The antiviral action of the RIG-I induced pathway of apoptosis (RIPA) is enhanced by its ability to degrade Otulin, which deubiquitinates IRF3

Mammalian innate immune response to virus infection is meditated by many cell-intrinsic pathways. RNA viruses, such as Sendai virus, which replicate in the cytoplasm, trigger the RIG-I-like

receptor pathway, which activates the transcription factor, IRF3. Activated IRF3 translocates to the nucleus and induces transcription of the genes which encode interferons, the major

antiviral cytokines. Interestingly, IRF3 activates another interferon-independent antiviral pathway, called RIG-I induced pathway of apoptosis (RIPA). For activating RIPA, IRF3 translocates

from the cytoplasm to mitochondria. RIPA requires linear polyubiquitination of IRF3 by the enzyme complex, LUBAC; ubiquitinated IRF3 binds to Bax and translocates it to mitochondria causing

the release of Cytochrome C, activation of caspases and apoptosis of the infected cell. Here, we report that Otulin, the deubiquitinase that removes linear polyubiquitin chains, inhibits

RIPA by deubiquitinating IRF3. Ablation of Otulin expression enhanced RIPA and its overexpression inhibited RIPA. In virus-infected cells, to overcome Otulin-mediated inhibition, RIPA

actively degrades Otulin. This degradation required sequential actions of RIPA-activated Caspase 3 and proteasomes. Caspase 3 cleaved Otulin at D31; the D31A mutant was not cleaved at all.

The caspase-cleaved fragment was totally degraded by proteasomes, which was preceded by its K48-linked ubiquitination. Mass spectrometric analysis of Otulin identified K64 and K197 as the

ubiquitinated residues. Otulin interacted with LUBAC after virus infection and the E3-ubiquitin ligase, HOIP, a component of LUBAC, ubiquitinated Otulin to trigger its proteasome-mediated

degradation. To assess the impact of Otulin degradation on RIPA-mediated antiviral action, we expressed, in Otulin-ablated cells, a non-degradable mutant of Otulin, in which D31, K64 and

K197 had been mutated. The cells expressing the Otulin mutant were less susceptible to virus-induced apoptosis, because RIPA was less active, and consequently virus replication was more

robust. Thus, our study has revealed an important positive feedback loop of RIPA.

Viral infection in host cells triggers antiviral response after recognition of the pathogen-associated molecular patterns (PAMPs) by different host-pathogen recognition receptors (PRRs),

such as RIG-I-like receptors (RLRs), cGAS/STING, and Toll-like receptors (TLRs) [1,2,3]. RLRs are cytoplasmic sensors of viral RNA having a DExD/H box helicase domain and a carboxyl-terminal

CARD domain [4, 5]. Many RNA viruses, such as Sendai virus, Dengue virus, and Influenza virus are recognized by the RLR pathway [6, 7]. After recognition of viral RNA, RLR signaling

activates transcription factors, including IRF3 and NF-ΚB, which then induce the synthesis of different interferons (IFN) and the IFN-stimulated genes (ISGs) to inhibit viral infection [8,

9]. IRF3, the major transcription factor that induces IFN, is activated through its phosphorylation by RLR signaling. Activation of RIG-I by viral RNA leads to recruitment of RIG-I to MAVS

to activate the cytoplasmic kinase TBK1. Activated TBK1 phosphorylates IRF3 at specific serine residues. Phosphorylated IRF3 dimerizes and translocates from the cytoplasm to the nucleus and

binds to IFN-stimulated response elements (ISRE) of target genes to induce type-I IFN production [10, 11].

