bioRxiv preprint doi: https://doi.org/10.1101/2020.08.30.274712; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 Loss of mitochondrial protease CLPP activates type I responses through the

2 mtDNA-cGAS-STING signaling axis.

3

4 Sylvia Torres-Odio*, Yuanjiu Lei*, Suzana Gispert†, Antonia Maletzko†, Jana Key†, Saeed

5 Menissy*, Ilka Wittig‡, Georg Auburger†, A. Phillip West*#

6

7 *Department of Microbial Pathogenesis and Immunology, College of Medicine, Texas A&M

8 University Health Science Center, Bryan, TX 77807, USA

9 †Experimental Neurology and ‡Functional Proteomics, Faculty of Medicine, Goethe University,

10 60590 Frankfurt am Main, Germany

11

12 # Correspondence to: [email protected]

13

14 Abstract

15 Caseinolytic mitochondrial matrix peptidase proteolytic subunit, CLPP, is a serine protease that

16 degrades damaged or misfolded mitochondrial . CLPP null mice exhibit growth

17 retardation, deafness, and sterility, resembling human Perrault syndrome (PS), but also display

18 immune system alterations. However, the molecular mechanisms and signaling pathways

19 underlying immunological changes in CLPP null mice remain unclear. Here we report the steady

20 state activation of type I interferon (IFN-I) signaling and antiviral expression in CLPP

21 deficient cells and tissues. Depletion of the cyclic GMP-AMP (cGAS)-Stimulator of Interferon

1 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.30.274712; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

22 (STING) DNA sensing pathway ablates heightened IFN-I responses and abrogates the

23 broad viral resistance phenotype of CLPP null cells. Moreover, we report that CLPP deficiency

24 leads to mitochondrial DNA (mtDNA) instability and packaging alterations. Pharmacological and

25 genetic approaches to deplete mtDNA or inhibit cytosolic release markedly reduce antiviral gene

26 expression, implicating mtDNA stress as the driver of IFN-I signaling in CLPP null mice. Our

27 work places the cGAS-STING-IFN-I innate immune pathway downstream of CLPP and may have

28 implications for understanding myriad human diseases involving CLPP dysregulation.

29

30

31 Introduction

32 Mitochondrial proteases are key modulators of several mitochondrial functions, including the

33 maturation of proteins, maintenance of quality control, control of mitochondrial gene

34 expression and biogenesis, mitophagy, and apoptosis (Quirós et al., 2015). CLPP, caseinolytic

35 mitochondrial matrix peptidase proteolytic subunit, is a highly conserved, processive serine

36 protease located in the mitochondrial matrix. CLPP is the protease component of the CLPXP

37 complex that cleaves and various proteins in an ATP-dependent process, together with

38 chaperone and ATPase CLPX (Baker and Sauer, 2012). While prokaryotic ClpXP has been

39 extensively studied, the role and function of CLPXP in mitochondria remain less clear, and there

40 is limited information regarding its specific substrates (Bhandari et al., 2018). Studies in C. elegans

41 have implicated CLPXP as a key component of the mitochondrial unfolded protein response

42 (UPRmt), and downstream signaling and transcriptional responses of the UPRmt are attenuated in

43 worms lacking CLPP activity (Haynes et al., 2007; Pellegrino et al., 2014). Mammalian CLPP is

44 involved in pleiotropic cellular functions such as myoblast differentiation, cell proliferation, and

2 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.30.274712; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

45 mitoribosome assembly, which controls the rate of mitochondrial protein synthesis (Deepa et al.,

46 2016; Szczepanowska et al., 2016). Moreover, CLPP has recently been implicated in cancer, and

47 hyperactivating CLPP was shown to alter mitochondrial function and selectively kill some cancer

48 cells (Ishizawa et al., 2019). Thus, CLPP is being studied as a biological target of anti-cancer drugs

49 in current clinical trials (Graves et al., 2019).

50 Studies performed in CLPP null mice (CLPP-KO) highlight the physiological and

51 pathological relevance of this model to the human disorder known as Perrault syndrome (PS).

52 CLPP-KO mice display profound phenotypic changes characterized by growth retardation,

53 deafness, and premature sterility, despite exhibiting only mild mitochondrial bioenergetic deficits

54 (Gispert et al., 2013). Similar phenotypes are also observed in PS, where the genetic component

55 involves autosomal recessive mutations in CLPP and other nuclear DNA-encoded mitochondrial

56 proteins (Jenkinson et al., 2013). Other phenotypes of this mouse include insulin resistance and

57 protection from diet-induced obesity (Bhaskaran et al., 2018), accelerated depletion of ovarian

58 follicular reserve (T. Wang et al., 2018), and impairment of adaptive thermogenesis due to a

59 decline in brown adipocytes (Becker et al., 2018).

60 Interestingly, CLPP-KO mice show activation of antiviral genes, with elevated expression

61 of interferon stimulated genes (ISGs) noted in many tissues and fibroblasts (Gispert et al., 2013;

62 Key et al., 2020). Activation of innate immune signaling downstream of mitochondrial dysfunction

63 has been reported in several mitochondrial mutant animals (Pellegrino et al., 2014; West et al.,

64 2015), as well as in animal models of human disease and patient samples (Nakahira et al., 2015;

65 West, 2017; West and Shadel, 2017). Emerging evidence localizes cGAS, Toll-like receptor 9

66 (TLR9), and the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3)

67 downstream of mitochondrial damage (Nakahira et al., 2015; West, 2017; West and Shadel, 2017).

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68 Eukaryotic mitochondria maintain prokaryotic features, including a double-stranded circular DNA

69 genome, inner membrane cardiolipin, and N-formylated proteins, which act as potent triggers of

70 cGAS, TLR9, NLRP3, and other innate immune sensors when released from mitochondria into

71 the or extracellular space. The release and accumulation of these so-called mitochondrial

72 called damage-associated molecular patterns (DAMPs) is increasingly implicated in the

73 inflammatory pathology of numerous human disorders including autoimmunity,

74 neurodegenerative diseases, and cancer (Nakahira et al., 2015; West and Shadel, 2017; Youle,

75 2019). The mitochondrial molecular mechanisms and innate immune signaling pathways

76 underlying activation of antiviral signatures in CLPP-KO mice remain uncharacterized, and the

77 biological significance of this phenotype is unclear. Moreover, the role of IFN-I signaling in

78 CLPP-KO mouse pathology and its possible implication in human PS remain to be examined.

79 Herein we show that enhanced ISG expression and IFN-I responses in CLPP-KO mice are

80 triggered by mtDNA stress and mediated by the cGAS-STING DNA sensing pathway. Deletion

81 of STING or the interferon a/b receptor (IFNAR) in CLPP-KO MEFs ablates steady state

82 expression of broadly antiviral ISGs and reduces the potent antiviral phenotypes observed during

83 RNA and DNA virus infection. These findings shed light on a new mitochondrial pathway

84 upstream of cGAS and STING, link mitochondrial proteostasis to mtDNA genome maintenance,

85 and may have important implications for understanding mitochondrial-innate immune crosstalk in

86 the context of human health and disease.

87

88 Results and Discussion

89 CLPP deficiency leads to steady state ISG expression and an antiviral signature in cells and

90 tissues

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91 We first expanded upon our prior findings that revealed increased steady state expression of

92 antiviral genes in CLPP deficient tissues (Gispert et al., 2013). Using Ingenuity Pathway Analysis

93 (IPA) software, we observed that predicted upstream regulators of the antiviral gene signature in

94 CLPP-KO tissues included factors governing IFN-I signaling (Irf7, Irf3 and Stat1), IFNa, and

95 IFNAR (Fig EV1A). Among the list of upregulated IRF7 and STAT1 target genes, we identified

96 canonical ISGs such as Usp18, Ifit1b, Ifi44, and Rtp4 (Fig EV1B). Individual analyses of heart and

97 liver tissues from 3-5-month-old mice revealed a significant enhancement of ISGs at the protein

98 level in both the liver and heart of CLPP-KO mice (Fig EV1C and D).

