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. Author manuscript; available in PMC: 2016 Aug 12.
Published in final edited form as: Cell Host Microbe. 2015 Aug 12;18(2):221–232. doi: 10.1016/j.chom.2015.07.007

RIP3 regulates autophagy and promotes Coxsackievirus B3 infection of intestinal epithelial cells

Katharine G Harris 1, Stefanie A Morosky 1, Coyne Drummond 1, Maulik Patel 2, Chonsaeng Kim 2, Donna B Stolz 4, Jeffrey M Bergelson 5, Sara Cherry 6, Carolyn B Coyne 1,*
PMCID: PMC4562276  NIHMSID: NIHMS715934  PMID: 26269957

Summary

Receptor Interacting Protein Kinase-3 (RIP3) is an essential kinase for necroptotic cell death signaling and has been implicated in antiviral cell death signaling upon DNA virus infection. Here, we performed high-throughout RNAi screening and identified RIP3 as a positive regulator of coxsackievirus B3 (CVB) replication in intestinal epithelial cells (IECs). RIP3 regulates autophagy, a process utilized by CVB for viral replication factory assembly, and depletion of RIP3 inhibits autophagic flux and leads to the accumulation of autophagosomes and amphisomes. Additionally, later in infection, RIP3 is cleaved by the CVB-encoded cysteine protease 3Cpro, which serves to abrogate RIP3-mediated necrotic signaling and induce a non-necrotic form of cell death. Taken together, our results show that temporal targeting of RIP3 allows CVB to benefit from its roles in regulating autophagy while inhibiting the induction of necroptotic cell death.

Graphical Abstract

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Introduction

Coxsackievirus B3 (CVB), a member of the enterovirus family, is associated with a variety of clinical outcomes that can range from mild febrile illness to more severe complications such as meningoencephalitis, myocarditis and dilated cardiomyopathy, or type I diabetes. CVB is transmitted via the fecal-oral route and encounters the polarized intestinal epithelial cells (IECs) lining the gastrointestinal tract early in infection. Despite serving as the primary cellular portal for CVB entry, very little is known regarding the specific molecular events that regulate CVB replication in and egress from the intestinal epithelium.

An important event in CVB pathogenesis is the induction of host cell death. CVB is a lytic virus and possesses few mechanisms for progeny release other than induction of cell death and subsequent destruction of the host cell membrane. The induction of cell death signaling by CVB in an infected cell must be precisely controlled as activating cell death prematurely or aberrantly could inhibit replication and/or induce inflammatory signaling. Whereas CVB induces apoptosis in non-polarized cells (Carthy et al., 1998), we have shown that CVB-infected polarized IECs undergo calpain-mediated necrosis, which is required for viral egress (Bozym et al., 2011). These results suggest that the cellular factors that facilitate and/or restrict CVB replication in polarized IECs may be unique to these specialized cells.

In addition to direct lysis of an infected cell, CVB may also egress via microvesicles that are associated with markers of autophagy (Robinson et al., 2014). Autophagy begins with the formation of an isolation membrane (which can be provided by an array of cellular organelles (Lamb et al., 2013)) to form the characteristic double-membrane vesicle called the autophagosome (AP). Once formed, APs can fuse with endosomes to form amphisomes (Berg et al., 1998), and APs or amphisomes can fuse with lysosomes to form autolysosomes, wherein the degradation of many AP-associated components (and any factors they may interact with) by lysosomal hydrolases occurs. Completion of this process and degradation of any autophagosomal cargo is referred to as autophagic flux (Klionsky et al., 2012). CVB replication is dependent on the induction of autophagy and the inhibition of this process both in vitro (Delorme-Axford et al., 2014; Wong et al., 2008) and in vivo (Alirezaei et al., 2012) greatly reduces viral replication.

In order to identify host cell factors that promote and/or restrict CVB replication, we previously performed genome-scale RNAi screening in polarized endothelial cells (Coyne et al., 2011). However, as this initial screening was conducted in polarized endothelial cells, it did not provide any information on the specific host cell factors involved in CVB replication in polarized IECs. In the current study, we conducted additional RNAi screening to identify factors required for CVB replication in IECs. Together, these screens provide an unbiased comparison of the gene products necessary for CVB infection of both epithelial and endothelial barriers. In the current study, we performed RNAi screening in Caco-2 IECs and identified receptor-interacting serine/threonine-protein kinase 3 (RIP3) as a gene product whose depletion restricted CVB replication. RIP3 is a nonreceptor serine/threonine kinase required for necroptotic cell death signaling downstream of tumor necrosis factor receptor (TNFR) (Cho et al., 2009; He et al., 2009; Zhang et al., 2009). RIP3 is activated via its phosphorylation upon recruitment to signaling complexes and subsequently phosphorylates the pseudokinase mixed lineage kinase domain-like protein (MLKL), which is required for necroptosis (de Almagro and Vucic, 2015). We show that RIP3 regulates CVB replication independently of its role in cell death signaling and instead identify a role for RIP3 in the regulation of autophagy. We show that RIP3 expression is restricted to many polarized IEC lines and that its RNAi-mediated silencing in these cells restricts an early post-entry event associated with CVB replication. Mechanistically, we show that IECs lacking RIP3 exhibit defects in autophagy and autophagic flux and are unable to survive nutrient deprivation. Furthermore, RIP3 interacts with p62/SQSTM1, an adaptor protein that links cargo destined for degradation to APs, is phosphorylated in response to serum starvation, and is then degraded after prolonged exposure to nutrient deprivation. Interestingly, we also show that at late stages of infection, the CVB virally-encoded cysteine protease 3Cpro proteolytically cleaves RIP3 to generate two RIP3 fragments, neither of which are capable of necrotic cell death signaling. Instead, the cleavage of RIP3 by CVB generates a C-terminal fragment that potently induces a non-necrotic form of cell death, suggesting that the virus targets RIP3 to induce a ‘switch’ between necrotic and non-necrotic forms of cell death. Thus, our current study not only identifies RIP3 as a host cell factor specifically required for CVB replication in polarized IECs, but also points to a direct role for RIP3 in the regulation of autophagy.