IRF3 can also trigger a non-transcriptional IFN-independent antiviral pathway, called RLR-induced IRF3 mediated Pathway of Apoptosis (RIPA) [12,13,14]. RIPA controls viral replication by

triggering apoptosis of virus-infected cells and does not require IFN or ISGs [15]. RIPA activation requires several RLR-transcriptional pathway components, such as MAVS, TRAF3, and TBK1, in

addition to TRAF2 and TRAF6 [12, 16]. In RIPA, IRF3 is recruited by the ubiquitinating LUBAC complex, consisting of HOIP, HOIL, and Sharpin. HOIP, an E3 ligase, attaches linear

polyubiquitin chains on IRF3, which activates it to interact with the pro-apoptotic protein, Bax. The activated IRF3-Bax complex translocates to mitochondria leading to the release of

Cytochrome C in the cytoplasm [12, 13, 16]. Cytoplasmic Cytochrome C activates the apoptosome complex, composed of APAF-1, pro-caspase 9, pro-caspase 3 and an inhibitor of apoptotic

caspases, such as XIAP, which prevents pro-caspase 9 cleavage and activation of the apoptotic cascade [17, 18]. The apoptotic activity of RIPA has been shown to be temporally regulated. In

the early phase of infection, RIPA is blocked by XIAP. Moreover, viruses activate the PI3K/AKT pathway in the early phase of infection to stabilize XIAP, thereby preventing early cell death.

However, during the late phase of infection, XIAP is degraded to relieve the inhibition of RIPA. If the PI3K/AKT axis is inhibited, RIPA causes early apoptosis of virus-infected cells [19].

Using cells and mice expressing an IRF3 mutant, which cannot be activated as a transcription factor, it was firmly established that RIPA mediates significant antiviral effects [15, 16, 20].

RIPA has been also implicated in metabolic liver diseases, such as alcoholic hepatitis (AH) and alcohol-related liver disease (ALD), where IRF3 transcription-independent functions have been

shown to differentially contribute to ethanol-induced liver injury and high-fat diet (HFD)-induced liver injury models. In Gao-binge liver injury model, RIPA- mediated apoptosis of

restorative Ly6Clow monocytes shifts the immune environment to more pro-inflammatory state thereby exacerbating the alcoholic hepatitis [21]. In contrast in HFD mediated liver injury model,

non-transcriptional IRF3 mice (IRF3S1/S1) were protected [22]. The differences have been shown to be associated with increased or decreased ubiquitination of IRF3.

RLR-signaling requires ubiquitination of RIG-I and other signaling proteins to promote activation of IRF3 as a transcription factor [23, 24]. This process is regulated by specific

deubiquitinases (DUB), such as CYLD and USP15, which remove the polyubiquitin chains from the signaling proteins [25,26,27]. We inquired whether IRF3 activation in RIPA, through its linear

polyubiquitination, was similarly regulated by a DUB. Otulin is a DUB that can remove linear polyubiquitin chains from signaling proteins, such as STAT1 and Nemo [28,29,30,31,32]. We report

here that Otulin inhibits RIPA, by deubiquitinating IRF3. However, to evade this negative regulation, enzymes activated in RIPA actively degrade Otulin, thereby creating a positive feed-back

loop to RIPA’s antiviral action.

In the RIPA pathway, linear polyubiquitin chains are added to IRF3 by the LUBAC complex and we were interested to learn whether RIPA can be negatively regulated by the removal of

polyubiquitin chains from IRF3 by a deubiquitinase. Otulin is a deubiquitinase that can cleave linear polyubiquitin chains from its substrates and we tested its ability to reduce IRF3 linear

polyubiquitination (polyUb). Sendai virus (SeV) infection, which activates RIPA, was performed in human HT1080 cells with or without ectopic expression of Otulin, followed by

immunoprecipitation of IRF3 from the extracts of these cells. We observed that in Otulin over-expressing cells, IRF3 linear polyUb was decreased significantly, indicating that Otulin

inhibited IRF3 linear polyUb (Fig. 1A). Total linear ubiquitination of the input was also assessed, with and without SeV infection, as shown in the right panel of Fig. 1A. We observed minor

changes in total linear ubiquitination of the input as expected; however the corresponding lanes of IRF3 linear ubiquitination (Fig. 1A, left panel) showed significant changes indicating the

effect of Otulin on IRF3 to be a specific effect. To further confirm this observation, we generated Otulin knockdown (OT-KD) cells (Supplementary Fig. S1A). We observed that IRF3 linear

polyUb was significantly enhanced in OT-KD cells, compared to WT cells, thereby indicating a physiological role of Otulin in regulating IRF3 polyUb (Fig. 1B). We used high salt washes (1 M