99 To better characterize this ISG response, we used mouse embryonic fibroblasts (MEFs)

100 and human foreskin fibroblasts deficient in CLPP. Expression profiles of CLPP-KO MEFs

101 revealed an enrichment of ISGs and antiviral signaling factors at both the RNA and protein level

102 (Fig 1A and B). Among the set of overexpressed ISGs, we observed those involved in direct

103 antiviral activity (VIPERIN/Rsad2, IFIT1, IFIT3); regulators of antiviral and IFN-I signaling

104 (USP18 and STAT1); as well as RNA and DNA sensors (RIG-I and ZBP1). Global proteome

105 profiling revealed that greater than 60 percent of the most differentially expressed proteins in

106 CLPP-KO MEFs were ISGs (Fig 1C). Transfection of CLPP-KO MEFs with poly(I:C), a dsRNA

107 analog agonist of the cytosolic RNA helicase MDA5, or challenge with LPS, a TLR4 ligand,

108 revealed elevated ISG expression compared to WT, which indicates potentiation/priming of

109 antiviral signaling pathways downstream of both cytosolic and membrane-bound pattern

110 recognition receptors (Fig 1D and E). To assess if this heightened ISG response was only observed

111 in mouse cells, we transduced human foreskin fibroblasts with short hairpin targeting CLPP

112 (shCLPP) or EGFP as a control (shEGFP). Similar to our findings in MEFs and murine tissues,

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113 reduction of CLPP in human fibroblasts led to the steady state upregulation of antiviral ISGs at the

114 protein level, including IFIT2, IFITM1, RIG-I, STAT1 and others (Fig 1F).

115

116 STING and IFNAR mediate the steady state ISG and antiviral signature observed in CLPP

117 deficient cells and tissues

118 Recent work has revealed that the release of mitochondrial nucleic acids into the cytosol is a potent

119 trigger of DNA and RNA sensors of the (Dhir et al., 2018; Rongvaux et al.,

120 2014; West et al., 2015). To next pinpoint downstream sensors mediating the antiviral response in

121 CLPP-KO cells, we transiently knocked down the DNA sensor cGAS or the cytosolic RNA

122 adaptor MAVS and quantified ISG responses in MEFs. Downregulation of cGAS profoundly

123 diminished the ISG response in CLPP-KO MEFs (Fig 2A) while MAVS depletion did not (Fig.

124 EV2A), implicating the cytosolic DNA sensing machinery as the main trigger of ISGs in CLPP

125 deficient cells. Using a parallel approach to bypass any off-target and immune stimulatory effects

126 of transient siRNA transfection, we crossed CLPP heterozygous mutant mice with Stinggt//gt and

127 Ifnar-/- strains to generate CLPP-KO/STINGgt/gt and CLPP-KO/IFNAR-KO mice, respectively,

128 and assessed the antiviral response in MEFs and tissues. Consistent with our siRNA data

129 implicating the cGAS pathway, the relative expression of ISGs Usp18, Isg15, and Cxcl10 was

130 markedly reduced in the absence of STING (Fig 2B), with this effect being more pronounced in

131 CLPP-KO/IFNAR-KO MEFs. Ablation of elevated IFN-I signaling was also observed in RNA

132 extracts from tissues of the double mutant crosses, as qRT-PCR analysis of heart (Fig 2C) and

133 liver (Fig EV2B) from double KO mice showed diminished ISG RNA compared to CLPP-KO

134 tissues. A similar effect was seen for antiviral proteins such as RIG-I, STAT1, ZBP1, and IFIT3

135 in heart extracts (Fig 2D), with marked decreases in all ISGs in CLPP-KO/STINGgt/gt and CLPP-

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136 KO/IFNAR-KO mice compared to CLPP-KO alone. Collectively, our findings indicate that loss

137 of CLPP in murine cells and tissues leads to exacerbated IFN-I signaling via engagement of the

138 cGAS-STING axis.

139 CLPP-KO mice exhibit reduced adiposity, increased whole‐body energy expenditure, and

140 protection from diet‐induced obesity, indicating a role of CLPP in metabolic rewiring (Bhaskaran

141 et al., 2018). Although ablation of STING and IFNAR was sufficient to blunt the steady state ISGs

142 signature in CLPP-KO cells and tissues, loss of STING or IFNAR signaling was not sufficient to

143 rescue the growth deficits of CLPP-KO mice (Fig EV2C). Moreover, STING depletion did not

144 significantly impact body composition changes (i.e. fat/lean ratio via EchoMRI) resulting from

145 loss of CLPP (Fig EV2D). Our study did not investigate the role of STING-IFN-I signaling in

146 infertility or deafness in CLPP-KO mice, which are also key features of human PS. However,

147 several studies have linked IFN-I to sterility in both transgenic mouse models and human patients

148 receiving exogenous IFN-a therapy (Hekman et al., 1988; Iwakura et al., 1988; Ulusoy et al.,

149 2004). Moreover, sudden hearing loss has been reported as an adverse effect of IFN-a

150 immunotherapy in patients with hepatitis C virus or cancer (Asal et al., 2014.; Formann et al.,

151 2004). Therefore, it will be interesting to explore whether ablation of STING or IFNAR in CLPP-

152 KO mice impacts the development or progression of sterility and/or deafness, which may have

153 implications for understanding the role of STING-IFN-I signaling in the diverse pathology of

154 human PS.

155

156 CLPP-KO cells are more resistant to viral infection owing to elevated activation of the

157 STING pathway

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158 To next assess if basal cGAS-STING-IFN-I signaling and increased steady state expression ISGs

159 in CLPP-KO cells is functionally relevant, we infected MEFs with recombinant Vesicular

160 Stomatitis Virus (VSV) expressing the viral protein VSV-G fused to GFP (VSV-G/GFP) (Dalton

161 and Rose, 2001). After infection at a MOI of 0.1 for 24h, microscopic analysis showed a striking

162 absence of GFP fluorescence in CLPP-KO cells compared to WT controls (Fig 3A). Quantification

163 of GFP (Fig EV3A) indicated that only 1.3% of CLPP-KO cells were positive for GFP compared

164 to 25% of WT cells. STINGgt/gt cells were more susceptible to VSV infection consistent with prior

165 results (Ahn and Barber, 2019; Franz et al., 2018), with more than 60% of cells staining GFP

166 positive. Similarly, 68% of CLPP-KO/STINGgt/gt cells were GFP positive, indicating that viral

167 resistance of CLPP-KO cells was completely lost in CLPP-KO/STINGgt/gt MEFs. Relative levels

168 of viral RNA transcripts for VSV-G and VSV-M were reduced greater than 95% in CLPP-KO cells

169 compared to WTs (Fig 3B). In contrast, viral transcript levels were equally high in VSV-infected

170 STINGgt/gt and CLPP-KO/STINGgt/gt cells. Similar results were found at earlier VSV infection

171 timepoints (i.e. 16 hpi, Fig EV3B), corroborating microscopic results demonstrating that antiviral

172 phenotypes of CLPP-KO cells are mediated by STING signaling.

173 To further evaluate the resistance of CLPP-KO cells to viral infection/replication, we

174 employed plaque assays to quantify the level of functional VSV virions released into the

175 supernatant of infected cells (Fig 3C). At 24hpi, there was a striking reduction of viral plaques in

176 CLPP-KO MEFs (2-log reduction) compared to WTs. This contrasted strongly with CLPP-

177 KO/STINGgt/gt cells, which exhibited only a small, 1.7-fold reduction compared to STINGgt/gt

178 littermate MEFs. The viral protection observed in CLPP-KO cells is likely due to both elevated

179 baseline and induced ISGs, as levels of antiviral genes Usp18 and Cmpk2 were further enhanced

180 in CLPP-KO over WT controls after VSV infection (Fig EV3C). Although the VSV-mediated

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181 upregulation of ISGs in CLPP-KO cells was greatly blunted by loss of STING, the fold induction

182 of ISGs in double mutant CLPP-KO/STINGgt/gt MEFs over infected STINGgt/gt MEFs was similar

183 to CLPP-KO over WT cells. This is an expected result considering that RNA viruses such as VSV

184 predominantly engage the RIG-I-MAVS pathway, which remains intact and active in the absence

185 of STING. Thus, the baseline antiviral priming in CLPP-KO cells driven by persistent STING-

186 IFN-I signaling, and not the potentiated induction of ISGs during infection, is likely the most

187 important factor governing the resistance of CLPP-KO cells to VSV.

