ABCE1 Regulates RNase L-Induced Autophagy during Viral Infections

Host response to a viral infection includes the production of type I interferon (IFN) and the induction of interferon-stimulated genes that have broad antiviral effects. One of the key antiviral effectors is the IFN-inducible oligoadenylate synthetase/ribonuclease L (OAS/RNase L) pathway, which is activated by double-stranded RNA to synthesize unique oligoadenylates, 2-5A, to activate RNase L. RNase L exerts an antiviral effect by cleaving diverse RNA substrates, limiting viral replication; many viruses have evolved mechanisms to counteract the OAS/RNase L pathway. Here, we show that the ATP-binding cassette E1 (ABCE1) transporter, identified as an inhibitor of RNase L, regulates RNase L activity and RNase L-induced autophagy during viral infections. ABCE1 knockdown cells show increased RNase L activity when activated by 2-5A. Compared to parental cells, the autophagy-inducing activity of RNase L in ABCE1-depleted cells is enhanced with early onset. RNase L activation in ABCE1-depleted cells inhibits cellular proliferation and sensitizes cells to apoptosis. Increased activity of caspase-3 causes premature cleavage of autophagy protein, Beclin-1, promoting a switch from autophagy to apoptosis. ABCE1 regulates autophagy during EMCV infection, and enhanced autophagy in ABCE1 knockdown cells promotes EMCV replication. We identify ABCE1 as a host protein that inhibits the OAS/RNase L pathway by regulating RNase L activity, potentially affecting antiviral effects.


Introduction
Degrading viral and cellular RNAs required for viral replication is an evolutionarily conserved antiviral mechanism. In higher vertebrates, this process is regulated by interferon (IFN), produced during a viral infection through the activation of the ubiquitous cellular latent endoribonuclease, ribonuclease L (RNase L). The 2 ,5 -oligoadenylate synthetase (OAS)/RNase L system is an innate immune pathway that responds to the double-stranded RNAs (dsRNAs) that serve as pathogen-associated molecular patterns (PAMPs) to induce the degradation of viral and cellular RNAs, thereby blocking the viral infection [1][2][3]. Type I IFN, produced and secreted by a virus-infected cell signal through the type I IFN receptor, activates JAK-STAT signaling and induces the expression of interferon-stimulated genes (ISGs), including oligoadenylate synthetases (OAS), that together establish the antiviral state [4,5]. OAS1-3 isoforms are expressed at varying levels in different cell types, and on activation by dsRNA PAMPs, certain OAS proteins produce 2-5A from cellular ATP [6][7][8]. 2-5A is a unique ligand that binds monomeric and latent RNase L with high affinity, causing RNase L dimerization and activation. Active RNase L cleaves diverse single-stranded RNA substrates, including viral genomes and cellular RNAs, directly impacting protein synthesis and limiting viral replication [3]. Activation of RNase L, through the generation of dsRNA cleavage products, amplifies IFN production, activates inflammasome, leads to autophagy, and promotes a switch from autophagy to apoptosis, affecting viral replication in cells [9][10][11][12][13].

Cell Culture and Transfections
The human fibrosarcoma cell line HT1080 (a gift from Ganes Sen, Cleveland Clinic, Cleveland, OH, USA), RNase L KO, ABCE1 KD, ABCE1 KD/RNase L KO, and the mouse fibroblast cell line L929 (a gift from Douglas Leaman, Wright State University) were cultured in Dulbecco's modified minimal essential medium with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 100 µg/mL penicillin/streptomycin, 2 mM L-glutamine, and nonessential amino acids. RNase L KO cells were generated with CRISPR/Cas9 gene editing as described previously [59]. The cells were maintained in 95% air, 5% CO 2 at 37 • C. Transfection of 2-5A (10 µM) or poly I:C (2 µg/mL) was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Briefly, the cells were plated one day before transfection so that the cells were 80-90% confluent at the time of transfection. Poly I:C or 2-5A was diluted into a serum-free medium and then mixed with Lipofectamine 2000 reagent for 20 min before being added to cells in growth media and incubated for indicated times.

Generation of ABCE1 Knockdown Cells
ABCE1 (RLI) was knocked down in HT1080 WT by transfecting pSUPER plasmid with short hairpin RNA homologous to ABCE1 (RLI) along with pBabe-puro as previously described [60]. Individual clones were generated by limiting dilution in a puromycin (1 µg/mL) containing medium, and gene knockdown was verified by immunoblot analysis using ABCE1 antibodies and normalized to β-actin levels for comparison. Two independent clones (clone 1 and clone 2) were used to determine RNase L activity, induction of autophagy, and cell viability assays to rule out clone-specific artifacts. The data obtained with clone 2 compared to clone 1 are shown in Figure S1. ABCE1 knockdown cells from early passages were used in this study as later passages showed reduced proliferation and viability, as observed in other studies [45]. To generate ABCE1 KD/RNase L KO cells, RNase L KO CRISPR/Cas9 gene edited cells were co-transfected with ABCE1 short hairpin Viruses 2021, 13, 315 4 of 16 RNA (shRNA) and pEGFP plasmid. GFP-positive individual clones obtained by limiting dilution were verified by immunoblot analysis as described above and used.