Results

RIP3 facilitates CVB infection in intestinal epithelial cells

In order to identify genes required for CVB infection of IECs, we performed a genome-scale RNAi screen in Caco-2 cells (schematic, Figure 1A). Our screening identified RIP3 as a gene product whose depletion significantly reduced CVB replication (robust z score of −2.08), which was confirmed in secondary follow up studies (Figures 1B, S1A). In addition, we confirmed that RNAi-mediated knockdown of RIP3 reduced CVB infection in HT29 cells, an independently derived IEC line (Figures 1C, S1B), and also found that CVB replication was restricted in HT29 cells stably expressing an shRNA targeting RIP3 (HT29shRIP3) (Figures 1D, 1E). In contrast, vesicular stomatitis virus (VSV) infection was significantly enhanced in HT29shRIP3 cells, indicating that RIP3 may be an enterovirus or CVB specific pro-viral factor (Figures 1D, 1E). Notably, treatment of cells with the RIP1 inhibitor necrostatin-1 did not restrict CVB replication in HT29 cells, suggesting that RIP3 may be acting independently of RIP1 to positively regulate CVB infection (Figure S1C).

Figure 1. RIP3 silencing restricts CVB replication.

Figure 1

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(A) Schematic depicting genome-scale RNAi screening in Caco-2 cells. (B) Caco-2 cells transfected with siRNA targeting RIP3 (RIP3si) or a control scrambled sequence (CONsi) were infected with CVB (5 PFU/cell) for ~8hrs and then immunostained for VP1 (green). DAPI-stained nuclei are shown in blue. Infection (%) is shown in white text at low right. Representative images from four independent experiments are shown. (C) HT29 cells transfected with siRNA targeting RIP3 or a control scrambled sequence were infected with CVB (0.5 PFU/cell) for 24hrs. Shown are viral RNA levels as assessed by RT-qPCR and averaged from three independent experiments. (D–E) WT HT29 or HT29shRIP3 cells infected with CVB (3 PFU/cell) or GFP-VSV (1 PFU/cell) for ~16hrs. Infected cells were analyzed for infection by either immunofluorescence microscopy to detect CVB VP1 or GFP-tagged VSV (D) or RT-qPCR to detect viral RNA (E). Inset in panel (E) shows immunoblotting for RIP1 (in green) and RIP3 (in red) in wild-type HT29 or HT29shRIP3 cells. Data shown are representative of at least 3 independent experiments. (F) The indicated cell lines were grown to confluence, then lysed and analyzed for RIP1 and RIP3 expression by immunoblotting. RIP1 is shown in red and RIP3 is shown in green. Lysates from rat cardiomyocyte HL-1 cells were used as a positive control. (G) HeLa-RIP3 Tet-Inducible cells with or without tetracycline were infected with CVB (0.5 PFU/cell) for 8hrs followed by immunofluorescence microscopy to detect CVB VP1. Shown is the percent infection normalized to untreated control cells. Data shown are an average of 3 independent experiments. Inset, HeLa-RIP3 Tet-Inducible cells with or without tetracycline were lysed and immunoblotted for RIP3 expression performed to confirm RIP3 protein expression upon tetracycline treatment. GAPDH (bottom) is included as a loading control. See also Figure S1.

Because RNAi-mediated knockdown of RIP3 restricted CVB infection in IECs, but its silencing had no effect on CVB infection in polarized endothelial cells by RNAi (Coyne et al., 2011), we next examined the expression of RIP3, as well as the related RIP family member RIP1, in a panel of IEC and non-IEC cell lines by immunoblotting. We found that every IEC cell line tested (Caco-2 (subclone BBE or ATCC), HCT116, HT-29, and T84) expressed RIP3, whereas a diverse panel of non-IEC lines (human brain microvascular endothelial cells (HBMEC), HEK293, HeLa, HT1080, and U2OS) did not (Figures 1F, S1D, S1E). In contrast, all cell lines expressed RIP1 (Figures 1F, S1D). Because we found that silencing of RIP3 in IECs restricted CVB replication, we next determined whether overexpression of RIP3 in HeLa cells, which do not endogenously express RIP3 (Figure 1F), would alter CVB replication. We found that induction of RIP3 in HeLa cells stably expressing a tetracycline inducible RIP3 expression vector had little effect on CVB replication (Figure 1G), although we did observe a significant reduction in RIP3 expression levels upon CVB infection (Figure 1G).

Taken together, these data imply that RIP3 is a specific regulator of CVB replication in polarized IECs.