188 To determine if the viral resistance extended to DNA viruses, we infected MEF lines with

189 HSV-1, a dsDNA virus that employs several diverse mechanisms to evade innate antiviral

190 responses. After infection at a MOI 0.001 and incubation for 72 hours, HSV-1 viral RNAs UL30

191 and Icp27 were reduced greater than 95% in CLPP-KO MEFs compared to WT controls (Fig 3D),

192 mirroring findings seen with VSV infections. The dramatic reduction in HSV-1 RNA expression

193 in CLPP-KO cells was lost in double KO cells, and in fact we observed that CLPP-KO/STINGgt/gt

194 MEFs exhibited five times more viral RNA transcripts than STINGgt/gt littermates (Fig 3D). This

195 marked increase in viral RNAs in the double KO cells was not observed in VSV infections, where

196 there was only a minor increase in viral transcripts in CLPP-KO/STINGgt/gt MEFs over infected

197 STINGgt/gt MEFs (Fig 3C). A similar trend was observed in quantification of viral particles by

198 plaque assays, as we observed a marked resistance of CLPP-KO cells to HSV-1 (1.5 log reduction

199 in plaques) compared to WT cells (Fig 3E). However, CLPP-KO/STINGgt/gt cells were more

200 susceptible to HSV-1 infection (0.5 log increase in plaques) compared to STINGgt/gt, consistent

201 with viral RNA expression data.

202 We next assessed whether the strong antiviral response was maintained at a higher and

203 more lytic MOI, where HSV-1 virulence and IFN-I-repressive mechanisms are more potent. At a

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204 MOI of 0.01 48hpi, CLPP-KO cells remained more resistant to HSV-1, albeit to a lesser extent

205 compared to infection with a lower MOI (Fig EV3D). CLPP-KO cells showed a 40% reduction in

206 UL30 and Icp27 expression compared to WTs. However, similar to results from MOI 0.001

207 infections, CLPP-KO/STINGgt/gt MEFs exhibited 5 times more viral RNA than STINGgt/gt cells

208 (Fig EV3D). The interesting finding that CLPP-KO/STINGgt/gt MEFs are more susceptible to

209 HSV-1 infection may be due to the fact that HSV-1 is a robust modulator of host mitochondrial

210 function and metabolism (Duarte et al., 2019; Reshi et al., 2018). We hypothesize that the complete

211 lack of cytosolic DNA sensing and antiviral IFN-I, combined with altered mitochondrial

212 homeostasis in CLPP-KO/STINGgt/gt cells, likely synergize to increase HSV-1 virulence

213 mechanisms and increase viral replication. Notably, HSV-1 can directly target host mtDNA

214 through the virally-encoded deoxyribonuclease UL12.5, resulting in nucleoid stress/enlargement

215 followed by rapid mtDNA depletion (Corcoran et al., 2009; Saffran et al., 2007; West et al., 2015).

216 Thus, increased interference with mtDNA homeostasis by UL12.5 or other encoded factors may

217 potentially explain the elevated susceptibility of double KO cells to HSV-1. In sum, our results

218 show that potentiated cGAS-STING-dependent ISG and IFN-I responses in CLPP-KO cells leads

219 to a robust antiviral state that is broadly restrictive to both RNA and DNA viruses.

220

221 CLPP deficiency results in altered mtDNA abundance and nucleoid morphology

222 Our prior work has documented that mtDNA stress and cytosolic release is a potent inducer of

223 IFN-I responses via the cGAS-STING DNA sensing axis (West et al., 2015). Since the ISG and

224 antiviral priming phenotypes in CLPP-KO cells and tissues were dependent on cGAS-STING, we

225 hypothesized that mtDNA might be the mitochondrial DAMP triggering this response.

226 Interestingly, we found that MEFs and tissues from CLPP-KO mice exhibit baseline elevations in

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227 mtDNA abundance by qPCR-based approaches (Fig EV4A), despite no coordinate upregulation

228 in the mtDNA packaging protein A mitochondrial (TFAM) (Gispert et al.,

229 2013) or full-length mtDNA genomes (Szczepanowska et al., 2016). We reasoned that this might

230 indicate the presence of mtDNA instability in CLPP deficient cells. In agreement with this

231 hypothesis, we observed that CLPP-KO MEFs (Fig 4A) and CLPP-depleted human fibroblasts

232 (Fig EV4B) displayed significant mtDNA nucleoid enlargement and aggregation by confocal

233 microscopy. These results are similar to the mtDNA stress phenotype observed in Tfam+/- MEFs,

234 which have enlarged mtDNA nucleoids and mitochondrial network hyperfusion that result in the

235 release and cytosolic accumulation of mtDNA, tonic cGAS-STING-IFN-I signaling, and antiviral

236 priming (West et al., 2015).

237 Of note, a recent clinical study revealed increased mtDNA copy number and decreased

238 CLPP mRNA levels in patient fibroblasts harboring disease-associated CLPP mutations

239 (Theunissen et al., 2016). Combined with our results in CLPP deficient cells, these findings

240 collectively suggest a conserved role for CLPP in regulation of mtDNA maintenance and a

241 potential role for mtDNA stress in the pathogenesis of PS. However, our CLPP knockdown and

242 knockout fibroblasts do not exactly phenocopy patient cells harboring CLPP missense mutations

243 associated with PS. These disease mutations are noted to alter key amino acids in a cluster near

244 the docking site for CLPX interaction or in the active site of the peptidase (Brodie et al., 2018).

245 Future work to re-introduce synonymous pathogenic mutant CLPP proteins into CLPP-KO mice

246 or to generate germline mutations in Clpp via CRISPR/Cas9 will help to determine how these

247 disease relevant mutations contribute to mtDNA instability, IFN-I responses, and disease

248 pathogenesis in PS.

249

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250 mtDNA mediates IFN-I responses in CLPP deficient fibroblasts

251 To next demonstrate that mtDNA is the DAMP triggering ISG responses in CLPP-KO MEFs, we

252 treated cells with dideoxycytidine (ddC), a nucleoside reverse transcriptase inhibitor that

253 specifically inhibits mtDNA replication and causes robust mtDNA depletion with little effect on

254 nuclear DNA (Kasashima et al., 2011). Notably, ddC treatment was sufficient to clear the

255 enlarged/aggregated nucleoids in CLPP-KO MEFs (Fig 4A), as well as robustly reduce mtDNA

256 levels (Fig EV4C). Consistent with the notion that mtDNA stress is driving cGAS-STING-IFN-I

257 signaling in CLPP-KO cells, the pronounced ISG signature observed in CLPP-KO MEFs was

258 markedly reduced at the RNA and protein levels after ddC treatment (Fig 4B and C). In a

259 complementary approach, we transfected MEFs with siRNA against Polg2 to knockdown the

260 accessory subunit of the mtDNA polymerase pol γ that is required for mtDNA replication (Young

261 and Copeland, 2016). Consistent with ddC results, we observed a significant reduction in ISG

262 transcripts in CLPP-KO cells when Polg2 was knocked down (Fig 4D).

263 Recently, it was described that VBIT-4, an inhibitor of voltage-dependent anion channel

264 (VDAC) oligomerization, blocks the formation of mitochondrial pores and prevents the release of

265 mtDNA fragments into the cytosol of Endog deficient MEFs (Kim et al., 2019). We next tested

266 this reagent in our MEF lines, and similar to the results with ddC treatment and Polg2 knockdown,

267 VBIT-4 treatment significantly diminished the strong ISG signature of CLPP-KO MEFs at

268 baseline (Fig 4E). VBIT-4 had no effect on ISG expression induced by recombinant mIFNb

269 treatment (Fig EV4D), indicating that the abrogation of steady state antiviral responses by VBIT-

270 4 was not due to a general suppression of IFN-I signaling in CLPP-KO cells. Collectively, these

271 results document that mtDNA stress and VDAC-mediated mtDNA release into the cytosol are key

272 triggers of cGAS-STING-IFN-I signaling and antiviral responses in CLPP-KO cells.

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273 While more than 50 nucleoid-associated proteins have been shown to participate in mtDNA

274 maintenance and gene expression, CLPP has not yet been implicated in either process (Lee and

275 Han, 2017). Our findings document that CLPP is necessary for maintaining mtDNA nucleoid

276 organization and distribution, as CLPP null cells exhibit markedly disrupted nucleoid architecture

277 and TFAM aggregation compared to WT cells. While it is unlikely that these effects result from

278 loss of direct CLPP-mtDNA interactions, CLPX was reported to maintain mtDNA nucleoid

279 distribution via TFAM and is involved in mtDNA segregation in human cells (Kasashima et al.,

280 2012). Although CLPX is upregulated in CLPP null cells, we found that downregulation of CLPX

281 by siRNA had no effect on nucleoid aggregation (data not shown) or steady state ISG responses

282 (Fig EV5A). It is also noteworthy that mono-allelic loss of another matrix protease and mtDNA

283 maintenance factor, LONP1, leads to steady state induction of ISGs in MEFs (Key et al., 2020).