Measuring RNase L Activity in Intact Cells
Cells were transfected with poly I:C (2 µg/mL) or 2-5A (10 µM) using Lipofectamine 2000 reagent and after 6 h, total RNA was isolated using Trizol reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Total RNA was resolved on RNA chips and analyzed with Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) as described previously [12]. RNase L activity was measured as the cleavage of total RNA by estimating the integrity by the RNA integrity number (RIN) [61].

Quantification of Autophagy
Cells were transfected with GFP-LC3 and 24 h later with 2-5A (1 0µM) using Lipofectamine 2000 and imaged at indicated times using an Olympus IX81 inverted fluorescence microscope. Cytoplasmic GFP-LC3 puncta (>10 puncta/cell) were considered autophagic and manually counted for at least 100 randomly selected cells per preparation from three independent experiments. Autophagy was quantified as a percent of GFP-LC3-positive cells showing puncta (>10 puncta/cell). The kinetics of the induction of autophagy in live cells was determined using the CYTO-ID autophagy detection kit (Enzo Life Sciences, Farmingdale, NY, USA) under a confocal microscope. Briefly, cells (1 × 10 5 ) were seeded on coverslips in a 12-well plate and transfected with 2-5A (10 µM) using Lipofectamine 2000 reagent. The Cyto-ID Green Autophagy Detection Reagent, which is excitable at 488 nm in autophagic vacuoles produced during autophagy, was added and imaged at indicated times. The percent of cells showing autophagic vacuoles from three random fields was counted. A minimum of 100 cells per preparation were quantified in three independent experiments.

Cell Viability and Caspase 3/7 Assays
The viability of cells was determined using the colorimetric CellTiter 96 Aqueous Cell Proliferation Assay (Promega, Madison, WI, USA). Briefly, cells (8 × 10 3 ) were seeded into 96-well plates and transfected with 2-5A (10 µM). At indicated times, 20 µL of tetrazolium salt (MTS reagent) was added and incubated at 37 • C. Absorbance was measured at 490 nm with a 96-well plate reader (model Spectra Max iD5; Molecular Devices, Menlo Park, CA, USA). Cell viability was normalized against mock-treated cells. The percent of viable cells was determined by staining in a 0.4% trypan blue solution (Life Technologies, Carlsbad, CA, USA) and estimating viable cells that exclude dye uptake using a hemocytometer and normalized to mock-treated cells. For caspase 3/7 assay, cells were grown in black-walled 96-well plates with transparent bottoms (Costar) and transfected with 2-5A (10 µM) using Lipofectamine 2000. At indicated times, caspase3/7 activity in lysates was measured using the ApoONE homogenous caspase-3 and -7 assay kit (Promega, Madison, WI, USA) and normalized to control mock-treated cells. Experiments were performed in triplicate and shown as mean ± SD.

Cell Death Assays
Cell death was analyzed in real time using dual dyes and an IncuCyte S3 Live-Cell imaging system (Essen BioScience, Ann Arbor, MI, USA). Cells (8 × 10 3 ) were seeded into a 96-well plate and transfected with 2-5A (10 µM) using Lipofectamine 2000 reagent. Cells were incubated with 250 nM Sytox-Green cell-impermeable nucleic acid dye (ThermoFisher Scientific, Waltham, MA, USA) that indicates dead cells and 250 nM of SytoTM 60-Red cellpermeable dye (ThermoFisher scientific, Waltham, MA, USA), which quantifies the total number of cells present in each field, and images were obtained in real time as indicated. Cell death was quantified as the percent of Sytox-Green-positive dead cells normalized to the total number of cells that were stained Sytox-Red positive at each time point using

Immunoblotting
Cells were washed in ice-cold PBS and lysed in an NP-40 lysis buffer containing 0.5% NP-40, 90 mM KCl, 5 mM magnesium acetate, 20 mM Tris (pH 7.5), 5 mM β-mercaptoethanol, 0.1 M phenylmethylsulfonyl fluoride (PMSF), 0.2 mM sodium orthovanadate, 50 mM NaF, 10 mM glycerophosphate, and a protease inhibitor (Roche Diagnostics). The lysates were clarified by centrifugation at 10,000× g (4 • C for 20 min). Proteins (15-60 µg per lane) were separated in polyacrylamide gels containing SDS and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) and probed with different primary antibodies according to the manufacturer's protocols. Membranes were washed and incubated with goat antimouse or goat anti-rabbit antibody tagged with horseradish peroxidase (Cell Signaling, Danvers, MA, USA) for 2 h. Immunoreactive bands were visualized using enhanced chemiluminescence reagents (Boston BioProducts, Ashland, MA, USA; Bio-Rad, Hercules, CA, USA). For determining the ratios of LC3-II/β-actin, P62/β-actin, and 3D Pol/β-actin, the intensity of each band was determined using Image J software (National Institutes of Health, Bethesda, MD, USA) and their relative intensities were calculated by normalizing to β-actin. Images were processed using Adobe Photoshop CS4 (Adobe, San Jose, CA, USA). In some instances, nonspecific lanes were cropped to generate the images and the boundaries are indicated in representative figures.