Silencing of RIP3 reduces CVB replication prior to viral egress

We previously showed that polarized IECs undergo necrotic cell death in response to CVB infection whereas non-IECs undergo apoptosis and that this necrotic cell death is required in IECs for viral egress (Bozym et al., 2011). Given that RIP3 expression has been associated with necrotic cell death in response to TNFα treatment, and was required for CVB replication in IECs, we next determined whether the decrease in CVB replication observed by RNAi silencing occurred early in the virus life cycle or could be attributed to deficiencies in viral egress. To do this, we performed a time course of CVB infection and assessed the levels of vRNA in HT29 cells transfected with a control or RIP3 targeting siRNA. Surprisingly, we found that RNAi-mediated knockdown of RIP3 led to dramatic reductions of vRNA by as early as 4 hours post-infection (p.i.) (Figures 2A, S2A) suggesting that the reduction in vRNA could not be directly attributed to late stages of the viral life cycle (such as egress, which occurs between 8 and 12 hours p.i., Figure S2B). In support of a necrosis-independent role for RIP3, we found that silencing of MLKL, which is required for necroptosis downstream of RIP3 (Cai et al., 2014; Chen et al., 2014; Sun et al., 2012), had no effect on CVB vRNA levels at 4 hours post-infection, although there was a slight decrease in vRNA levels at late stages (24 hours p.i.) of infection (Figures S2C, S2D).

Figure 2. RIP3 silencing restricts CVB replication prior to viral egress but is not required for viral entry.

Figure 2

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(A) HT29 cells transfected with siRNA targeting RIP3 or a control scrambled sequence were infected with CVB (0.5 PFU/cell) for 2–12hrs and viral RNA levels determined by RT-qPCR at the indicated times. Data shown are representative of 3 independent experiments (4–12hrs p.i.) or average of 3 independent experiments (2hrs p.i.) (B) Schematic depicting NR-CVB replication assay. (C) HT29 cells transfected with siRNAs targeting RIP3 or a control scrambled sequence were infected with NR-CVB (10 PFU/cell) as described in (B) and infection determined by immunofluorescence microscopy to detect VP1. Data shown are displayed as percent infection and are representative of 3 independent experiments. See also Figure S2.

RIP3 is not required for CVB entry into IECs

Because we found that silencing of RIP3 expression impacted CVB vRNA levels early in infection (between 2–4 hours p.i.), we next directly tested whether RIP3 plays a role in CVB entry into IECs. In order to address this question, we performed a neutral red (NR) CVB infection assay. When propagated in the presence of NR, CVB becomes photosensitive due to damage of the viral RNA induced by photon emission from NR upon illumination, thus inhibiting further replication. However, once NR-CVB has entered the cell and uncoated, the virus becomes light insensitive due to dilution of the neutral red dye in the cytoplasmic compartment (schematic, Figure 2B) (Crowther and Melnick, 1961). HT29 cells transfected with a control or RIP3 siRNA were infected with NR-CVB under dark and illuminated (light) conditions. As expected, we found that both control siRNA- and RIP3 siRNA-transfected-cells exposed to light at 0 hours p.i. (prior to entry and uncoating), were extremely photosensitive and exhibited very low levels of CVB replication (Figure 2C). In addition, we found that cells transfected with RIP3 siRNA and infected in the dark exhibited a reduction in CVB vRNA levels, confirming the role of RIP3 as a positive regulator of CVB infection (Figures 2C, S2E). Importantly, we found that light exposure at 2 hours p.i. (allowing sufficient time for entry and uncoating to occur) did not further reduce CVB infection levels in cells transfected with siRIP3 compared to cells infected in the dark (Figures 2C, S2E), indicating that entry of CVB in IECs was not delayed or restricted by RNAi-mediated RIP3 knockdown.

RIP3 regulates autophagy in IECs

As RIP3 was not required for CVB entry into IECs, we reasoned that RIP3 must be required for a viral and/or cellular event that takes place between 2 and 4 hours p.i., as we found similar levels of CVB vRNA at 2 hours p.i., but alterations in vRNA by 4h hours p.i. (Figure 2A). CVB, like many positive-stranded RNA viruses, assembles viral replication factories on scavenged host-cell membranes. These host-cell membranes can be derived from diverse locations within the cell, but there is evidence to support a prominent role for autophagy in the generation of CVB viral replication factories (Suhy et al., 2000). Given that our data suggested a role for RIP3 in an early post-entry event associated with CVB replication, we examined whether RNAi-mediated knockdown of RIP3 impacted nutrient depletion-induced autophagy and/or interfered with CVB-induced autophagy. First, we investigated the impact of RIP3 silencing in IECs under basal states and in cells subjected to serum starvation, which induces the canonical form of autophagy (macroautophagy), by transmission electron microscopy (TEM). For TEM studies, we utilized a cell line stably expressing an shRNA targeting RIP3 in order to ensure that a high percentage of cells exhibited RIP3 silencing. We observed a pronounced loss of cell viability in HT29shRIP3 cells subjected to serum starvation compared to wild-type controls, suggesting that these cells are unable to survive periods of starvation due to alterations in autophagy (Figures 3A, S3A, S3B). In addition, we found that knockdown of RIP3 increased the basal levels of p62 as assessed by immunoblotting (Figures 3B, 3C) and led to an accumulation of large p62-positive punctae as assessed by immunofluorescence microscopy (Figures 3D, 3E, S3C), without a change in the total number of punctae (Figure S3D). Additionally, we noted an accumulation of amphisome-like compartments in untreated HT29shRIP3 cells when compared to wild-type HT29 cells by TEM that resembled those observed in wild-type HT29 cells treated with Bafilomycin-A1 (BafA1) (Figures 3F, 3G, S3E, S3F), which prevents the maturation of autolysosomes and thereby the degradation of any contents therein. These data suggest that RIP3 may regulate an aspect of autophagic flux that occurs following initiation/induction. In support of this, silencing of RIP3 expression did not block conversion of LC3B-I to LC3B-II, a hallmark of autophagy initiation, upon serum starvation (Figure S3G).