284 Collectively, these results suggest that perturbations in mitochondrial matrix protease levels, which

285 disrupt mtDNA homeostasis and stability, generally engage IFN-I signaling, thus linking

286 mammalian UPRmt factors to antiviral innate immunity (Melber and Haynes, 2018; Pellegrino et

287 al., 2014; S. Wang et al., 2018).

288 Finally, we explored other factors that modulate mtDNA stress and/or signaling to IFN-I

289 in order to rule in or out their involvement in the enhanced ISG signature of CLPP-KO cells. We

290 employed siRNA approaches to target both mitochondrial fusion and fission proteins and

291 examined ISG expression in CLPP-KO MEFs, since mitochondrial dynamics facilitate proper

292 nucleoid distribution and removal of damaged mtDNA (Ban-Ishihara et al., 2013; Malena et al.,

293 2009). However, we found that transient downregulation of mitofusin 1 (Mfn1) or the fission factor

294 -related protein 1 (Drp1) did not significantly alter the ISG signature in CLPP-KO cells

295 (Fig EV5B). In addition, another study identified ERAL1, a putative 12S rRNA chaperone , as a

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296 CLPXP substrate, implicating CLPP in mitoribosomal assembly and mitochondrial translation

297 (Szczepanowska et al., 2016). Although mitoribosomal maturation/assembly was not analyzed

298 carefully in this study, treatment with several concentrations of chloramphenicol (CAM), a

299 mitochondrial translation inhibitor, yielded no effect on the elevated steady state ISG signature of

300 CLPP-KO cells the aberrant nucleoid morphology in CLPP-KO cells (Fig EV5C and data not

301 shown). However, CAM treatment significantly inhibited mitochondrial translation in MEFs, as

302 seen by the significant reductions in cytochrome c oxidase subunit I (mt-COI) protein levels.

303 Future studies aimed at uncovering the molecular mechanisms by which CLPP integrates

304 mitoribosomal assembly, proteostasis, mitochondrial nucleoid organization, and mtDNA release

305 should yield new insight into crosstalk between the mammalian UPRmt and the innate immune

306 system.

307 In conclusion, Caseinolytic peptidase XP (ClpXP) is an evolutionarily conserved,

308 heteromeric, mitochondrial matrix localized serine protease complex, and despite extensive studies

309 on prokaryotic ClpXP, the diverse functions of CLPXP in mitochondria remain less well defined

310 (Levytskyy et al., 2016). Our results place the cGAS-STING-IFN-I innate immune pathway

311 downstream of CLPP and illuminate new links between mitochondrial proteostasis, mtDNA

312 genome maintenance, and antiviral immunity. Imbalances in mitochondrial protease function,

313 mtDNA damage, and innate immunity are associated with several pathological conditions, such as

314 neurodegenerative disorders, infertility, metabolic syndromes, and cancer (Bárcena et al., 2017;

315 Levytskyy et al., 2016). This work sheds new light on the biology of mammalian CLPP, further

316 advances our understanding of mitochondrial-innate immune crosstalk, and may have implications

317 for understanding PS pathogenesis and other diseases involving CLPP dysregulation.

318

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319 Materials and Methods

320 Viral stocks and infections

321 VSV-G/GFP and HSV1/GFP were maintained as described previously (Dalton and Rose, 2001;

322 Desai and Person, 1998). MEFs were plated in 12-well plates at 7x104 cells/well, 16hrs before

323 infection in DMEM+10% FBS. The next morning, virus stocks were diluted at indicated MOIs in

324 serum-free DMEM (D5756-500ML, Sigma). 300 μl serum-free media containing virus (or DMEM

325 alone for the control wells) was gently added to wells. Plates were incubated at 37°C 5% CO2 and

326 rocked gently every 15 min for 1hr, after which the supernatant was removed, bleached, and

327 discarded. Then, 0.5 mL of fresh DMEM+10% FBS was added to each well, and the cells were

328 allowed to incubate for the duration of the experiment. At the indicated times post infection,

329 supernatant was collected and cleared by centrifugation at 6,000 rpm, 4oC, then kept at -80°C until

330 further processing. Cells were then fixed and stained for microscopy or lysed in RNA Lysis buffer.

331 For plaque assay: BHK-1 or Vero cells were plated in 6-well plates at 95% confluency in 3%

332 Methylcellulose containing DMEM media. Next, different dilutions of supernatant from VSV or

333 HSV1 infected MEFs were added to the cells and incubated until plaques were visible, between

334 24-72hrs. Plaques were stained with Cresyl Violet and counted with Image J software.

335

336 Immunofluorescence

337 Cells were grown on 12mm or 18mm coverslips and treated or infected as described. After washing

338 in PBS, cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton

339 X-100 in PBS for 5 min, blocked with PBS containing 10% FBS for 30 min, stained with primary

340 antibodies for 60 min, and stained with secondary antibodies for 60 min. Cells were washed with

341 PBS between each step. Coverslips were mounted with Prolong Gold anti-fade reagent containing

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342 DAPI (Molecular Probes). For viral infections, viral GFP fluorescence images were captured using

343 the LionHeart FX fluorescent microscope at 20X and tiling images at 2x2. For mtDNA nucleoid

344 staining, a 60X oil-immersed objective was used and images were taken with a Confocal Laser

345 Scanning Microscope Olympus FV3000. Images were processed in Image J software.

346

347 Quantitative PCR

348 To measure relative gene expression by qRT-PCR, total cellular RNA was isolated using the

349 Quick-RNA Micro Prep Kit (R1051, Zymo Research). Approximately 250-500ng RNA was

350 normalized across samples and cDNA was generated using the qScript cDNA Synthesis Kit

351 (101414-098, VWR). cDNA was then subjected to qPCR using PerfecTa SYBR Green FastMix

352 (84069, Quantabio) and primers as indicated in Table 1. Three technical replicates were performed

353 for each biological sample, and expression values of each replicate were normalized against Rp137

354 using the 2 -Dddct method. For relative expression (fold), control samples were centered at 1; for

355 relative expression (%), control samples were centered at 100%. Mitochondrial DNA abundance

356 analysis was performed as described above using primers specific to mtDNA regions and

357 normalized against nuclear Tert.

358

359 Table 1. Primer sequences

mCmpk2 AAAGAATCAACCAACTTT GGCCTCCACTCACCTCAGTA mCxcl10 CCAAGTGCTGCCGTCATTTTC GGCTCGCAGGGATGATTTCAA mDrp1 TCCCAATTCCATTATCCTCGC CATCAGTACCCGCATCCATG mIfit1 CAAGGCAGGTTTCTGAGGAG GACCTGGTCACCATCAGCAT

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mIfit3 TTCCCAGCAGCACAGAAAC AAATTCCAGGTGAAATGGCA mIrgm1 CCAGGAAGGCCACTAACATC TGGTTCCTTCGAATGCCTTA mIsg15 CTAGAGCTAGAGCCTGCAG AGTTAGTCACGGACACCAG mMfn1 ATGGCAGAAACGGTATCTCCA GCCCTCAGTAACAAACTCCAGT mPolg2 TGGAAATGTGTCTACGATACAGG GCTGGAAGGAATCGTAGAGGT mRnf213 TTTGTACCGTTCCCCCAAT GTTCACTGCCTCCAATTGCT mRsad2 ATAGTGAGCAATGGCAGGCCT AACCTGCTGATGCAAGCTGT mRp137 CATCCTTTGGTAAGCGTCGCA TGGCACTCCAGTTATACTTCCT mUsp18 GAGAGGACCATGAAGAGGA TAAACCAACCAGACCATGAG mZbp1 TCAAAGGGTGAAGTCATGGA GTGGAGTGGCTTCAGAGCTT m.mtATP6 CCTTCCACAAGGAACTCCAATTTCAC CTAGAGTAGCTCCTCCGATTAGGTG m.mtCO3 GACGTAATTCGTGAAGGAACCTACC GATAGAACGCTCAGAAGAATCCTGC m.mtCytb GCTTTCCACTTCATCTTACCATTTA TGTTGGGTTGTTTGATCCG m.mtDLoop1 AATCTACCATCCTCCGTGAAACC TCAGTTTAGCTACCCCCAAGTTTAA m.mtND6 TTTAGCATTAAAGCCTTCACC CCAACAAACCACTTAACAAT m.nucTert CTAGCTCATGTGTCAAGACCCTCTT GCCAGCACGTTTCTCTCGTT HSV1 ICP27 RNA TTTCTCCAGTGCTACCTGAAGG TCAACTCGCAGACACGACTCG HSV1 UL30 RNA CGCGCTTGGCGGGTATTAACAT TGGGTGTCCGGCAGAATAAAGC VSV-G CAAGTCAAAATGCCCAAGAGTCACA TTTCCTTGCATTGTTCTACAGATGG VSV-M TATGATCCGAATCAATTAAGATATG GGGACGTTTCCCTGCCATTCCGATG 360