Virus Infections and Plaque Assays
The cells were plated into 6-well plates, and the next day, the cells were washed twice in PBS and infected with EMCV (strain k) at MOI = 1.0. After 1 h, the virus was removed and the cells were washed with PBS and replaced with a complete growth medium. In experiments with inhibitors, the cells were pretreated with 3-methyladenine (5 mM) or bafilomycin A1 (100 nM) for 1 h and infected with EMCV, and then fresh complete media with 10% FBS was added. At indicated times post infection, EMCV containing supernatants and cell pellets were harvested for virus titration. Serial dilution of supernatants or clarified cell lysates containing intracellular virus were added to the confluent monolayer of L929 cells in 12-well plates. The plates were incubated for 1 h at 37 • C. The cells were washed with PBS and overlaid with DMEM containing 0.5% carboxymethylcellulose and incubated for 24 h. The cells were fixed with 10% formaldehyde, and plaques were stained using 0.1% crystal violet and counted. The assays were performed in triplicate, and the fold change in virus titers was determined from three independent experiments and shown as mean ± SD. The expression of the viral antigen was determined on Western blots using the 3D Pol antibody (Santa Cruz Biotechnology, Dallas, TX, USA).

Statistical Analysis
All values are presented as mean ± SD and are representative of at least three independent experiments. Two-way ANOVA (or one-way ANOVA for a single time point) was used for determining statistical significance between groups, using Prism 8 (GraphPad) software, and p values of <0.05 were considered significant.

ABCE1 (RLI) Regulates RNase L Enzyme Activity
ABCE1, also known as RLI, was identified as an inhibitor of IFN-regulated endoribonuclease, RNase L. To determine if ABCE1 regulates RNase L enzyme activity, ABCE1 knockdown stable cell lines were generated in HT1080 cells by transfecting shRNA-targeting ABCE1 and stable clones with greater than 90% reduced expression were screened and used in this study ( Figure 1A). Two independent clones were evaluated for effect on RNase L enzyme activity (Figure 1 and Figure S1A). As reported in other studies, prolonged ABCE1 knockdown caused reduced cell proliferation and early passage cells were expanded and used [45]. The effect of ABCE1 on RNase L enzyme activity was determined by transfecting 2-5A, a unique ligand and activator of RNase L, and rRNA cleavage characteristic of RNase L activity was quantitated and analyzed on RNA chips. Control cells produced 8.4% rRNA cleavage, while ABCE1 knockdown (ABCE1 KD) had 3-fold increased rRNA cleavage (25%). Transfecting cells with synthetic dsRNA (poly I:C), which binds oligoadenylate synthetase (OAS) to produce 2-5A from cellular ATP, produced 22.4% rRNA cleavage in WT cells compared to 59% in ABCE1 KD cells ( Figure 1B,C). As expected, RNase L KO cells generated by CRISPR/Cas9 technology showed no cleavage of rRNA with 2-5A or poly I:C transfection [58,59]. To further test that the impact of ABCE1 on RNase L activity was mediated by RNase L, we knocked down ABCE1 in RNase L KO cells using shRNA and monitored rRNA cleavage by 2-5A or poly I:C transfection ( Figure 1A-C). No rRNA cleavage was observed in the ABCE1 KD/RNase L KO cells, indicating that the effect of ABCE1 was mediated by regulating RNase L activity ( Figure 1B,C). These results suggest that ABCE1 regulates RNase L enzyme activity and cells with reduced levels of ABCE1 show enhanced RNase L activity in the presence of the 2-5A ligand.
knockdown stable cell lines were generated in HT1080 cells by transfecting shRNA-targeting ABCE1 and stable clones with greater than 90% reduced expression were screened and used in this study ( Figure 1A). Two independent clones were evaluated for effect on RNase L enzyme activity ( Figure 1 and Figure S1A). As reported in other studies, prolonged ABCE1 knockdown caused reduced cell proliferation and early passage cells were expanded and used [45]. The effect of ABCE1 on RNase L enzyme activity was determined by transfecting 2-5A, a unique ligand and activator of RNase L, and rRNA cleavage characteristic of RNase L activity was quantitated and analyzed on RNA chips. Control cells produced 8.4% rRNA cleavage, while ABCE1 knockdown (ABCE1 KD) had 3-fold increased rRNA cleavage (25%). Transfecting cells with synthetic dsRNA (poly I:C), which binds oligoadenylate synthetase (OAS) to produce 2-5A from cellular ATP, produced 22.4% rRNA cleavage in WT cells compared to 59% in ABCE1 KD cells ( Figure 1B,C). As expected, RNase L KO cells generated by CRISPR/Cas9 technology showed no cleavage of rRNA with 2-5A or poly I:C transfection [58,59]. To further test that the impact of ABCE1 on RNase L activity was mediated by RNase L, we knocked down ABCE1 in RNase L KO cells using shRNA and monitored rRNA cleavage by 2-5A or poly I:C transfection ( Figure 1A-C). No rRNA cleavage was observed in the ABCE1 KD/RNase L KO cells, indicating that the effect of ABCE1 was mediated by regulating RNase L activity ( Figure 1B,C). These results suggest that ABCE1 regulates RNase L enzyme activity and cells with reduced levels of ABCE1 show enhanced RNase L activity in the presence of the 2-5A ligand.