Figure 3. RIP3 is required for autophagic flux in IECs.

Figure 3

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(A) Transmission electron micrographs of WT HT29 or HT29shRIP3 cells grown to confluence in 12 well plates were subjected to serum starvation (SS) in Hank’s Buffered Saline Solution (HBSS) for 9hrs. Black square denotes zoomed image shown below. * indicates nuclei of nonviable cells. (B–C) HT29 cells were transfected with siRNAs targeting RIP3 or a control scrambled sequence and immunoblotted for p62 (top) and RIP3 (middle). GAPDH (bottom) is included as a loading control. Shown are two replicates immunoblotted in parallel, non-adjacent lanes from the same gel are separated with a black bar (B). Densitometry was performed and averaged from three independent experiments and is shown as the fold change in p62 expression normalized to control siRNA (C). (D–E) HT29 cells transfected with siRNA targeting RIP3 or a control scrambled sequence were analyzed by immunofluorescence microscopy for p62. Representative images are shown (D) and punctae size was quantified from at least 28 individual cells per condition (E). (F–G) Transmission electron micrographs of WT HT29 or HT29shRIP3 cells are shown, black arrows denote amphisomes, hatched box denotes zoomed image shown in inset (F). Number of amphisomes per cell under mock or Bafilomycin A1-treated conditions for 5hrs were quantified (G). (H) HT29 cells transfected with siRNAs targeting RIP3 or a control scrambled sequence were infected with CVB for 7hrs. 2hrs p.i. cells were mock- or Bafilomycin A1-treated. Following infection (with or without BafilomycinA1 treatment), cells were lysed and immunoblotted for LC3B (top) and RIP3 (middle). GAPDH (bottom) is included as a loading control. Densitometry was performed to obtain a ratio of LC3B-II/LC3B-I and is shown below. See also Figure S3.

Next, we determined whether loss of RIP3 impacted CVB-induced autophagy. We found that RIP3 knockdown in IECs did not prevent conversion of LC3B-I to LC3B-II in response to CVB infection (Figure 3H). In contrast, LC3B-II levels were enhanced upon RIP3 knockdown, suggesting a defect in post-initiation autophagic flux in the absence of RIP3 (Figure 3H). In support of this, we also observed the accumulation of LC3B during the course of CVB infection in HT29shRIP3 cells when compared to WT HT29 cells (Figure S3H). Collectively, these data support a direct role for RIP3 in the regulation of a post-initiation step of autophagic flux in IECs and suggest that the decrease in CVB replication in IECs lacking RIP3 may be related to alterations in this pathway.

RIP3 is phosphorylated in response to autophagy induction and interacts with p62

The activity of RIP3 is tightly controlled by its phosphorylation (Cho et al., 2009; Sun et al., 2012; Wu et al., 2014), which can be observed through a size shift by immunoblotting. We found that RIP3 became phosphorylated when cells were subjected to serum starvation for 4 hours in both mock- and BafA1-treated conditions (Figure 4A), supporting a role for its activation during the induction of autophagy.

Figure 4. RIP3 associates with p62.

Figure 4

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(A) HT29 cells grown to confluence were subjected to serum starvation (SS) in HBSS for 4hrs and were mock- or Bafilomycin A1-treated. Cells were then lysed and subjected to immunoblotting for RIP3 (top panel). GAPDH (bottom) is included as a loading control. Grey arrow indicates phosphorylated RIP3 as detected by shift in electrophoretic mobility. (B–C) HT29 cells in nutrient rich (Ctrl) or depleted (SS) conditions for 12hrs were immunoblotted for RIP3. Control lysates are also immunoblotted in panel 3B (B). Densitometry was performed and is representative of eight independent experiments (C). (D) HT29 cells grown to confluence were subjected to serum starvation (SS) in HBSS for 5hrs, lysed and subjected to immunoprecipitation for p62, and then immunoblotted for RIP3 (top). In parallel, whole cell lysates were immunoblotted for RIP3 (middle) and GAPDH (bottom) to control for protein loading.

LC3B-associated proteins, such as p62, can bind to cargo proteins that become internalized into APs and are degraded by autophagic flux (Kirkin et al., 2009; Lippai and Low, 2014). Surprisingly, we found that similar to p62, RIP3 protein levels were diminished upon long periods of serum starvation of IECs (Figures 4B, 4C), suggesting that RIP3 may be incorporated into APs through a physical association with a component of the autophagic pathway. Indeed, we found that endogenous RIP3 co-immunoprecipitated with endogenous p62 in HT29 IECs under resting conditions and when subjected to serum starvation for 5 hours (Figure 4D). These findings are consistent with a recent publication that found an association between RIP3 and p62 upon overexpression of these proteins in 293T cells (Matsuzawa et al., 2015). Taken together, these data suggest that RIP3 is activated in response to the induction of autophagy and is required for autophagy in IECs, which may be facilitated by its association with p62.