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361

362 Proteomics

363 CLPP-KO MEF and their matching controls (n=3) were analyzed by quantitative mass

364 spectrometry essentially as described in Key et al., 2019. Mass spectrometry data were analyzed

365 by MaxQuant (1.5.3.30) (Cox and Mann, 2008) using a mouse proteome Uniprot database

366 (Download 2/2016) and a false discovery rate (FDR) less than 1%. For quantification, proteins

367 were quality filtered according to a minimum of three valid values in one group (n=3) using

368 Perseus software (v. 1.5.2.6). All missing values from this reduced matrix were replaced by

369 background value from normal distribution. For statistical comparison, students t-tests were used.

370 The full datasets will be deposited in the Proteomics Identification Database (PRIDE) upon

371 acceptance of the manuscript.

372

373 Immunoblotting

374 Proteins from either homogenized tissue (<50mg) or cells (<1x107) were lysed in 1% NP40 buffer,

375 and spun down at 15,000 rpm for 10 min at 4°C. The supernatant was collected as protein lysate

376 and quantified with micro-BCA assay (23235, Proteintech). Between 20-30μg of protein were

377 separated on 10-20% polyacrylamide gradient gels, then transferred onto PVDF membranes at

378 100V for 1hr. Membranes were dried for 30 min and incubated with primary antibodies overnight

379 at 4°C. Membranes were washed with 1X PBS for 30 min and incubated with HRP-conjugated

380 secondary antibodies for 1hr. Membranes were washed with PBS for 1 hr before developing with

381 Millipore Luminata Crescendo HRP Substrate (WBWR0500).

382

383 Table 2. List of Antibodies

18 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.30.274712; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Name Company Catalog number Dilution

CLPX Abcam ab168338 [EP8772] 1:1,000

IFIT3 gift from G.Sen at Cleveland Clinic 1:10,000

ZBP1 Adipogen AG-20B-0010

aTUBULIN DSHB 12G10 1:5,000

STAT1 Cell Signaling 9172S 1:1,000

cGAS Cell Signaling 31659 1:1,000

RIG-I Cell Signaling 4200S 1:1,000

IRGM1 Cell Signaling 14979 1:1,000

MAVS Cell Signaling 4983 1:1,000

GFP DSHB 8H11 1:30

TFAM Millipore ABE483 1:1,000

ANTI-DNA Millipore CBL-186 (AC-30-10) 1:300

IFITM1 Proteintech 60074-1-Ig 1:5,000

IFIT2 Proteintech 12604-1 1:1,000

CALNEXIN Proteintech 10427-2-AP 1:1,000

b-ACTIN Proteintech 66009-1-Ig 1:5,000

CLPP Proteintech 15698-1 1:1,000

GAPDH Proteintech 60004-1-LG 1:5,000

HSP60 Santa Cruz sc-1052 1:5,000

mt-COI ThermoFisher 459600 (ID6E1A8) 1:1,000

384

385 Mouse Strains

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386 Embryonal stem cell line IST13563G11 (line G), with heterozygous GeneTrap insertions at Clpp

387 intron 2, was re-derived at the Texas Institute for Genomic Medicine (TIGM) on a C57BL/6N

388 background. Heterozygous breeders were mated to generate Clpp-null (CLPP-KO) mutant and

389 littermate WT control offspring. Stinggt/gt (C57BL/6J-Sting1gt/J, strain 017537) and Ifnar-/-

390 (B6(Cg)-Ifnar1tm1.2Ees/J, strain 028288) were obtained from the Jackson Laboratory. CLPP

391 heterozygous mutant mice were crossed with Stinggt/gt and Ifnar-/- for several generations to obtain

392 double KO mice on a C57BL/6NJ mixed background. All animal experiments were conducted in

393 compliance with the guidelines established by the Texas A&M University Institutional Animal

394 Care and Use Committee (IACUC).

395

396 EchoMRI

397 Body composition analysis on live mice was completed using the EchoMRI™-100H at the Rodent

398 Preclinical Phenotyping Core at Texas A&M University. This analyzer delivers precise body

399 composition measurements of fat, lean, free water, and total water masses in live animals weighing

400 up to 100g.

401

402 Cell Culture

403 Primary wild-type, CLPP-KO, STINGgt/gt, IFNAR-/- (IFNAR-KO), CLPP-KO/STINGgt/gt and

404 CLPP-KO/IFNAR-KO MEFs were generated from E12.5-14.5 embryos. Cells were maintained in

405 DMEM (D5756-500ML, Sigma) supplemented with 10% FBS (VWR), and sub-cultured for no

406 more than five passages before experiments. Transfection of MEFs with siRNA was performed

407 with Lipo-RNAi Max (13778-150, Invitrogen) in Opti-MEM media (11058021, ThermoFisher

408 Scientific) and the following siRNAs were used at a final concentration of 25nM: sicGAS

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409 (MMC.RNAI.N173386.12.1 IDT), siRNA mPolg2 (NM_015810 Sigma-Aldrich), siMfn1

410 (NM_024200 Sigma-Aldrich), siDrp1 (MMC.RNAI.N152816.12.1), siClpX

411 (MMC.RNAI.N011802.12.2 IDT) and siRNA NTC (Negative Control DsiRNA, 51-01-14-03,

412 IDT) according to manufacturer’s instructions. Cells were harvested for RNA or protein between

413 48-72hrs later. For Rig-I/MDA5 challenge, cells were transfected with Poly(I:C) (P1530, SIGMA)

414 complexed in polyethylenimine (PEI, 43896, Alfa Aesar) in Opti-MEM media. For LPS, cells

415 were treated with LPS (LPS-B5 Ultrapure, InvivoGen tlrl-pb5lps) at 1µg/ml and harvested for

416 RNA 4hrs later. For mtDNA depletion, ddC (D5782, SIGMA) was resuspended in PBS, added to

417 MEFs at a final concentration of 150uM and refreshed every 48hrs for 4 days. For

418 Chloramphenicol (CAM) treatment, cells were treated with CAM (R4408, SIGMA) at different

419 concentrations (50-200uM) and harvested for protein between 24-48hrs later. For VBIT-4

420 treatment, VBIT-4 (HY-129122, MedChem Express) was resuspended in DMSO at 10mM and

421 added to the media at a final concentration of 10uM during 48hrs while control cells were treated

422 with DMSO. For VBIT-4 treatment plus challenge, cells were treated with VBIT-4 and 6hrs before

423 the 48hrs harvesting time mIFNb (8234-MB-010/CF R&D) was added to the cells at a final

424 concentration of 1ng/ml. All cells were harvested 48hrs after challenge for RNA analysis. To

425 generate CLPP knockdown human foreskin fibroblasts (HFF-1), MISSION shRNA Lentiviral

426 Transduction Particles against human CLPP (TRCN0000291174) or eGFP (RHS4459) were

427 purchased from Sigma-Aldrich and Horizon Discovery respectively. HFF-1 were transduced with

428 the shRNA encoding lentivirus stocks in the presence of polybrene (8 μg/ml) and stably selected

429 with puromycin.

430

431 Statistical analyses

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432 Error bars displayed throughout the manuscript represent the mean +/- s.e.m. unless otherwise

433 indicated and were calculated from triplicate or quadruplicate technical replicates of each

434 biological sample. Sample sizes were chosen by standard methods to ensure adequate power, and

435 no randomization or blinding was used for animal studies. No statistical method was used to

436 predetermine sample size. Statistical analysis was determined using GraphPad software and

437 statistics tests include: unpaired Student’s t-tests, ordinary one-way ANOVA and two-way

438 ANOVA with Tukey's post-hoc. Significance was stablished as *p<0.05; **p<0.01; ***p<0.001;

439 ****p<0.0001; or not significant (ns) when p>0.05. Data shown are representative of 2-3

440 independent experiments (unless otherwise specified) including microscopy images, Western

441 blots, and viral challenges.