ABCE1 Modulates RNase L-Induced Autophagy
Previous work from our and other groups have shown that RNase L activation induces autophagy [11,12]. We used several assays to determine the impact of ABCE1 on RNase L-induced autophagy [62]. First, WT, ABCE1 KD, RNase L KO, and ABCE1 KD/RNase L KO cells expressing GFP-LC3 were transfected with 2-5A and the formation of distinct GFP-LC3 puncta that correspond to autophagosomes and induction of autophagy was quantified at indicated times ( Figure 2A). We observed a significant increase in GFP-LC3 puncta (35.3% of GFP+ cells) as early as 4 h post transfection in ABCE1 KD cells compared to WT cells (10.4%). A similar increase in GFP-LC3 puncta was observed in another ABCE1 KD clone 2 ( Figure S1B). In comparison, GFP-LC3 puncta were observed KD/RNase L KO cells were transfected with 2-5A (10 µM) or 2 µg/mL of poly I:C for 6 h, and the RNase L-mediated cleavage of rRNA (arrows) was analyzed on RNA chips using Agilent Bioanalyzer 2100. (C) The cleavage of rRNA was quantitated and normalized to mock-treated samples. Data are representative of at least three independent experiments and expressed as means ± SD; WT: wild-type; ** p < 0.01; *** p < 0.001.

ABCE1 Modulates RNase L-Induced Autophagy
Previous work from our and other groups have shown that RNase L activation induces autophagy [11,12]. We used several assays to determine the impact of ABCE1 on RNase L-induced autophagy [62]. First, WT, ABCE1 KD, RNase L KO, and ABCE1 KD/RNase L KO cells expressing GFP-LC3 were transfected with 2-5A and the formation of distinct GFP-LC3 puncta that correspond to autophagosomes and induction of autophagy was quantified at indicated times ( Figure 2A). We observed a significant increase in GFP-LC3 puncta (35.3% of GFP+ cells) as early as 4 h post transfection in ABCE1 KD cells compared to WT cells (10.4%). A similar increase in GFP-LC3 puncta was observed in another ABCE1 KD clone 2 ( Figure S1B). In comparison, GFP-LC3 puncta were observed in only 3.7% of RNase L KO and 5.1% of ABCE1 KD/RNase L KO cells at the same time point. After 8 h, the number of GFP-LC3 puncta increased to 64.3% in ABCE1 KD cells and 18.9% in WT cells. However, the numbers remained largely unchanged in RNase L KO and ABCE1 KD/RNase L KO cells ( Figure 2B). We then measured the accumulation of autophagic vacuoles in live cells transfected with 2-5A by staining with a fluorescent dye that selectively labels autophagic vacuoles. Compared to WT cells, ABCE1 KD cells showed a 2-fold increase in autophagic vacuole accumulation  Figure 2C). During autophagy, LC3-I is cleaved and lipidated to LC3-II, which is associated with autophagosomes. We found that ABCE1 KD enhanced autophagy in 2-5A-transfected cells, as shown by increased levels of LC3-II on immunoblots as early as 8 h compared to WT cells ( Figure 2D). As shown previously, cells lacking RNase L did not induce autophagy with 2-5A treatment [12,13], and cells lacking both ABCE1 and RNase L did not induce autophagy by 2-5A transfection. Consistent with enhanced and early onset of autophagy, 2-5A treatment of ABCE1 KD cells caused pronounced degradation of p62 (SQSTM1), an LC3-binding protein that is degraded on autophagosome-lysosome fusion, compared to WT, RNase L KO, or ABCE1 KD/RNase L KO cells ( Figure 2D-F). Taken together, these results suggest that the knockdown of ABCE1 induced early autophagy in RNase L-activated cells and these effects of ABCE1 are mediated by a direct effect on RNase L. in only 3.7% of RNase L KO and 5.1% of ABCE1 KD/RNase L KO cells at the same time point. After 8 h, the number of GFP-LC3 puncta increased to 64.3% in ABCE1 KD cells and 18.9% in WT cells. However, the numbers remained largely unchanged in RNase L KO and ABCE1 KD/RNase L KO cells ( Figure 2B). We then measured the accumulation of autophagic vacuoles in live cells transfected with 2-5A by staining with a fluorescent dye that selectively labels autophagic vacuoles. Compared to WT cells, ABCE1 KD cells showed a 2-fold increase in autophagic vacuole accumulation 4 h and 8 h post 2-5A treatment, while RNase L KO and ABCE1 KD/RNase L KO cells showed very low levels (Figure 2C). During autophagy, LC3-I is cleaved and lipidated to LC3-II, which is associated with autophagosomes. We found that ABCE1 KD enhanced autophagy in 2-5A-transfected cells, as shown by increased levels of LC3-II on immunoblots as early as 8 h compared to WT cells ( Figure 2D). As shown previously, cells lacking RNase L did not induce autophagy with 2-5A treatment [12,13], and cells lacking both ABCE1 and RNase L did not induce autophagy by 2-5A transfection. Consistent with enhanced and early onset of autophagy, 2-5A treatment of ABCE1 KD cells caused pronounced degradation of p62 (SQSTM1), an LC3-binding protein that is degraded on autophagosome-lysosome fusion, compared to WT, RNase L KO, or ABCE1 KD/RNase L KO cells ( Figure 2D-F). Taken together, these results suggest that the knockdown of ABCE1 induced early autophagy in RNase L-activated cells and these effects of ABCE1 are mediated by a direct effect on RNase L.  were monitored on immunoblots and normalized to β-actin levels. (E,F) The band intensity was calculated using Image J software, and the ratio of LC3-II/β-actin or p62/β-actin was determined and the levels were compared to WT cells. Results are representative of three independent experiments. WT: wild-type; ** p < 0.01; *** p < 0.001.