NaCl) to detect IRF3 linear poly-ubiquitination in HT0180 WT and OT-KD cells during RIPA activation to rule out the contribution of bait-interacting proteins and we found similar results as

observed for Fig. 1B (data not shown). To demonstrate the dependence of IRF3 linear polyUb on the enzymatic activity of Otulin, OT-KD cells were reconstituted with either Otulin WT or the

enzymatically inactive Otulin C129A mutant. SeV infection of cells expressing Otulin C129A showed an IRF3 linear polyUb level similar to that in OT-KD cells, whereas in cells expressing WT

Otulin, IRF3 linear polyUb was much reduced (Fig. 1C). After confirming the role of Otulin on IRF3 linear ubiquitination, we performed a co-immunoprecipitation assay to investigate

Otulin-IRF3 interaction during RIPA activation. The result showed strong interaction of IRF3 with Otulin (Fig. 1D). Because RIPA activation induces apoptosis, we analyzed, as a measure of

apoptosis, cleaved PARP and cleaved Caspase 3 levels in HT1080 WT and OT-KD cells, post-SeV infection. We found that OT-KD cells had increased levels of cleaved PARP and cleaved Caspase 3,

indicating enhanced apoptosis (Fig. 1E). We also analyzed apoptotic cell death, in HT080 WT and OT-KD cells post-SeV infection, by Annexin-V staining. OT-KD cells showed more cell death than

HT080 WT (Supplementary Fig. S2E) whereas IFN production was similar in both cell lines (Supplementary Figs. S2F, S2G, and S2H). This enhanced apoptosis in OT-KD cells inhibited virus

replication, as

measured by SeV-P mRNA (Supplementary Fig. S1B) and protein expression (Fig. 1F). As expected, these cells produced less infectious virus (Fig. 1G). RIPA requires an interaction of IRF3 with

Bax and its translocation to mitochondria. We observed that IRF3 interaction with Bax and its mitochondrial localization were more pronounced in infected OT-KD cells, compared to WT cells

(Supplementary Fig. S1C, D). We also tested whether the anti-apoptotic effect of Otulin was dependent on IRF3. We observed that the absence of Otulin expression enhanced apoptosis and

inhibited viral replication in HT1080 WT cells, but not in HT1080sh.IRF3 cells, in which IRF3 expression had been knocked down (Supplementary Fig. S1E). The above results indicate that

Otulin specifically attenuates RIPA pathway to support viral replication.

A IRF3 linear ubiquitination was analyzed in HT1080 cell line transfected with either empty vector (lanes 1, 2, 3 and 4) or HA-Otulin (lane 5) followed by Sendai virus (SeV) infection (lanes

2, 4 and 5) for 24 h (MOI 10). Immunoprecipitation was performed with IRF3 antibody and immunoblotting was done with LUB9 antibody for detection of linear ubiquitination. Right panel shows

the total linear ubiquitination of the input. B IRF3 linear ubiquitination was assessed in HT1080 WT and HT1080 Otulin-knockdown (OT-KD) cells post-SeV infection, using LUB9 antibody. C

HT1080 Otulin knockdown cells (OT-KD) were reconstituted with either Otulin WT or enzymatically inactive mutant Otulin C129A and IRF3 linear ubiquitination was assessed by LUB9 antibody post

SeV infection. D Otulin-IRF3 interaction was assayed by their co-immunoprecipitation from extracts of HT1080 cells that had been infected with SeV for 1 h and 3 h. E HT1080 WT and HT1080

OT-KD cells were either mock or SeV infected and C-PARP and C-caspase 3 levels were measured for assessing apoptotic cell death at the indicated time points. F SeV-C protein levels were

assessed post-SeV infection in WT and OT-KD cells as a measure of viral protein synthesis at the indicated time points. G SeV yields were measured after infection of WT and OT-KD cells with

SeV for 24 h (MOI 10). Quantification of western blots and statistical analyses are provided in Supplementary Fig. 8 (mean ± SEM, n = 3; ns > 0.05; *P