The CVB-encoded cysteine protease 3Cpro cleaves RIP3

CVB is known to extensively interact with and antagonize host cell pathways through various mechanisms, including via the activity of two virally-encoded proteases, 2Apro and 3Cpro (Harris and Coyne, 2013, 2014). Although we found that RIP3 was involved in the facilitation of CVB replication in IECs by promoting autophagy, RIP3 is also a key component in the pro-inflammatory necrotic cell death pathway, which may be detrimental to CVB replication. Additionally, we observed a reduction in RIP3 expression levels upon CVB infection in HeLa cells overexpressing RIP3 (Figure 1G). Therefore, we determined whether the loss of RIP3 expression in response to CVB infection was a direct result of CVB-mediated proteolytic cleavage. We analyzed the primary protein sequence of RIP3 for the presence of consensus cleavage sites for 2Apro or 3Cpro (Blom et al., 1996) and found that there were two possible cleavage sites for 3Cpro located at Q134 and Q430 of RIP3 (schematic, Figure 5A). Indeed, CVB infection of 293T cells ectopically expressing RIP3 resulted in cleavage of RIP3 as detected by immunoblotting (Figure 5B), which occurred at ~8–10hrs p.i. (Figure S4A), well after the establishment of CVB replication organelles, which occurs >5 hrs p.i. in Caco-2 cells (Figure S4B). Cleavage of RIP3 in 293T cells was also observed upon ectopic expression of 3Cpro, but not when a catalytically inactive 3Cpro mutant (3Cpro C147A) was expressed (Figure 5C), implicating 3Cpro in the cleavage of RIP3 during CVB infection. By mutating the consensus 3Cpro cleavage sites of RIP3, we found that mutation of residue Q430 resulted in complete blockage of CVB- and 3Cpro expression-mediated RIP3 cleavage (Figures 5D, 5B), indicating that 3Cpro-mediated cleavage occurs exclusively after RIP3 Q430.

Figure 5. The CVB virally-encoded 3C protease cleaves RIP3 after residue Q430.

Figure 5

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(A) Schematic depicting RIP3 domains, consensus 3Cpro cleavage sites (in red), and fragments generated by CVB infection. (B) 293T cells ectopically expressing Flag-tagged WT or Q430A RIP3 were infected with CVB (2 PFU/cell) for 9hrs followed by immunoblotting for RIP3 (top), VP1 (middle), and GAPDH (bottom). (C) 293T cells ectopically expressing Flag-tagged RIP3 and Myc-tagged WT or C147A 3Cpro were lysed and lysates were immunoblotted for Flag(top), Myc (middle), and GAPDH (bottom). (D) 293T cells ectopically expressing Flag-tagged WT, Q430A, Q134A, or Q134A/Q430A RIP3 and GFP-tagged WT or C147A 3Cpro were lysed and lysates were immunoblotted for Flag (top), GFP (middle), and GAPDH (bottom). Grey arrow indicates RIP3 cleavage fragment (B–D). (E) Confocal micrographs of U2OS cells transiently transfected with GFP-tagged RIP3, RIP3NT, or RIP3CT. See also Figure S4.

Interestingly, 3Cpro-mediated cleavage of RIP3 after residue Q430 cleaves RIP3 in close proximity to its RHIM domain, a protein-protein interaction motif necessary for recruitment to cell death signaling complexes for the induction of necrosis (schematic, Figure 5A). Given this, we investigated the function of the CVB-induced cleavage fragments of RIP3 that contain either the kinase domain (RIP3NT) or the RHIM domain (RIP3CT). First, we assessed the localization of these fragments in U2OS cells, which do not express endogenous RIP3 (Figure 1F). Upon ectopic expression, we found that RIP3NT localized diffusely throughout the cytoplasm, whereas RIP3CT localized either to punctate structures that resembled those observed upon expression of full length RIP3 or, less frequently, to the nucleus (Figure 5E).

3Cpro-mediated cleavage fragments are incapable of inducing necrotic cell death

The formation of the RIP1-RIP3 complex (termed the ‘necrosome’) is mediated by the RHIM domains present in both molecules and is required for the induction of necroptosis downstream of TNFα (Moriwaki and Chan, 2013). Consistent with the RHIM-dependent role of RIP3 in the formation of RIP1/RIP3 necrosomes, we found that RIP3CT co-immunoprecipitated with RIP1, whereas RIP3NT did not (Figure 6A). Given the role of RIP3 in necrotic signaling, and the ability of RIP3CT to interact with RIP1, we next assessed the ability of either RIP3NT or RIP3CT to mediate cell death signaling. We found that ectopic expression of full-length RIP3 and RIP3CT, but not RIP3NT, led to the enhanced uptake of propidium iodide (PI) uptake as measured by flow cytometry, consistent with an induction of cell death (Figures 6B, 6C). In addition, cells transfected with full-length RIP3 and RIP3CT exhibited classic signs of cell death and distress, including cell rounding and disruption of the cell monolayer (Figure 6D). However, although both full-length RIP3 and RIP3CT induced pronounced PI uptake, only full-length RIP3 was capable of inducing necrosis as determined by the nuclear release of HMGB1 by immunostaining and ELISA (Figures 6E, 6F), despite its localization into punctate structures and co-immunoprecipitation with RIP1. Similarly, RIP3NT expression had no effect on HMGB1 nuclear release (Figures 6E, 6F). Thus, although RIP3CT is incapable of inducing necrotic cell death, these results suggest that this fragment potently induces a non-necrotic form of cell death.