RNase L Activation Sensitizes ABCE1 Knockdown Cells to Apoptosis
Activation of RNase L by 2-5A induces cell death by apoptosis, and the byproducts of RNase L enzyme activity promote a switch from autophagy to apoptosis [13,63]. To determine if the increased RNase L enzyme activity in ABCE1 KD cells affected cell viability, WT, ABCE1 KD, RNase L KO, and ABCE1 KD/RNase L KO cells were transfected with 2-5A. At indicated times, cell viability was determined by MTS assay and trypan blue exclusion assays. After 8 h, ABCE1 KD cells showed 72% cell viability compared to minimal loss of viability in WT, RNase L KO, and ABCE1 KD/RNase L KO cells. The viability of ABCE1 KD cells was further reduced at 16 h to 57%, compared to 88% in WT cells, and the loss of viability continued to decrease at 24 h to 44% in ABCE1 KD cells while 62% of the WT cells were viable. Similarly, ABCE1 KD clone 2 showed 48% cell viability at 24 h ( Figure S1C). No significant loss of viability was observed in RNase L KO and ABCE1 KD/RNase L KO cells ( Figure 3A).

RNase L Activation Sensitizes ABCE1 Knockdown Cells to Apoptosis
Activation of RNase L by 2-5A induces cell death by apoptosis, and the byproducts of RNase L enzyme activity promote a switch from autophagy to apoptosis [13,63]. To determine if the increased RNase L enzyme activity in ABCE1 KD cells affected cell viability, WT, ABCE1 KD, RNase L KO, and ABCE1 KD/RNase L KO cells were transfected with 2-5A. At indicated times, cell viability was determined by MTS assay and trypan blue exclusion assays. After 8 h, ABCE1 KD cells showed 72% cell viability compared to minimal loss of viability in WT, RNase L KO, and ABCE1 KD/RNase L KO cells. The viability of ABCE1 KD cells was further reduced at 16 h to 57%, compared to 88% in WT cells, and the loss of viability continued to decrease at 24 h to 44% in ABCE1 KD cells while 62% of the WT cells were viable. Similarly, ABCE1 KD clone 2 showed 48% cell viability at 24 h ( Figure S1C). No significant loss of viability was observed in RNase L KO and ABCE1 KD/RNase L KO cells ( Figure 3A). ABCE1 KD cells showed increased cell death from 26% to 67% in trypan blue exclusion assay over time compared to WT cells (16% to 59% cell death), while over 80% of RNase L KO and ABCE1 KD/RNase L KO cells remained viable ( Figure 3B). ABCE1 KD clone 2 showed 68% cell death at 24 h after 2-5A transfection ( Figure S1D). We then assessed the effects of 2-5A on cell survival in real time of WT, ABCE1 KD, RNase L KO, or ABCE1 KD/RNase L KO cells using dual dyes and an IncuCyte real-time monitoring system for 30 h (described in Methods). The quantitation of cell survival showed that only 10% of ABCE1 KD cells survived after 30 h compared to 47% of WT cells, while RNase L KO and ABCE1 KD/RNase L KO cells were mostly unaffected ( Figure 3C,D). These results show that activation of RNase L by 2-5A in cells lacking ABCE1, which increases RNase L activity, also enhances apoptosis compared to WT cells. ABCE1 KD cells showed increased cell death from 26% to 67% in trypan blue exclusion assay over time compared to WT cells (16% to 59% cell death), while over 80% of RNase L KO and ABCE1 KD/RNase L KO cells remained viable ( Figure 3B). ABCE1 KD clone 2 showed 68% cell death at 24 h after 2-5A transfection ( Figure S1D). We then assessed the effects of 2-5A on cell survival in real time of WT, ABCE1 KD, RNase L KO, or ABCE1 KD/RNase L KO cells using dual dyes and an IncuCyte real-time monitoring system for 30 h (described in Methods). The quantitation of cell survival showed that only 10% of ABCE1 KD cells survived after 30 h compared to 47% of WT cells, while RNase L KO and ABCE1 KD/RNase L KO cells were mostly unaffected ( Figure 3C,D). These results show that activation of RNase L by 2-5A in cells lacking ABCE1, which increases RNase L activity, also enhances apoptosis compared to WT cells.