442

443

444 Acknowledgements

445 We would like to thank our colleagues in the West Lab for feedback on the manuscript, as well as

446 Dr. Malea Murphy in the Integrated Microscopy and Imaging Laboratory at the Texas A&M

447 College of Medicine for assistance with confocal imaging. S.T.O was partially financed by the

448 Arthur Merx Stiftung Frankfurt. This research was supported by awards W81XWH-17-1-0052 and

449 W81XWH-20-1-0150 to A.P.W. from the Office of the Assistant Secretary of Defense for Health

450 Affairs through the Peer Reviewed Medical Research Programs. Additional support was provided

451 by NIH grant R01HL148153 to A.P.W. and NIEHS P30 ESES029067. Opinions, interpretations,

452 conclusions, and recommendations are those of the author and are not necessarily endorsed by the

453 NIH or Department of Defense.

454

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455

456 Author Contributions

457 S.T-O. and A.P.W. designed the experiments, analyzed the data, and wrote the manuscript. Y.L.,

458 A.M., J.K., and S.M. assisted with experiments, sample processing, and/or data analysis. I.W.

459 performed proteomic analyses. G.A. and S.G. contributed datasets, provided experimental advice,

460 and edited the paper. A.P.W. conceived the project and provided overall direction.

461

462

463 Conflict of Interest

464 The authors declare no conflicts of interest.

465

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585 Melber, A., Haynes, C.M., 2018. UPRmt regulation and output: a stress response mediated by 586 mitochondrial-nuclear communication. Cell Res. 28, 281–295. https://doi.org/10.1038/cr.2018.16

587 Nakahira, K., Hisata, S., Choi, A.M.K., 2015. The Roles of Mitochondrial Damage-Associated 588 Molecular Patterns in Diseases. Antioxid. Redox Signal. 23, 1329–1350. 589 https://doi.org/10.1089/ars.2015.6407

590 Pellegrino, M.W., Nargund, A.M., Kirienko, N.V., Gillis, R., Fiorese, C.J., Haynes, C.M., 2014. 591 Mitochondrial UPR-regulated innate immunity provides resistance to pathogen infection. Nature 592 516, 414–417. https://doi.org/10.1038/nature13818

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595 Reshi, L., Wang, H.-V., Hong, J.-R., 2018. Modulation of Mitochondria During Viral Infections. 596 Mitochondrial Dis. E. Taskin, C. Guven and Y. Sevgiler, IntechOpen. 597 https://doi.org/10.5772/intechopen.73036

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612 Abnormalities Reveal Severe Perrault Syndrome due to CLPP Defects. Front. Neurol. 7, 203. 613 https://doi.org/10.3389/fneur.2016.00203

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620 Wang, T., Babayev, E., Jiang, Z., Li, G., Zhang, M., Esencan, E., Horvath, T., Seli, E., 2018. 621 Mitochondrial unfolded protein response gene Clpp is required to maintain ovarian follicular 622 reserve during aging, for oocyte competence, and development of pre-implantation embryos. 623 Aging Cell 17, e12784. https://doi.org/10.1111/acel.12784

624 West, A.P., 2017. Mitochondrial dysfunction as a trigger of innate immune responses and 625 inflammation. Toxicology 391, 54–63. https://doi.org/10.1016/j.tox.2017.07.016

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636

29 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.30.274712; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

637 Figure Legends

638 Figure 1. CLPP deficiency leads to steady state interferon stimulated gene (ISG) expression

639 in mouse and human cells.

640 A, B Quantitative real-time PCR (A) and western blots (B) of baseline ISGs of littermate WT

641 and CLPP-KO mouse embryonic fibroblasts lines (n=2).

642 C Heat map from the top 40 significantly (p<0.05) upregulated proteins from baseline

643 proteomic data of littermates WT and CLPP-KO MEFs lines. Data represented as Label-free

644 quantification (LFQ) Fold Change of each CLPP-KO MEFs over average WT (n=3).

645 D, E Quantitative real-time PCR of ISGs in WT and CLPP-KO MEFs, 4h after transfection with

646 poly(I:C) (D) or LPS challenge (E).

647 F Human foreskin fibroblasts were transduced with the shRNA against CLPP and EGFP (as

648 control) and selected with puromycin (2µg/ml). After selection, cells were plated in 12-well dishes

649 and harvested for western blots of baseline ISGs.

650 Data information: Data are presented as mean ± s.e.m. of triplicate technical replicates and are

651 representative of three independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

652 In A, two-way ANOVA Tukey’s post-hoc. In D, E Student’s t-test

653

30 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.30.274712; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Fig 1 CLPP deficiency leads to steady state interferon stimulated gene (ISG) expression in mouse and human cells.

Basal MEF proteomics Top significantly upregulated proteins MEFS (ISGs in bold) A B C 40 KO1 KO2 KO3 WT WT1 WT2 CLPP-KO1CLPP-KO2 IFIT3 **** CLPP-KO RIG-I ISG15 30 IRGM3 STAT1 GVIN1 ISGS TGTP **** ZBP1 20 ** SDHAF2

ex pression (fold) *** IFIT3 GBP4 IFI204 10 CLPX IRGM2 Relative BST2 CLPP DTX3L 0 GRSF1 αTubulin Rsad2 Ifit1 Cmpk2 Usp18 MALSU1 LIAS D E MDA5/IFIH1 3000 ** 5000 LGALS3BP BCKDK ** 4000 * STAT1 2000 STAT2 3000 MRPL39 1000 PSMB8 ex pression (fold)

400 ex pression (fold) 2000 * * ** PLA2G7 200 1000 AURKAIP1

Relative RNF213 Relative 0 0 PARP9 Ifit1 Cmpk2 Rsad2 Cxcl10 Rsad2 Cxcl10 MTHFD2 Transfected Poly(I:C) 4h LPS (1µg/ml) 4h MNDAL H2-D1 Human Fibroblasts GBP2 F MRPL43 EGFP CLPP GABARAP RIG-I LGALS9 25 STAT1 GFM1 20 ISGS LMCD1 IFIT2 15 IRGM1 IFITM1 10 TAP2 5 MHC CLPX 0.08 IFI35 CLPP LFQ Fold Change PGM5 KO/WT CLPP β-Actin

31 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.30.274712; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

654 Figure 2. STING and IFNAR mediate the steady state ISG and antiviral signature in CLPP

655 deficient cells and tissues.

656 A Western blots of ISGs in WT and CLPP-KO MEFs transfected with siRNA Control (Ctrl)

657 and sicGAS for 72hrs.

658 B Quantitative real-time PCR of baseline ISGs in WT, CLPP-KO, and CLPP-KO/STINGgt/gt

659 or CLPP-KO/IFNAR-KO double mutant MEFs (n=2 MEF lines). Real-time PCR data was

660 represented as relative expression percentage (%) with CLPP-KO set to 100%.

661 C, D Quantitative real-time PCR (C) and western blots (D) of baseline ISGs from heart tissue

662 from 12-month old male mice (n=3).

663 Data information: In B and C data are presented as mean ± s.e.m. of triplicate technical replicates.

664 ***p<0.001, ****p<0.0001, ns, non-significant. (One-way ANOVA, Tukey’s post-hoc)

665

32 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.30.274712; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Fig 2 STING and IFNAR mediate the steady state ISG and antiviral signature in CLPP deficient cells and tissues

MEFs

A WT CLPP-KO B siRNA Mock Ctrl cGAS Mock Ctrl cGAS 120 RIG-I **** **** **** CLPP-KO 100 CLPP-KO/STINGgt/gt STAT1 80 CLPP-KO/IFNAR-KO

ZBP1 60 40 IFIT3 20 cGAS 20

Relative expression (%) 10 CLPP 0 Calnexin Usp18 Isg15 Cxcl10

HEART C WT 8 6 4 CLPP-KO **** STINGgt/gt **** **** CLPP-KO/STINGgt/gt 6 3 IFNAR-KO 4 CLPP-KO/IFNAR-KO

4 2 ex pression (fold) 2 ns 2 ns 1 ns ns ns

Relative ns 0 0 0 Usp18 Zbp1 Ifit1

HEART

D CLPP-KO CLPP-KO gt/gt WT CLPP-KO STING STINGgt/gt IFNAR-KO IFNAR-KO 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 RIG-I

STAT1

ZBP1

IFIT3

CLPP

αTubulin

33 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.30.274712; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

666 Figure 3. CLPP-KO fibroblasts are more resistant to viral infection owing to elevated

667 activation of the STING pathway.