Figure 6. 3Cpro-mediated cleavage of RIP3 alters host cell signaling pathways.

Figure 6

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(A) 293T cells transfected with vector or GFP-tagged RIP3, RIP3NT, or RIP3CT and HA-tagged RIP1 were lysed, subjected to immunoprecipitation for GFP, and immunoblotted for RIP1 (top) or GFP (middle). In parallel, whole cell lysates were immunoblotted for RIP1 to control for protein loading (bottom). (B–C) 293T cells transfected with GFP-tagged RIP3, RIP3NT, or RIP3CT were analyzed for propidium iodide (PI) uptake by flow cytometry. Data shown are representative of 3 experiments. (D) Differential interference contrast (DIC) and wide-field fluorescence images were obtained from 293T cells transfected with GFP-tagged RIP3, RIP3NT, or RIP3CT. (E) HeLa cells transfected with GFP-tagged RIP3, RIP3NT, or RIP3CT were fixed, permeabilized, and HMGB1 localization was determined by immunofluorescence microscopy. White arrows indicate nuclei of interest. (F) Supernatants from HeLa cells transfected with GFP-tagged RIP3, RIP3NT, or RIP3CT were analyzed for HMGB1 content by ELISA 48 hours post-transfection. Results shown are average of 3 independent experiments. See also Figure S5.

Given that 3Cpro-mediated cleavage of RIP3 might also alter its role in autophagy in a manner that may impact CVB replication,, we examined the ability of RIP3NT or RIP3CT to induce autophagy. Indeed, we found that ectopic expression of RIP3 or RIP3CT, but not RIP3NT, in U2OS cells, which do not express endogenous RIP3, led to an accumulation of LC3B+ punctae (Figure S5A). However, as we previously found that ectopic expression of RIP3 or RIP3CT led to cell death, the resultant autophagy may be due to cross-talk between cell death and autophagic pathways, not due to a direct role of RIP3 or RIP3CT in autophagy. Additionally, we found that RIP3CT but not RIP3NT remains associated with p62/SQSTM1, as assessed by co-immunoprecipitation studies (Figure S5B).

Taken together, these data suggest that the cleavage of RIP3 by 3Cpro directly alters the ability of RIP3 to induce necrotic cell death signaling while promoting an alternative non-necrotic cell death pathway. Additionally, RIP3CT, but not RIP3NT, may remain associated with p62/SQSTM1 and continue to promote autophagy. This suggests that CVB actively manipulates the balance of cell death and cell survival signaling pathways in infected host cells through the cleavage of RIP3.

Discussion

Here we show that RIP3 positively regulates CVB infection. Our data suggest that RIP3 contributes to the formation of CVB replication organelles via its regulation of autophagy. Strikingly, we also found that once CVB establishes its replication and produces significant levels of virally-encoded 3Cpro, this protease directly targets RIP3 for proteolytic cleavage. The CVB-mediated cleavage of RIP3 dampens its ability to promote necrotic cell death, but promotes a non-necrotic form of cell death. Thus, CVB manipulates the function of RIP3 to enhance its own replication at both early and late stages of the viral life cycle (schematic, Figure 7).

Figure 7.

Figure 7

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Schematic depicting role of RIP3 during CVB infection. Approximately 2–4hrs post-infection, RIP3 contributes to the establishment of viral replication factories (green dashed arrows) through its role as a positive regulator of autophagy. Later in infection (>4hrs p.i., black dashed box), large amounts of the viral protease 3Cpro are produced, leading to the cleavage of RIP3 into two distinct cleavage fragments, RIP3NT and RIP3CT. These fragments are incapable of inducing necrosis, however RIP3CT retains its ability to associate with RIP1 and leads to a non-necrotic form of cell death.

We have shown previously that necrotic cell death pathways are activated in polarized IECs upon CVB infection (Bozym et al., 2011). Our data now show a positive correlation between necrotic cell death upon CVB infection and RIP3 expression. However, our data suggest that RIP3 plays a role independent of its function in the induction of necroptosis during CVB replication in IECs, which is supported by the finding that silencing of MLKL did not affect early events in CVB replication. Instead, our findings support a functional link between RIP3 and autophagy. Autophagy serves to promote cell survival upon conditions of cellular stress including starvation and organelle damage. However, there have been many reports of high levels of autophagy accompanying cell death (Marino et al., 2014), and a subset of cases in which cell death is actually dependent upon autophagy (Elgendy et al., 2011; Liu et al., 2013; Yu et al., 2004). Inhibition of caspase-8 induces autophagy-dependent cell death in L929 cells (Yu et al., 2004), and cell death upon caspase-8 inhibition in L929 cells was later shown to be dependent on RIP3 (Kaiser et al., 2011). Although this loss of viability was attributed to the role of RIP3 in necroptotic cell death, in light of our data it is possible that inhibition of caspase-8, a known negative regulator of RIP3 (Oberst et al., 2011), may cause an increase in RIP3-dependent autophagic flux, resulting in autophagic cell death.