ABCE1 Knockdown Augments RNase L-Induced Apoptosis by Enhancing Caspase-3 Activity and Beclin-1 Proteolytic Cleavage
In previous studies, we showed that small dsRNAs generated by RNase L enzyme activity promote a switch from autophagy to apoptosis by the caspase-mediated cleavage of Viruses 2021, 13, 315 9 of 16 key autophagy protein, Beclin-1 [13]. Here, we show increased RNase L activity accompanied by early onset of autophagy correlating with a switch to apoptotic cell death in cells lacking ABCE1. Together, these events suggest the involvement of caspase cascade and Beclin-1 cleavage in events leading to enhanced cell death in ABCE1 KD cells. To investigate the activation of caspase-3, we monitored the cleavage of caspase-3 and PARP, both hallmarks of apoptosis, in WT, ABCE1 KD, RNase L KO, and ABCE1 KD/RNase L KO cells after 2-5A transfection at indicated times on immunoblots. The appearance of cleaved PARP was evident by 8 h in ABCE1 KD cells (9-fold more than WT) and remained sustained at 24 h compared to WT cells. As expected, PARP cleavage was barely detectable in RNase L KO and ABCE1 KD/RNase L KO cells. Caspase-3 activity, measured by levels of cleaved caspase-3 on immunoblots, showed a similar temporal profile in ABCE1 KD cells (2.5-fold more than WT at 8 h and 3.6-fold more at 24 h) compared to WT cells and low to no cleavage in RNase L KO cells and ABCE1 KD/RNase L KO cells ( Figure 4A,B). We measured caspase-3 enzyme activity in cell lysates using a fluorescent substrate following RNase L activation at indicated times and observed a 2-fold to 3.6-fold increase in ABCE1 KD cells compared to WT cells. No significant caspase-3 enzyme activity was detected in the lysates of RNase L KO cells and ABCE1 KD/RNase L KO cells ( Figure 4C). Based on our previous results, we hypothesized that increased apoptosis due to RNase L activation in ABCE1 KD cells could be attributed to caspase-3-mediated cleavage of Beclin-1. Subsequently, using immunoblot analysis, we determined increased cleavage of Beclin-1 (3.8-fold more than WT) 8 h after RNase L activation in ABCE1 KD cells compared to WT cells, which overlapped with increased caspase-3 activity ( Figure 4D). No significant modulation of caspase-3 activity or Beclin-1 cleavage was observed in RNase L KO cells and ABCE1 KD/RNase L KO cells. These results suggest that compared to WT cells, increased RNase L activity in ABCE1 KD cells enhances apoptosis by the increased caspase-3-mediated cleavage of Beclin-1 and cells lacking both ABCE1 and RNase L are resistant to apoptosis by 2-5A treatment.

ABCE1 Knockdown Augments RNase L-Induced Apoptosis by Enhancing Caspase-3 Activity and Beclin-1 Proteolytic Cleavage
In previous studies, we showed that small dsRNAs generated by RNase L enzyme activity promote a switch from autophagy to apoptosis by the caspase-mediated cleavage of key autophagy protein, Beclin-1 [13]. Here, we show increased RNase L activity accompanied by early onset of autophagy correlating with a switch to apoptotic cell death in cells lacking ABCE1. Together, these events suggest the involvement of caspase cascade and Beclin-1 cleavage in events leading to enhanced cell death in ABCE1 KD cells. To investigate the activation of caspase-3, we monitored the cleavage of caspase-3 and PARP, both hallmarks of apoptosis, in WT, ABCE1 KD, RNase L KO, and ABCE1 KD/RNase L KO cells after 2-5A transfection at indicated times on immunoblots. The appearance of cleaved PARP was evident by 8 h in ABCE1 KD cells (9-fold more than WT) and remained sustained at 24 h compared to WT cells. As expected, PARP cleavage was barely detectable in RNase L KO and ABCE1 KD/RNase L KO cells. Caspase-3 activity, measured by levels of cleaved caspase-3 on immunoblots, showed a similar temporal profile in ABCE1 KD cells (2.5-fold more than WT at 8 h and 3.6-fold more at 24 h) compared to WT cells and low to no cleavage in RNase L KO cells and ABCE1 KD/RNase L KO cells ( Figure  4A,B). We measured caspase-3 enzyme activity in cell lysates using a fluorescent substrate following RNase L activation at indicated times and observed a 2-fold to 3.6-fold increase in ABCE1 KD cells compared to WT cells. No significant caspase-3 enzyme activity was detected in the lysates of RNase L KO cells and ABCE1 KD/RNase L KO cells ( Figure 4C). Based on our previous results, we hypothesized that increased apoptosis due to RNase L activation in ABCE1 KD cells could be attributed to caspase-3-mediated cleavage of Beclin-1. Subsequently, using immunoblot analysis, we determined increased cleavage of Beclin-1 (3.8-fold more than WT) 8 h after RNase L activation in ABCE1 KD cells compared to WT cells, which overlapped with increased caspase-3 activity ( Figure 4D). No significant modulation of caspase-3 activity or Beclin-1 cleavage was observed in RNase L KO cells and ABCE1 KD/RNase L KO cells. These results suggest that compared to WT cells, increased RNase L activity in ABCE1 KD cells enhances apoptosis by the increased caspase-3-mediated cleavage of Beclin-1 and cells lacking both ABCE1 and RNase L are resistant to apoptosis by 2-5A treatment.  The band intensity was calculated using Image J software and the ratio of cleaved PARP/β-actin or cleaved caspase-3/β-actin was determined and the levels were compared to WT cells. (C) Caspase-3/7 enzyme activity was measured in HT1080 WT, ABCE1 KD, RNase L KO, and ABCE1 KD/RNase L KO cells transfected with 10 µM of 2-5A for indicated times and the cleavage of fluorescent caspase-3 substrate was determined. Results shown represent mean ± SD for the experiment performed in triplicate and representative of three independent experiments. (D) Immunoblot analysis of HT1080 WT, ABCE1 KD, RNase L KO, and ABCE1 KD/RNase L KO cells transfected with 10 µM of 2-5A for indicated times and probed with Beclin-1 antibodies and normalized to β-actin levels. The band intensity was calculated using Image J software and the ratio of cleaved Beclin-1/β-actin was determined and the levels were compared to WT cells. Results are representative of three independent experiments. WT: wild-type; ** p < 0.01; *** p < 0.001; ns: not significant.

