.01, ∗∗∗p < 0.001 (Student's t test). For (C) and (D), data represent fold change according to wells transfected with empty vector (set as 1). WT, wild-type; mt, mutant; Hs, Homo sapiens; Md, Myotis davidii; Pa, Pteropus alecto; Rs, Rhinolophus sinicus; pIC, poly I:C. See also Figure S2 and Table S2 To test this, we compared the functional difference in the induction of IFNs between bat and mouse by cGAMP. Splenocytes from three individual Rhinolophus sinicus (Rs) and three laboratory mice were stimulated by cGAMP. Rs has been reported to be the reservoir host of the lethal severe acute respiratory coronavirus (SARS-CoV) (). In contrast to a strong induction of IFNβ and IRF7 (an IFN-stimulated gene [ISG]) in mouse, transfection of cGAMP induced a much lower level of IFNβ and IRF7 mRNA in Rs bats from qPCR analysis ( Figure 2 A). As controls, both poly I:C and Sendai virus treatment resulted in a comparable induction level of IFNβ and IRF7 in both cells ( Figure 2 A). The qPCR results were corroborated by RNA high-throughput sequencing (RNA-seq). As shown in Figure 2 B, a number of mouse ISGs were strongly upregulated upon cGAMP treatment, whereas the upregulation of bat ISGs was much less, both in number and fold change. Between the two Rs bats, there were subtle differences in ISG induction, which was not unexpected considering wild-caught outbred bats were used in this study.

Phylogenetic analysis showed bat STING clustered with known mammalian STING ( Figure S1 A). qPCR analysis of mRNA levels in a range of Rs, Myotis davidii (Md), and Pteropus alecto (Pa) primary organs revealed an expression pattern not dissimilar to that found in mouse: STING was found to be expressed in a variety of tissues in bats, with highest levels in spleen and lung ( Figure S1 B). It can thus be concluded that phylogenetic divergence or difference in gene expression patterns between bats and mice is unlikely to be responsible for the observed reduction in STING-mediated IFN production.

Sun et al., 2013 Sun L.

Wu J.

Du F.

Chen X.

Chen Z.J. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the Type I interferon pathway. Yi et al., 2013 Yi G.H.

Brendel V.P.

Shu C.

Li P.W.

Palanathan S.

Kao C.C. Single nucleotide polymorphisms of human STING can affect innate immune response to cyclic dinucleotides. Zhong et al., 2008 Zhong B.

Yang Y.

Li S.

Wang Y.Y.

Li Y.

Diao F.

Lei C.

He X.

Zhang L.

Tien P.

et al. The adaptor protein MITA links virus-sensing receptors to IRF3 transcription factor activation. Zhou et al., 2016 Zhou P.

Tachedjian M.

Wynne J.W.

Boyd V.

Cui J.

Smith I.

Cowled C.

Ng J.H.J.

Mok L.

Michalski W.P.

et al. Contraction of the type I IFN locus and unusual constitutive expression of IFN-alpha in bats. We then examined whether the dampening of bat STING function by the change in residue 358 is common in other bats. In HEK293T cells, which lack endogenous cGAS and STING expression (), STING from three representative bats, Rs, Pa, and Md, and human was overexpressed together with human cGAS and IFNβ promoter plasmids. Although polymorphism has been observed in human STING, a previous study indicated that the three variants (RGR, AQ, and HAQ) have different but comparable ability in IFN induction activities depending on the experimental conditions (). In this study, we have confirmed their observation and used the AQ variant for further studies. Mutant human STING S358A significantly reduced STING-induced IFNβ production as reported previously (). Conversely, mutant bat STING X358S (X = N, H, or D) significantly restored their ability for IFNβ induction ( Figure 2 C). To exclude the possibility that the human cell system may affect bat STING function, we repeated the experiment in PakiT03, a Pa bat cell line that expresses reasonable amount of cGAS but a very low level of STING (). The pattern was essentially identical to that observed in HEK293T, with wild-type bat STING showing dampened induction of IFN and ISGs compared with the X358S mutants ( Figure S2 A). We also tested this dampening function by cGAMP. HEK293T cells stably expressing wild-type (D358) or mutant S358 Rs STING were stimulated with cGAMP in digitonin permeabilization solution. The S358 STING induced significantly higher IFN ( Figure 2 D). These results suggest that the S358 replacement is mainly responsible for dampened STING-dependent IFN activation with cGAS co-expression or cGAMP stimulation.

Schountz, 2014 Schountz T. Immunology of bats and their viruses: challenges and opportunities. Wynne and Wang, 2013 Wynne J.W.

Wang L.F. Bats and viruses: friend or foe?. It is proposed that bat's exceptional ability to host viruses with few or no clinical disease is likely the result of an intricate balance between the host immune system and virus infection (). We hypothesized that the dampened STING-IFN responses could be partially responsible for that intricate balance. In assessing the effect of different STING on herpes simplex virus (HSV) infection in PakiT03 cells, it was found that the wild-type human STING was about 2.5-fold more effective in blocking HSV replication than the S358A mutant. However, this was reversed for bat STING, in which the wild-type bat STING was less effective than the mutant STING X358S with a reduction of approximately 3-, 2.5-, and 2-fold, respectively, for Md, Pa, and Rs STING ( Figure 2 E).

Tanaka and Chen, 2012 Tanaka Y.

Chen Z.J. STING specifies IRF3 phosphorylation by TBK1 in the cytosolic DNA signaling pathway. In human STING, residues S366 and S358 are important for IRF3, but not TBK1, binding and activation (). To understand the detailed mechanism of dampened STING-dependent IFN activation, we investigated whether this bat-specific S358 replacement universally affects IRF3 and TBK1 activation. When HEK293T cells were transfected with human or bat STING-expressing plasmids, phosphorylation of IRF3, but not TBK1, was markedly higher in cells transfected with S358 STING ( Figure S2 B). Similar findings were observed in bat PakiT03 cells ( Figure S2 C), which eventually contributed to a different downstream IFN response and in turn the observed difference in modulating HSV replication. Taken together, these results demonstrated that while bat STING maintained its antiviral defense similar to human STING, the dampening likely contributed in part to the long-term co-existence of bats and viruses.

We hypothesized that excessive exposure to cytosolic DNA in bat cells during flight and/or viral infection would pose a strong natural selection pressure to reduce activation of bat DNA sensors. In this report, we have provided genetic and functional data to support this hypothesis. We have demonstrated that bat STING is less active in IFN induction and pinpointed residue 358 as the key site of difference between bat and human STING. Experimentally, we have demonstrated the replacement of the S358 residue in different bat STING resulted in dampened downstream IFN production via IRF3 phosphorylation. To our knowledge, this is the most conclusive experimental demonstration of a key innate defense pathway that is functionally different between bats and non-bat mammals with implications that bats are more effective in peaceful co-existence with a large number of viruses.