key: cord-0687214-ry5pnu9h authors: Kim, Yu-Jin; Witwit, Haydar; Cubitt, Beatrice; de la Torre, Juan C. title: Inhibitors of anti-apoptotic Bcl-2 family proteins exhibit potent and broad-spectrum anti-mammarenavirus activity via cell cycle arrest at G0/G1 phase date: 2021-08-17 journal: bioRxiv DOI: 10.1101/2021.08.16.456587 sha: 038f645a0bcca070976f5b48e2e0d112ae574a22 doc_id: 687214 cord_uid: ry5pnu9h Targeting host factors is a promising strategy to develop broad-spectrum antiviral drugs. Drugs targeting anti-apoptotic Bcl-2 family proteins that were originally developed as tumor suppressors have been reported to inhibit multiplication of different types of viruses. However, the mechanisms whereby Bcl-2 inhibitors exert their antiviral activity remain poorly understood. In this study, we have investigated the mechanisms by which obatoclax (OLX) and ABT-737 Bcl-2 inhibitors exhibited a potent antiviral activity against the mammarenavirus lymphocytic choriomeningitis virus (LCMV). OLX and ABT-737 potent anti-LCMV activity was not associated with their pro-apoptotic properties, but rather their ability of inducing cell arrest at G0/G1 phase. OLX and ABT-737 mediated inhibition of Bcl-2 correlated with reduced expression levels of thymidine kinase 1 (TK1), cyclin A2 (CCNA2), and cyclin B1 (CCNB1) cell cycle regulators. In addition, siRNA-mediated knock down of TK1, CCNA2, and CCNB1 resulted in reduced levels of LCMV multiplication. The antiviral activity exerted by Bcl-2 inhibitors correlated with reduced levels of viral RNA synthesis at early times of infection. Importantly, ABT-737 exhibited moderate efficacy in a mouse model of LCMV infection, and Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and SARS-CoV-2. Our results suggest that Bcl-2 inhibitors, actively being explored as anti-cancer therapeutics, might be repositioned as broad-spectrum antivirals. IMPORTANCE Anti-apoptotic Bcl-2 inhibitors have been shown to exert potent antiviral activities against various types of viruses via mechanisms that are currently poorly understood. This study has revealed that Bcl-2 inhibitors mediated cell cycle arrest at the G0/G1 phase, rather than their pro-apoptotic activity, plays a critical role in blocking mammarenavirus multiplication in cultured cells. In addition, we show that Bcl-2 inhibitor ABT-737 exhibited moderate anti-mammarenavirus activity in vivo, and that Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and SARS-CoV-2. Our results suggest that Bcl-2 inhibitors, actively being explored as anti-cancer therapeutics, might be repositioned as broad-spectrum antivirals. Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different 30 mammarenaviruses and SARS-CoV-2. Our results suggest that Bcl-2 inhibitors, actively being 31 explored as anti-cancer therapeutics, might be repositioned as broad-spectrum antivirals. Anti-apoptotic Bcl-2 inhibitors have been shown to exert potent antiviral activities 39 against various types of viruses via mechanisms that are currently poorly understood. This study 40 has revealed that Bcl-2 inhibitors mediated cell cycle arrest at the G0/G1 phase, rather than their 41 pro-apoptotic activity, plays a critical role in blocking mammarenavirus multiplication in 42 cultured cells. In addition, we show that Bcl-2 inhibitor ABT-737 exhibited moderate anti-43 mammarenavirus activity in vivo, and that Bcl-2 inhibitors displayed broad-spectrum antiviral 44 activities against different mammarenaviruses and SARS-CoV-2. Our results suggest that Bcl-2 45 inhibitors, actively being explored as anti-cancer therapeutics, might be repositioned as broad-46 spectrum antivirals. anti-apoptotic Bcl-2 inhibitors have been reported to exert antiviral activities against various 80 types of viruses (6-12). However, the mechanisms by which Bcl-2 inhibitors exert their antiviral 81 activities are not well understood. Several Bcl-2 inhibitors can arrest cells at the G0/G1 phase of 82 the cell cycle, which disrupts normal proliferation and migration of tumor cells (13) (14) (15) . Notably, 83 several viruses encode proteins that interact with cell cycle regulating proteins and alter cell 84 cycle progression in different ways (16), but how these interactions can impact virus 85 multiplication remains to be elucidated. In a previous drug screening, we identified OLX, an antagonist of anti-apoptotic Bcl-2 87 proteins, as a potent anti-mammarenaviral drug (17). To investigate the mechanisms whereby 88 Bcl-2 inhibitors could exert their anti-mammarenavirus activity, we selected the Bcl-2 inhibitors 89 OLX and ABT-737 due to their potent inhibitory effect on the activity of anti-apoptotic Bcl-2 90 proteins in vitro (18, 19) and in vivo (20-24). We found that the antiviral activity exerted by OLX Effect of Bcl-2 inhibitors on LCMV multiplication in cultured cells. 