Recently, it has been shown that RIP1 negatively regulates basal levels of autophagy in a kinase-independent manner (Yonekawa et al., 2015). The negative regulation of autophagy by RIP1 was accomplished through the phosphorylation and consequent negative regulation of TFEB, a transcription factor that induces expression of autophagy-related genes (Yonekawa et al., 2015). Whereas RIP1 is required for TNFα-induced RIP3-dependent necroptosis, recent evidence has shown that RIP1 negatively regulates TRIF-mediated, IFN-mediated and spontaneous RIP3-dependent necroptosis in a kinase independent fashion (Dillon et al., 2014; Kaiser et al., 2014; Rickard et al., 2014). Here, we show that RIP3 positively regulates autophagy. Taken together, these data suggest that negative regulation of autophagy by RIP1 may rely on its constitutive negative regulation of RIP3.

The relationship between host cell autophagy and viral pathogens is complex. Autophagy can serve as an antiviral defense for the host cell, degrading intracellular pathogens and preventing establishment of an infection (Kirkegaard et al., 2004). Indeed, here we note that depletion of RIP3, which we show to be a positive regulator of autophagy, enhances VSV infection in IECs, a virus whose replication can be restricted by autophagy (Shelly et al., 2009). In contrast, autophagy is conducive to enterovirus replication, possibly through the provision of membranes for the assembly of viral replication factories. Whether flux through the autophagic pathway is necessary or autophagic initiation alone is sufficient to promote CVB replication remains unclear. Studies in non-polarized cells have suggested that whereas autophagosome formation and initiation of the autophagic pathway is utilized by CVB for replication, autolysosomal fusion is dispensable (Wong et al., 2008). However, recent studies from our laboratory have shown that in polarized endothelial cells, complete flux through the autophagic pathway may be beneficial for CVB infection (Delorme-Axford et al., 2014). Here, our data suggest that a post-initiation and RIP3-regulated step of autophagic flux may be involved in the regulation of CVB infection in polarized IECs, consistent with the notion that complete autophagic flux promotes CVB replication.

Recently it was reported that exogenously expressed RIP3 and p62 associated in 293T cells, a line that does not express any endogenous RIP3 (Matsuzawa et al., 2015). Here, we show that endogenous RIP3 exists in complex with endogenous p62 in IECs under resting conditions. As p62 is necessary for selective autophagy through its role in binding to both the autophagic membrane associated protein LC3B and cellular components destined for degradation (Stolz et al., 2014), it is possible that the endogenous association of RIP3 with p62 serves to recruit RIP3 to the inside of APs, where it acts to promote flux. Consistent with this, we found that RIP3 is degraded during autophagy, likely due to its localization within the AP. Interestingly, we observed a change in electrophoretic mobility of RIP3 by immunoblot indicative of RIP3 phosphorylation soon after transition of cells to serum starvation conditions, indicating that RIP3 phosphorylation and activation may be important in its role in promoting autophagic flux.

We propose that RIP3 may be a tissue-specific regulator of autophagy. It is known that autophagy is an especially important pathway in IECs for maintaining intestinal homeostasis. Genome wide association studies have identified a role for autophagy related genes in inflammatory bowel disease (Anderson et al., 2011) and autophagy is known to be important for the maintenance of the intestinal epithelium as a barrier against intestinal pathogens (Benjamin et al., 2013). Thus, RIP3 may function as an IEC-specific regulator of autophagy. In support of this, we found that ectopic expression of RIP3 in HeLa cells had little impact on CVB replication. Additionally, autophagy is known to play a key role in embryonic development and mice deficient in various genes required for autophagy display embryonic lethality (Mizushima and Levine, 2010). As RIP3 deficient mice do not exhibit such defects (Newton et al., 2004), and RIP3 is expressed in tissue-specific patterns (Yu et al., 1999), it is unlikely that RIP3 is globally required for autophagy.

The function of RIP3 in necrosis is conserved between mice and humans. However, residue Q430 in human RIP3, which is targeted by CVB 3Cpro, is not conserved in mouse RIP3 (Figure S4C). Interestingly, sequence comparisons of RIP3 from several primates revealed that RIP3 in Old World and New World monkeys contains a Histidine at position 430, whereas the evolution of a Glutamine at position 430 arose in the common ancestor of the apes. (Figure S4C). Indeed, we found that neither African Green Monkey nor Colobus Monkey RIP3 constructs were targeted by 3Cpro for cleavage (Figure S4D). The viral protease 3Cpro is well-conserved among all enteroviruses. The ubiquity of these viruses suggests extensive co-evolution of enteroviruses with their hosts, perhaps made possible in part by their ability to take advantage of the newly evolved cleavage site in RIP3 of ancestral apes. Indeed, limiting necrotic cell death through cleavage of RIP3 at residue Q430 by 3Cpro could serve to lessen the damaging inflammatory effects of intestinal necrosis; such attenuations of pathogenic effects of infection are common in viral pathogens that are well-adapted to their hosts (Longdon et al., 2014).

