Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press DUSP11-mediated control of 5′-triphosphate RNA regulates RIG-I sensitivity Joon H. Choi, James M. Burke,1 Kayla H. Szymanik, Upasana Nepal, Anna Battenhouse, Justin T. Lau, Aaron Stark, Victor Lam,2 and Christopher S. Sullivan Department of Molecular Biosciences, LaMontagne Center for Infectious Disease, The University of Texas at Austin, Austin Texas 78712, USA Deciphering the mechanisms that regulate the sensitivity of pathogen recognition receptors is imperative to un- derstanding infection and inflammation. Here we demonstrate that the RNA triphosphatase dual-specificity phos- phatase 11 (DUSP11) acts on both host and virus-derived 5′-triphosphate RNAs rendering them less active in inducing a RIG-I-mediated immune response. Reducing DUSP11 levels alters host triphosphate RNA packaged in extracellular vesicles and induces enhanced RIG-I activation in cells exposed to extracellular vesicles. Virus in- fection of cells lacking DUSP11 results in a higher proportion of triphosphorylated viral transcripts and attenuated virus replication, which is rescued by reducing RIG-I expression. Consistent with the activity of DUSP11 in the cellular RIG-I response, mice lacking DUSP11 display lower viral loads, greater sensitivity to triphosphorylated RNA, and a signature of enhanced interferon activity in select tissues. Our results reveal the importance of con- trolling 5′-triphosphate RNA levels to prevent aberrant RIG-I signaling and demonstrate DUSP11 as a key effector of this mechanism. [Keywords: DUSP11; RIG-I signaling; inflammation; innate immunity; noncoding RNA; tumor–stromal interaction; virus infection] Supplemental material is available for this article. Received June 18, 2020; revised version accepted September 28, 2020. Life is a great balancing act with overlapping and unique lar, many host transcripts generated by RNA polymerase molecular controls required to maintain homeostasis III (RNAP III) lack a 5′ capping mechanism (Dieci et al. (Kotas and Medzhitov 2015; tenOever 2016). Host detec- 2013). A series of recent studies indicate that some of tion of pathogens must be properly tuned to promote rapid these endogenous RNAP III transcripts can function as and effective responses when an actual pathogen is detect- damage-associated molecular patterns (DAMPs), which ed, but must not be so sensitive as to trigger an inflamma- are recognized by RIG-I (Boelens et al. 2014; Nabet et al. tory response when no pathogen is present (Medzhitov 2017; Chiang et al. 2018; Zhao et al. 2018). To avoid auto- and Janeway 2002; Roers et al. 2016). RIG-I is a pattern rec- inflammatory pathology, the RIG-I response must there- ognition receptor (PRR) that triggers the antiviral cyto- fore be carefully regulated. Indeed, several upstream and kine type I interferon (IFN) response (Yoneyama et al. downstream protein modulators of RIG-I signaling have 2004; Kato et al. 2005; Gack et al. 2007). Major patho- already been identified (Chiang et al. 2014). However, gen-associated molecular patterns (PAMPs) that activate host mechanisms that directly control the 5′-triphosphate RIG-I are structured or double-stranded RNAs that pos- proinflammatory capacity of PAMP or DAMP transcripts sess a 5′-triphosphate moiety, a hallmark of viral RNA-de- are not well understood. pendent RNA polymerase (RdRP) transcripts that are DUSP11 is an RNA triphosphatase that we and others expressed during the course of infection (Hornung et al. have previously demonstrated to be active on the 5′ ends 2006; Pichlmair et al. 2006). However, host transcripts, of structured host and viral transcripts (Deshpande et al. at least initially, also possess a 5′-triphosphate. In particu- 1999; Burke et al. 2016; Burke and Sullivan 2017; Kincaid et al. 2018; Vabret et al. 2019). The 5′-triphosphates of hep- atitis C virus (HCV) RNAs are converted to monophos- Present addresses: 1Department of Biochemistry, University of Colorado, Boulder, CO 80309, USA; 2Department of Pharmaceutical Chemistry, phates rendering them susceptible to exonuclease XRN- University of California at San Francisco, San Francisco, CA 94158, USA. mediated attack (Amador-Cañizares et al. 2018; Kincaid Corresponding author: [email protected] Article published online ahead of print. Article and publication date are online at http://www.genesdev.org/cgi/doi/10.1101/gad.340604.120. Free- © 2020 Choi et al. This article, published in Genes & Development,is ly available online through the Genes & Development Open Access available under a Creative Commons License (Attribution 4.0 Internation- option. al), as described at http://creativecommons.org/licenses/by/4.0/. GENES & DEVELOPMENT 34:1–16 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/20; www.genesdev.org 1 Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Choi et al. et al. 2018). A different outcome arises when triphos- (Supplemental Fig. S1A,B). Combined with our previous phorylated RNAP III viral precursor microRNAs (miR- work (Burke et al. 2016), these data do