ABCE1 Regulates Autophagy during EMCV Infection
Previous work has shown that EMCV infection can induce autophagy in an RNase Ldependent manner and at later times of viral growth, inhibiting autophagy reduced EMCV yield [11,12]. Our results show increased autophagy in ABCE1 KD cells, so we determined the impact of ABCE1 on EMCV infection. HT1080 WT and ABCE1 KD cells were infected with EMCV at multiplicity of infection (MOI) of 1. Cells were harvested at 4 h and 8 h post infection, proteins were separated by denaturing polyacrylamide gel electrophoresis, and immunoblots were probed with LC3 and P62 antibodies to monitor autophagy induction. Lysates were also probed with anti-3D-Pol antibody to detect accumulation viral of the viral protein ( Figure 5A). lot analysis of HT1080 WT, ABCE1 KD, RNase L KO, and ABCE1 KD/RNase L KO cells transfected with 10 μM of 2-5A for indicated times and probed with Beclin-1 antibodies and normalized to β-actin levels. The band intensity was calculated using Image J software and the ratio of cleaved Beclin-1/β-actin was determined and the levels were compared to WT cells. Results are representative of three independent experiments. WT: wild-type; ** p < 0.01; *** p < 0.001; ns: not significant.

ABCE1 Regulates Autophagy during EMCV Infection
Previous work has shown that EMCV infection can induce autophagy in an RNase L-dependent manner and at later times of viral growth, inhibiting autophagy reduced EMCV yield [11,12]. Our results show increased autophagy in ABCE1 KD cells, so we determined the impact of ABCE1 on EMCV infection. HT1080 WT and ABCE1 KD cells were infected with EMCV at multiplicity of infection (MOI) of 1. Cells were harvested at 4 h and 8 h post infection, proteins were separated by denaturing polyacrylamide gel electrophoresis, and immunoblots were probed with LC3 and P62 antibodies to monitor autophagy induction. Lysates were also probed with anti-3D-Pol antibody to detect accumulation viral of the viral protein ( Figure 5A).   Figure 5F,G). Taken together, these results indicate that ABCE1 plays an important role in regulating EMCV-induced autophagy and that the enhanced autophagy induction in ABCE1 KD cells supports EMCV infection.