Here, we show that RIP3 serves as a specific pro-viral regulator of CVB replication in polarized IECs via its role in the regulation of autophagy. Although the polarized intestinal epithelium is likely to be the primary cell type targeted by CVB during host invasion, the mechanisms by which CVB establishes its infection in these cells have remained elusive. Our findings thus advance our understanding of intestinal epithelial cell pathways that specifically facilitate CVB replication and the pathways used by the virus to attenuate these defenses and promote intestinal infection.

Experimental Procedures

Cells and Viruses

Human colorectal adenocarcinoma Caco-2 cells (ATCC HTB-37) were cultured in minimal essential media (MEM) supplemented with 20% fetal bovine serum (FBS), sodium pyruvate, and nonessential amino acids. Human colorectal adenocarcinoma HT29 cells (ATCC HTB-38) were cultured in McCoy’s 5A (modified) media supplemented with 10% FBS. HT29 cells stably expressing an shRNA vector targeting RIP3 and RIP3 tet-inducible HeLa cells were kindly provided by Dr. Xiaodong Wang (National Institute of Biological Sciences, Beijing), as previously described (He et al., 2009; Sun et al., 2012).

Experiments were performed with CVB3-RD or VSV-GFP (Indiana), as previously described (Coyne and Bergelson, 2006). Neutral red-labeled CVB3-RD was prepared as previously described (Delorme-Axford et al., 2013b). For time courses of infection in HT29 cells, CVB was pre-adsorbed to cells for 1 hr at 16°C to synchronize infection.

Immunofluorescence and electron microscopy

Confluent monolayers were grown in 8-well chamber slides (BD Biosciences), then fixed and permeabilized with an ice-cold mixture of 3:1 methanol:acetone or 4% paraformaldehyde followed by 0.1% Triton X-100. Fixed and permeabilized cells were incubated with primary antibody for 1 hour followed by appropriate secondary antibody for 30 minutes. Slides were mounted in Vectashield containing DAPI (Vector Labs). Differential interference contrast (DIC) images were obtained from live cells grown to confluence in MatTek glass-bottomed dishes. Images were captured using an IX83 inverted widefield microscope (Olympus) or a FV1000 confocal laser scanning microscope (Olympus), analyzed using Imaris (Bitplane) or ImageJ, and contrasted and merged using Photoshop (Adobe). Electron microscopy was performed as described (Delorme-Axford et al., 2013a). For measurements of amphisome size and number by TEM, at least 40 individual amphisomes were measured using Image J from at least 10 unique cells.

Neutral Red CVB Entry Assay

Confluent cell monolayers were grown in 8-well chamber slides (BD Biosciences) and then incubated with neutral red-labeled CVB3-RD (described above) (MOI=10) for one hour at 16°C in semi-dark conditions in McCoy’s 5A (modified) media supplemented with 20 mM HEPES. Monolayers were then washed in PBS and fresh media was added. Monolayers were incubated at 37°C, 5% CO2 and illuminated for 20 minutes on a light box at 0 or 120 minutes p.i., or kept in semi-dark conditions. 16–18 hours post-infection, infection levels were assessed by immunofluorescence microscropy to detect VP1 as described above.

Immunoblots

Confluent monolayers were grown in 24-well plates. Monolayers were lysed in RIPA Buffer or EBC Buffer (Figure 4D only). Lysates were briefly sonicated and nonsoluble cell debris was removed through centrifugation. Protein lysates in SDS containing sample buffer (Boston BioProducts) were heated at 95°C for 10 minutes, then electrophoresed in polyacrylamide gradient gels (4–20% or 10–20%, Bio-Rad). Protein was transferred onto nitrocellulose membranes and probed with indicated primary antibodies in 5% non-fat dry milk and the appropriate secondary antibodies conjugated to horseradish peroxidase (HRP, Santa Cruz) or infrared dyes (Li-Cor) and protein was detected using chemiluminescent HRP substrates (Pierce) or by the Odyssey CLx imaging system (Li-Cor).

Statistical Analysis

All statistical analysis was performed using GraphPad Prism. Students t-test or one-way ANOVA were performed as appropriate. * indicates p<0.05. ** indicates p<0.01. *** indicates p<0.001. Pooled data is displayed as mean +/− SD.

Acknowledgments

We thank Elizabeth Delorme-Axford (University of Michigan) and Rebecca Bozym (LaRoche College) for technical assistance, Saumendra Sarkar (University of Pittsburgh) and Harmit Malik (Fred Hutchinson Cancer Center) for reagents and advice, and Kwang Sik Kim (Johns Hopkins University) for the HBMEC used in the study. This work was supported by the National Institutes of Health (R01AI081759[CBC], R01AI072490 [JMB and SC], T32AI049820[KGH], and T32AI060525[KGH]). Additionally, CBC and SC are supported by the Burroughs Wellcome Investigators in the Pathogenesis of Infectious Disease award, JMB by the Plotkin Endowed Chair in infectious Diseases at the Children’s Hospital of Philadelphia, and KGH is a recipient of an ARCS Foundation Scholar Award.

Footnotes

Author Contributions

CK, JMB, and SC designed and performed the RNAi screen. KGH and CBC designed experiments and wrote the manuscript. KGH, SAM, CD, and CBC performed experiments. DBS provided support for electron microscopy. MP provided essential reagents related to the cleavage of nonhuman RIP3.

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