not support a back- NAs) are processed by DUSP11. These viral miRNAs ground of strong antiviral response in cultured cells with require DUSP11 for full silencing activity via stable load- reduced DUSP11, but are consistent with a subtle en- ing of 5′-monophosphate miRNAs into the RNA-induced hanced predisposition of these cells to undergo RIG-I silencing complex (RISC) (Burke et al. 2016). Additionally, signaling. the 5′-triphosphate status of several host RNAP III non- We next studied the effects of DUSP11 on exogenously coding RNA transcripts including vault RNAs (vtRNAs), introduced in vitro transcribed RNAs (Fig. 1A). We used Alu SINE RNAs, Y RNAs, and RMRP RNA are also mod- the 5′ untranslated region (UTR) of the HCV RNA, as its ulated by DUSP11 (Burke et al. 2016; Zhao et al. 2018; expression is immunostimulatory (Saito et al. 2007). Addi- Vabret et al. 2019). The ability of DUSP11 to convert the tionally, we and others had previously demonstrated tri- 5′ end of diverse structured and partially double-stranded phosphatase activity of DUSP11 on HCV RNAs during host and viral transcripts into monophosphates opens up infection (Amador-Cañizares et al. 2018; Kincaid et al. the possibility that DUSP11 can alter the immunostimu- 2018). Consistent with RIG-I induction, cationic-lipid-me- latory activity of endogenous and exogenous triphos- diated transfection of HCV 5′-triphosphate RNA (referred phorylated transcripts (Burke and Sullivan 2017). to here as “5′-ppp-RNA”) into the A549 adenocarcinoma Although DUSP11 can convert structured triphosphate human lung epithelial cell line resulted in the induction RNAs into monophosphates (Burke and Sullivan 2017), of both IFNB1 cytokine and ISG15 mRNA transcripts and DUSP11 has been implicated as being reduced during (Fig. 1B). Under conditions where DUSP11 protein levels infection by some viruses (Zhao et al. 2018; Vabret et al. are abolished due to CRISPR-mediated knockout, trans- 2019), the physiological role of DUSP11 in the innate im- fection of 5′-ppp-RNA results in enhanced induction of mune response is poorly understood. Here we test the hy- IFNB1 and ISG15 mRNA (Fig. 1B). Consistent with RIG- pothesis that DUSP11 is a modulator of the RNA-induced I being an interferon-stimulated gene (Matsumiya and innate immune response. Our data demonstrate that tun- Stafforini 2010), RIG-I protein levels were also further in- ing the 5′-triphosphate levels of both endogenous host and duced in cells with reduced DUSP11 (Supplemental Fig. exogenous viral transcripts is a regulator of RIG-I signal- S1C). To determine whether this regulation was depen- ing and that DUSP11 is a key mediator of 5′-triphosphate dent on the catalytic triphosphatase activity of DUSP11, balance. we used A549 DUSP11 knockout cells stably reconstitut- ed with a control empty vector (vector), wild-type DUSP11 (D11), or the catalytically inactive mutant DUSP11 Results (Cys152Ser, D11-CM) (Fig. 1A; Yuan et al. 1998; Desh- pande et al. 1999; Burke et al. 2016). Upon transfection of DUSP11 modulates RIG-I signaling sensitivity ′ 5 -ppp-RNA, expression of wild-type DUSP11, but not to liposomal 5′-triphosphate RNAs the empty vector or catalytic mutant DUSP11, reduced Among the host defense machineries that sense invading ISG15 mRNA induction (Fig. 1C). Consistent with pathogens, RIG-I is the key PRR that recognizes the 5′-tri- DUSP11 directly reducing the visibility of RNAs to RIG- phosphate or diphosphate moiety of double-stranded I, pretreatment of 5′-ppp-RNA with calf intestinal phos- RNA transcripts and in turn activates the antiviral im- phatase (CIP) or the purified catalytic core of DUSP11 mune defense signaling pathway (Hornung et al. 2006). repeatably resulted in reduced induction of IFNB1 and Previous work demonstrated that DUSP11 sequentially ISG15 transcripts (Fig. 1D). To test the requirement of removes the γ and β phosphates of triphosphorylated RIG-I for ISG induction, we used siRNA-mediated knock- structured host and viral transcripts leaving 5′-monophos- down of RIG-I. Cells with reduced RIG-I displayed signifi- phates (Deshpande et al. 1999; Burke et al. 2016; Kincaid cantly reduced induction of ISG15 transcripts (Fig. 1E). et al. 2018). This led us to hypothesize that DUSP11 can Together, these results indicate that DUSP11 modulates modulate the immunogenicity of triphosphorylated tran- RIG-I signaling sensitivity via its catalytic phosphatase ac- scripts (Burke and Sullivan 2017) and alter the sensitivity tivity on 5′-triphosphate PAMP RNA. of these transcripts to detection by RIG-I. To begin to test this hypothesis, we first looked for evidence of enhanced DUSP11 alleviates the interferon response mediated interferon-mediated gene expression. Although our previ- by tumor–fibroblast cell interaction ous RNA-seq experiments (Burke et al. 2016) did not dis- play a strong signal of interferon response in HEK293T We next wanted to determine whether DUSP11 alters cel- or A549 cells lacking DUSP11, we re-examined this possi- lular susceptibility to endogenous RIG-I DAMP RNAs.