Discussion
The OAS/RNase L antiviral pathway is activated by IFN, produced during viral infections, and the activity of RNase L is regulated by 2-5A synthesized by certain OAS isoforms from cellular ATP. Many RNA and DNA viruses are susceptible to the antiviral effects of this pathway. Accordingly, viruses encode for proteins that antagonize or inhibit one or many steps of this pathway [17,18]. In addition, host cells express proteins that keep the activation of this pathway in check as RNase L cleaves cellular RNAs and inhibits protein synthesis that can be detrimental to cellular homeostasis. This study focused on one of the earliest identified cellular inhibitors of RNase L, ABCE1, also known as RNase L inhibitor (RLI). RLI was characterized as a negative regulator of the OAS/RNase L pathway by antagonizing 2-5A binding and nuclease activity of RNase L [38]. Our results show that the knockdown of ABCE1 in HT1080 fibrosarcoma cells results in increased RNase L activity in cells transfected with its activator, 2-5A, or synthetic dsRNA (poly I:C), which activates OAS to produce 2-5A. Two independent ABCE1 KD clones yielded similar results in the assays we studied, ruling out clone-specific artifacts ( Figure S1). A similar increase in RNase L activity in RLI KD cells was observed in prostate cancer cells activated with 2-5A [60]. A decrease in the inhibition of RNase L activity was also observed in Hela cells expressing RLI antisense cDNA [42]. We quantified the differences in RNase L activity across cell lines and samples using the RNA integrity number (RIN) to minimize variability [61]. The effects of ABCE1 on inhibiting RNase L is specific as cells lacking both proteins show no nucleolytic activity. In contrast, another recent study showed the interaction of ABCE1 with Dom34 (Pelota) and RNase L to function as a positive regulator of exogenous RNA decay [40]. It is not clear if transfected exogenous RNAs are processed differently from the endogenous RNAs cleaved in the cellular context and during viral infections. In line with other studies on RNase L activation, in our studies, the ligand 2-5A was delivered by complexing with lipid reagents, in contrast to the electroporation-mediated delivery used by Nogimori et al. [40]. Furthermore, basal levels of OAS proteins and RNase L vary by cell types and are determinants of IFN induction during viral infection, including EMCV [64]. Hela M cells are deficient in endogenous RNase L activity and have been used to reconstitute the expression of RNase L mutants [65][66][67]. It is not clear if the variable levels of endogenous RNase L in cell lines, along with differences in the dose and mode of delivery of 2-5A, can explain the differences in the two studies, and further investigation will be required. ABCE1 is evolutionarily conserved in archaea and eukaryotes, compared to the limited function of RNase L in higher vertebrates, suggesting more fundamental roles. Accordingly, recent studies have shown ABCE1 function in regulating translation, ribosome recycling, and ribosome homeostasis. Depletion of ABCE1 in yeast, Drosophila, and mammalian cells have shown a marked reduction in polysomes and accumulation of mRNA-free 80S monomers [51,68]. It is likely that in cells depleted of ABCE1, cellular RNAs inclusive of mRNAs are more accessible to RNase L for cleavage, causing enhanced degradation. Similar to other results, our data support the role of ABCE1 as an inhibitor of RNase L activity.
Activation of RNase L during a viral infection or directly by 2-5A transfection induced autophagy only in cells expressing WT RNase L and not in RNase L KO Mouse embryonic fibroblasts (MEFs) or cells with RNase L knockdown. Reconstituting RNase L KO cells with a RNase L mutant lacking enzyme activity did not restore autophagy, implicating RNA cleavage as a triggering event [11,12]. Our results show that RNase L activation in ABCE1 knockdown cells significantly enhanced autophagy at earlier time points that correlate with RNase L activity compared to parental WT cells or cells lacking RNase L or both proteins. RNase L cleaves single-stranded regions of viral and cellular RNAs, including rRNA, to produce short dsRNAs with signaling roles. RNase L-mediated rRNA cleavage can inhibit protein translation and disassembly and turnover of polysomes similar to observations made in ABCE1-depleted yeast, Drosophila, and mammalian cells. Therefore, the activation of RNase L in ABCE1 knockdown cells can synergistically enhance the accumulation of non-translating ribosomes that may be sequestered in autophagosomes by specialized autophagy, ribophagy, and serve as a recycling pathway.
RNase L participates in the cross-talk between autophagy and apoptosis. Depending on the cell type and the expression levels of apoptotic and anti-apoptotic proteins, RNase L activates caspase-3 to induce apoptosis [13,60,63,64]. Our previous studies showed that dsRNAs produced by RNase L induces caspase-3, which in turn cleaves autophagy protein, Beclin-1, to promote a switch from autophagy to apoptosis [13]. Here, we have demonstrated that RNase L activation in ABCE1 knockdown sensitizes cells to apoptosis and shows significantly reduced cell proliferation. Our results show a close correspondence of increased RNase L activity with caspase-3 enzyme activity, as observed previously in prostate cancer cells with ABCE1 knockdown. Here, we have shown that caspase-3 activity and cleavage of PARP is consistent with caspase-3-mediated cleavage of Beclin-1 observed at the same time points after RNase L activation. The knockdown of ABCE1 expression in human esophageal cancer, breast cancer, and small cell lung cancer cells causes reduced proliferation, increased apoptosis, migration, and invasion; however, the role of RNase L inhibition in mediating these effects remains to be determined [53][54][55]. It is likely that mammals may have evolved additional roles of ABCE1 in regulating the tumor suppressor roles of RNase L by regulating RNase L enzyme activity. ABCE1 has diverse roles during viral infections. The initial characterization of ABCE1 supported an inhibition of antiviral role of IFN during infection with EMCV and HIV. In these studies, ABCE1 was transcriptionally induced by virus infection and not IFN. In both instances, overexpression of ABCE1 inhibited RNase L activity and enhanced virus production; however, decreasing ABCE1 levels correlated with decreased viral yield [41,42]. Based on recent studies, ABCE1 may serve dual roles in HIV pathogenesis. Overexpression causes a decrease in RNase L activity and increased HIV production. In contrast, antisense ABCE1 (RLI) construct expression reverses inhibition of RNase L activity, with a corresponding decrease in HIV titers [41]. Additionally, ABCE1 (HP68) was shown to be required for immature HIV-1 capsid assembly [44]. Recently, ABCE1 has been shown to interact with gag and cellular protein, DDX6, to facilitate HIV-1 immature capsid assembly using RNA granules [43,69,70]. In other studies, ABCE1 was identified in a genome-wide screen as a proviral host protein needed for the efficient translation of measles (MeV), mumps (MuV), and RSV mRNA and not cellular RNAs [45]. Many viruses induce translation shut-off by perturbing mRNA or protein synthesis. However, paramyxovirus relies on ongoing protein synthesis and requires the ribosome recycling function of ABCE1. The polycistronic paramyxovirus mRNA could cause altered termination of ribosome, and hence the rigid requirement of ABCE1 to recycle ribosomes in sustaining viral replication. The initiation of translation during EMCV infection is cap independent due to its internal ribosome entry sites (IRES) that bind eIFs to directly recruit the 40S ribosomal subunit [71]. The precise role of ABCE1 in the translation of EMCV proteins will need further investigation. However, we observe an increase in the accumulation of EMCV proteins in ABCE1 KD cells detected on immunoblots by 3D polymerase antibodies ( Figure 5A). Previous studies have shown that autophagy induced by RNase L during SeV, EMCV, or VSV infections is antiviral in the early stages of infection and at later time points, subversion of autophagy promotes viral growth [11,12]. In our studies, early onset of autophagy in ABCE1 knockdown cells during EMCV infection increased virus yield. These observations support studies that indicate that autophagy promotes EMCV replication and the RNA replication during infectious cycle occurs on autophagosome-like membranes. The effect of autophagy on EMCV replication varies by viral strain, cell type, and multiplicity of infection (MOI) and length of infection. At lower MOI and single replication cycle, autophagy induced in WT cells has antiviral roles and at later times promotes viral growth [11,12]. In ABCE1 knockdown cells, early onset of autophagy facilitates replication, at levels higher than WT cells, and a switch to apoptosis possibly contributes to viral dissemination and perpetuation. While not explored here, additional roles of ABCE1 in ribosome recycling, regulating translation, and ribosome homeostasis may together contribute to EMCV pathogenesis. Our studies identify ABCE1 as a regulator of the OAS/RNase L pathway by balancing and fine-tuning the RNase L activity that can impact the outcomes of viral infections.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.