668 A-C Analysis of viral load and viral plaques in MEFs after 24hrs of VSV infection at

669 multiplicity of infection (MOI) of 0.1. (A) Microscopy images of viral GFP expression in MEFs

670 infected with VSV. DAPI was used for nuclear staining. (B) Quantitative real-time PCR of viral

671 RNA transcripts, VSV-G and VSV-M, after VSV infection (n=3 MEF lines). (C) Quantification of

672 plaque assays from VSV infected MEFs.

673 D Quantitative real-time PCR of viral RNA in MEFs infected with HSV-1 at 72hrs post

674 infection (hpi) and MOI of 0.001 (n=3 MEF lines).

675 E Quantification of plaque assay from HSV-1 infected MEFs. Plaque figure is representative

676 of 10-2 supernatant dilution and graphed as pfu/ml (n=2).

677 Data information: In (C, E), plaque figure is representative of 10-5 supernatant dilution and graphed

678 as pfu/ml. In (B, D), Real-time PCR data was represented as relative expression percentage (%)

679 comparing CLPP-KO vs WT and CLPP-KO/STINGgt/gt vs STINGgt/gt MEFs. In (B-E), data are

680 presented as mean ± s.e.m. of triplicate technical replicates. **p<0.01, ***p<0.001,

681 ****p<0.0001, ns non-significant (Student’s t-test)

682

34 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.30.274712; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Fig 3 CLPP-KO fibroblasts are more resistant to viral infection owing to elevated activation of the STING pathway

VSV MOI 0.1 (24hpi) A WT CLPP-KO STINGgt/gt CLPP-KO STINGgt/gt

VSV-G GFP

300 um

DAPI

Merge

VSV MOI 0.1 (24hpi) HSV1 MOI 0.001 (72hpi) B D 120 700 120 150 ns *** ** **** *** 600 **** ns 100 100 500 **** 100 80 80 400 300 60 50 60 10 4 200 5 2 100 Relative expression (%) Relative expression (%) Relative expression (%) Relative expression (%) 0 0 0 0 VSV-G VSV-M VSV-G VSV-M UL30 Icp27 UL30 Icp27

6 C 109 E 10 *** * WT CLPP-KO 106 WT CLPP-KO 104 WT CLPP-KO pfu/ml pfu/ml 103 STINGgt/gt 102 CLPP-KO/ STINGgt/gt 100 100 9 6 10 * 10 **

gt/gt gt/gt 6 STING CLPP-KO STING gt/gt gt/gt 10 104 STING CLPP-KO STING pfu/ml pfu/ml 103 102

100 100

35 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.30.274712; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

683 Figure 4. Altered mtDNA homeostasis mediates IFN-I responses in CLPP-KO fibroblasts.

684 A WT and CLPP-KO MEFs were cultured in the presence of ddC (150μM) for 4 days. Cells

685 were harvested after 4 days for confocal microscopy (A) of WT and CLPP-KO MEFs stained with

686 anti-DNA (DNA), anti-Tfam (mtDNA nucleoid marker) and anti-HSP60 (mitochondrial matrix

687 protein).

688 B Quantitative real time PCR of ISG expression after culture in ddC (n=3 MEF lines).

689 C Western blots of ISG protein expression after culture in ddC.

690 D Quantitative real time PCR of ISGs in CLPP-KO MEFs transfected with control or siPolg2

691 siRNA for 72hrs (n=2 MEF lines).

692 E Quantitative real time PCR of ISGs of WT and CLPP-KO MEFs treated with VBIT-4

693 (10μM) or Control (DMSO) for 48hrs (n=2 MEF lines).

694 Data information: In D, Real-time PCR data are presented as relative expression percentage (%)

695 considering siControl as 100%. Error bars indicate ± s.e.m. of triplicate technical replicates

696 (Student’s t-test). In B and E, data represented as mean ± s.e.m. of triplicate technical replicates,

697 two-way ANOVA Tukey’s post-hoc. *p<0.05, **p<0.01, ****p<0.0001

698

36 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.30.274712; this version posted August 31, 2020. The copyright holder for this preprint Fig 4 Altered(which mtDNA was not certified homeostasis by peer review) mediates is the author/funder. type I Allinterferon rights reserved. responses No reuse allowed in CLPP-KO without permission. fibroblasts.

A DNA Tfam Hsp60 Merge

WT

Control

CLPP-KO

WT

ddC

CLPP-KO

B Ifit1 Usp18 C WT CLPP-KO ddC - + - + 25 20 WT CLPP-KO RIG-I 20 **** **** ZBP1 15 15 10 IFIT3 3

ex pression (fold) 10

ex pression (fold) 4 TFAM 2 1 2 CLPP Relative Relative 0 0 αTubulin Control ddC Control ddC

D E Ifit1 Isg15 Usp18 140 siControl siPolg2 15 15 10 *** 120 ** * ** ** *** 8 100 * 10 10 80 6

ex pression (%) 60

ex pression (fold) 4 40 5 5 2 Relative 20 Relative 0 0 0 0 Usp18 Ifit3 Rsad2 Polg2 DMSO VBIT-4 DMSO VBIT-4 DMSO37 VBIT-4 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.30.274712; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

699 Figure 5. Loss of CLPP activates IFN-I through mtDNA-cGAS-STING- signaling.

700 Absence of CLPP protease leads to mtDNA stress (packaging alterations and cytosolic release),

701 triggering the cGAS-STING-IFN-I pathway, which leads to elevated ISG expression and

702 resistance to DNA and RNA virus infection. Future study should investigate whether chronic

703 activation of this pathway contributes to Perrault syndrome (PS) phenotypes.

704

38 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.30.274712; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Fig 5 Loss of CLPP activates IFN-I through mtDNA-cGAS-STING- signaling

CLPP deficiency

VDAC DNA + RNA Type I virus infection interferon responses cGAS/STING ?

Perrault syndrome phenotypes

39 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.30.274712; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

705 Expanded View Figure Legends

706 Figure EV1. CLPP-KO mice exhibit elevated ISG expression and protein abundance in

707 brain, heart, muscle and liver.

708 A, B Microarray profiling from results reported in Gispert S et al, 2013, GEO Accession

709 GSE40207. Heatmaps of predicted upstream regulators (A) and putative IRF7 and STAT1 target

710 genes of the antiviral gene signature (B) in CLPP-KO brain, heart, muscle and liver tissues using

711 Ingenuity Pathway Analysis (IPA) (n=3).

712 C-D Heatmaps showing most significantly upregulated ISGs from microarray data

713 (GSE40207). Western blots of baseline ISGs in heart (C) and liver (D) tissues of WT and CLPP-

714 KO male mice, between 3-5 months old (n=3).

715

40 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.30.274712; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Fig EV1 CLPP-KO mice exhibit elevated ISG expression and protein abundance in brain, heart, muscle and liver.

GSE Accession number : GSE40207 Microarray Profiling

A Top IPA B Top IPA Top IPA Upstream Regulators IRF7 target genes STAT1 target genes

BrainHeartMuscleLiver BrainHeartMuscleLiver BrainHeartMuscleLiver IRF7 Usp18 Usp18 Ifit1b Interferon alpha Ifit1b Ifit3 Ifit3 TRIM24 Ifi44 Ifi44 IFNAR Irf7 Gbp6 Poly rI:rC-RNA Rtp4 Irf7 Rtp4 IRF3 Oas1 Rsad2 Oas1 IFNA2 Oasl2 Rsad2 PNPT1 Stat1 Oasl2 STAT1 Ifi47 Stat1 Ifi47 IFNL1 Activation z-score Ifit2 Exp. Fold Change KO/WT -6.1 6.9

-2.7 4.9

C HEART D LIVER

WT CLPP-KO WT CLPP-KO WT CLPP-KO WT CLPP-KO Usp18 1 2 3 1 2 3 Rsad2 1 2 3 1 2 3 Ifit1 STAT1 Xaf1 RIG-I ISGS Ifit3 ZBP1 Trim30a STAT1

Irf7 IFIT3 Ifit3 ZBP1 ISGS

Ifit2 CLPP Ifi44 IRGM1

Ifi44 αTubulin Oas1a IFIT3

Oasl2 Usp18 CLPP Stat1 Ifit1 αTubulin Gbp6 Gbp6

Microarray Microarray Bi-weight Avg. Signal (log2) Bi-weight Avg. Signal (log2)

Low High Low High

41 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.30.274712; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

716 Figure EV2. STING and IFNAR signaling drive elevated ISG responses in CLPP deficient

717 cells and tissues, but ablation of IFN-I signaling does not lessen growth retardation of CLPP

718 KO mice.

719 A Western blots of ISGs of WT and CLPP-KO MEFs transfected with siRNA Control (Ctrl)

720 and siMavs for 72hrs.

721 B Quantitative real-time PCR of baseline ISGs of liver tissue from 12-month old male mice

722 (n=3).

723 C Body weight analysis of 5-7-month old male mice from 6 different genotypes (n= 5-8).

724 D Fat mass and lean mass measurements in 7-10-month old female WT, CLPP-KO,

725 STINGgt/gt and CLPP-KO/STINGgt/gt mice, as assessed by EchoMRI and normalized to body

726 weight (n= 6-10).

727 Data information: In (B-D), data are presented as mean ± s.e.m. of triplicate technical replicates.

728 In (B, C) **p<0.01, ****p<0.0001, ns non-significant (One-way ANOVA Tukey’s post-hoc). In

729 D, ***p<0.001, ****p<0.0001, ns non-significant (Two-way ANOVA Tukey’s post-hoc).

730

42 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.30.274712; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Fig EV2 STING and IFNAR signaling drive elevated ISG responses in CLPP deficient cells and tissues, but ablation of IFN-I signaling does lessen growth retardation of CLPP KO mice

MEF LIVER A B

WT CLPP-KO 15 siRNA Ctrl Mavs Ctrl Mavs 5 **** ** STAT1 4 **** ZBP1 10 3 IFIT3 ex pression (fold) 2 ns 5 CLPP 1 ns

MAVS Relative ns ns 0 0 αTubulin Usp18 Zbp1

C D 100 ns *** WT 50 **** ns CLPP-KO **** STINGgt/gt 80 gt/gt 40 CLPP-KO/STING **** IFNAR-KO **** CLPP-KO/IFNAR-KO 60 30

40 20 **** **** % of Body weight ns Body weight (gr) 10 20

0 0 Body weight Fat mass Lean mass

43 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.30.274712; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

731 Figure EV3. CLPP-KO fibroblasts are more resistant to viral infection owing to elevated

732 activation of the STING pathway.

733 A Quantification of GFP staining in MEFS at MOI 0.1 24hrs after VSV infection.

734 B Quantitative real-time PCR of viral RNA at MOI 0.1 16hrs after VSV infection.

735 C Quantitative real-time PCR of ISGs 24hrs after VSV infection at MOI of 0.1.

736 D Quantitative real-time PCR of viral RNA in MEFs infected with HSV-1 at 48hpi and MOI

737 of 0.01.

738 Data information: In (B, C) Real-time PCR data are presented as relative expression percentage

739 (%) comparing CLPP-KO vs WT and CLPP-KO/STINGgt/gt vs STINGgt/gt MEFs. Error bars

740 indicate ± s.e.m. of triplicate technical replicates (Student’s t-test). In (A, C) data are presented as

741 mean ± s.e.m. of triplicate technical replicates (One-way ANOVA Tukey’s post hoc).

742 *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns non-significant.

743

44 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.30.274712; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Fig EV3 CLPP-KO firboblasts are more resistant to viral infection owing to elevated activation of the STING pathway

A 100 B VSV MOI 0.1 (16hpi) 80 ****ns 150 150 WT CLPP-KO 60 ce ll s (%) ** ns STINGgt/gt gt/gt 40 ** 100 100 CLPP-KO/STING 20 po sitive 4 50 50 DA PI 2 Relative expression (%) GFP/ 0 0 0 VSV-G VSV-G

C VSV MOI 0.1 (24hpi) WT 40 25 CLPP-KO STINGgt/gt ** *** ***** gt/gt 20 30 CLPP-KO/STING

15 20 10 ex pression (fold) 10 5

Relative 0 0 Usp18 Cmpk2

D HSV1 MOI 0.01 (48hpi)

gt/gt WT 800 STING 120 gt/gt * * CLPP-KO 700 CLPP-KO/STING 100 **** 600 80 500 *** 60 400 300 40 200 20 100 Relative expression (%) Relative expression (%) 0 0 UL30 Icp27 UL30 Icp27

45 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.30.274712; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

744 Figure EV4. Altered mtDNA homeostasis mediates IFN-I responses in mouse and human

745 CLPP deficient fibroblasts.

746 A Quantification of mtDNA abundance relative to nuclear DNA (Tert) in WT and CLPP-KO

747 MEFs at baseline.

748 B Human foreskin fibroblasts (HFF-1) were transduced with the shRNA encoding CLPP and

749 EGFP (as control) and selected with puromycin (2µg/ml). After selection, cells were plated in 12-

750 well dishes, fixed, and stained with anti-DNA (DNA), anti-HSP60 (mitochondria) and DAPI

751 before imaging on a confocal microscope with a 60X oil immersion objective.

752 C Quantification of mtDNA abundance relative to nuclear DNA (Tert) in WT and CLPP-KO

753 MEF before and after ddC treatment.

754 D Quantitative real time PCR of ISGs of WT and CLPP-KO MEFs treated with VBIT-4

755 (10μM) and challenged with mIFNb (1ng/ml) for 6hrs.

756 Data information: In (A, C) data are presented as mean ± s.e.m. of triplicate technical replicates

757 (Student’s test). In D, data are presented as mean ± s.e.m. of triplicate technical replicates (Two-

758 way ANOVA Tukey’s post hoc). *p<0.05, **p<0.01, ***p<0.001,****p<0.0001.

759

46 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.30.274712; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Fig EV4 Altered mtDNA homeostasis mediates IFN-I responses in mouse and human CLPP deficient fibroblasts

MEFs Human fibroblasts A B DNA HSP60 MERGE 2.5 WT CLPP-KO 2.0 ** * HFF-1 ** * shEGFP 1.5

1.0 mtDNA abundance

0.5 Relative 0.0 HFF-1 shCLPP

mt-Nd6 mt-CoIII mt-Atp6 mt-DLoop1

MEFs

C WT WT+ ddC D CLPP-KO CLPP-KO + ddC 2300 WT+mIFNβ **** **** WT+mIFNβ+VBIT-4 1800 2.0 CLPP-KO+mIFNβ **** CLPP-KO+mIFNβ+VBIT-4 **** **** 1300 **** 750 1.5 ns

1.0 ex pression (fold) 500 ns

mtDNA abundance ns 0.5 250 ns Relative

Relative 0.0 0 mt-DLoop1 mt-Cytb Ifit1 Usp18 Isg15

47 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.30.274712; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

760 Figure EV5. mtDNA stress-mediated IFN-I responses in CLPP-KO fibroblasts are

761 independent of mitochondrial dynamics and translation.

762 A, B Quantitative real time PCR of ISGs in CLPP-KO MEFs transfected with siRNA Control

763 (Ctrl) and (A) siClpX, or (B) siMfn1 and siDrp1 for 72hrs (n=3 MEF lines).

764 C ISG protein levels of WT and CLPP-KO MEFs treated with chloramphenicol (CAM, 50-

765 200μM) over a 24hr to 48hr time course (n=2 MEF lines).

766 Data information: In (A, B) Real-time PCR data are presented as relative expression percentage

767 (%) considering CLPP-KO siControl as 100%. Data are presented as mean ± s.e.m. of triplicate

768 technical replicates. In A, Student’s t-test and in B, One-way ANOVA Tukey’s post hoc. *p<0.05,

769 **p<0.01, ***p<0.001, ****p<0.0001, ns non-significant.

770

48 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.30.274712; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Fig EV5 mtDNA stress-mediated IFN-I responses in CLPP-KO fibroblasts are independent of mitochondrial dynamics and translation.

A B siControl siControl 160 siMfn1 150 siClpX ns 140 siDrp1 ns *** ns ns * ns * 120 ns *** 100 100

80

50 60

Relative expression (%) 40 Relative expression (%)

20 0 Cxcl10 Isg15 ClpX 0 Cxcl10 Rnf213 Irgm1 Mfn1 Drp1

C 24h 48h 24h 48h 0 50 100 200 50 100 200 0 50 100 200 50 100 200 CAM (uM) ZBP1

IFIT3 mt-COI

CLPP

αTubulin

WT CLPP-KO

49