110 To investigate whether OLX-mediated anti-mammarenavirus activity was also observed 111 with other inhibitors of anti-apoptotic Bcl-2 family proteins, we compared the effect of the Bcl-2 Effect of Bcl-2 inhibition on LCMV RNA synthesis. 171 We previously reported that OLX treatment caused a significant reduction on LCMV and of viruses (6-12). However, the mechanisms by which these Bcl-2 inhibitors exerted their 213 antiviral activity are not well understood. In this study, we examined the mechanisms whereby 214 the Bcl-2 inhibitors OLX and ABT-737 exerted their potent anti-LCMV activity. We found that 215 OLX and ABT-737 potent anti-LCMV activity was not associated with their pro-apoptotic 216 properties, but rather their ability of inducing cell cycle arrest at G0/G1 phase. Small molecules targeting the Bcl-2 family proteins were first developed for cancer 218 treatment, as apoptotic cell death caused by inhibition of anti-apoptotic Bcl-2 proteins 219 contributes to reduce tumor progression (4, 5). We therefore examined whether apoptosis 220 induced by Bcl-2 inhibitors contributed to OLX and ABT-737 anti-LCMV activity. Our finding 221 that OLX and ABT-737 anti-LCMV activity was not affected in the presence of the pan-caspase 222 inhibitor zVAD-FMK or the caspase-3-specific inhibitor zDEVD-FMK (33, 34), indicated that 223 apoptosis did not contribute to the anti-LCMV activity exhibited by the Bcl-2 inhibitors OLX 224 and ABT-737. In addition to their apoptosis inducing property, Bcl-2 inhibitors have been reported to 226 regulate different cellular processes via mechanisms that are currently poorly understood (29). (Fig. 3B ). In contrast, treatment with dinaciclib, known to promote G2/M phase arrest, 240 resulted in highly increased TK1 expression. Our finding that OLX and ABT-737 treatment 241 significantly decreased expression levels of CCNA2 and CCNB1 (Fig. 3B) , further supported 242 that expression of G2/M phase regulators was prevented in the presence G0/G1 promoting Bcl-2 243 inhibitors. Our finding that siRNA-mediated knock-down of cell cycle regulators TK1, CCNA2, 244 and CCNB1 resulted in reduced levels of virus multiplication, suggested that these factors likely 245 play a role in OLX and ABT-737 mediated anti-LCMV activity (Fig. 3C) . We found that siRNA-246 mediated knock-down of Bcl-2 resulted in the strongest reduction on levels of LCMV 247 multiplication, which was associated also with reduced levels of TK1, CCNA2, and CCNB1 (Fig 248 3D ), suggesting that Bcl-2 is an upstream regulator of TK1, CCNA, and CCNB expression. progression or Bcl-2 inhibitor-mediated G0/G1 arrest (Fig. 3E) , and in contrast to IAV and CoVs, 256 cell cycle arrest at G0/G1 generates a cellular environment that contributes to limiting LCMV 257 multiplication. Intriguingly, treatment with ribavirin resulted also in increased numbers of cells 258 at the G0/G1 phase (Fig. 3E ), suggesting that cell cycle modulation might be another mechanism, 259 in addition to those previously documented (30, 41) by which ribavirin exerts its broad-spectrum 260 antiviral activity. 261 We previously showed that OLX did not significantly affect mammarenavirus cell entry 262 or budding, but strongly inhibited steady state levels of mammarenavirus RNA at 48 hpi (17). In 263 the present work we have shown that OLX and ABT-737 did not significantly affect levels of 264 LCMV replication at 2 hpi, but both compounds significantly inhibited viral genomic and anti-265 genomic RNA synthesis at 4 hpi (Fig. 4) . Under our experimental conditions, anti-genomic RNA OLX and ABT-737 exhibited antiviral activity also against two other mammarenaviruses, 287 ML29, and the most distantly related New World JUNV. In addition, consistent with recent 288 published findings, we found that OLX exerted a strong inhibitory effect on SARS-CoV-2 289 multiplication (Fig. 6) . These results suggest the possibility that Bcl-2 inhibitors actively being 290 explored as anti-cancer therapeutics, could be repositioned as broad-spectrum antivirals. Cells were plated and incubated at 37 °C and 5% CO 2 in DMEM containing 2% FBS. After 20 hours, each compound was added at the indicated concentration and for indicated times. (A-B) Adult C58BL/6 mice (n = 4/group) were treated with ABT-737 (20mg/kg/day; intraperitoneal route) 463 or vehicle for 17 days. At day 0, mice were infected with rCl-13 (2 x 10 6 pfu/mouse). Body weight 464 changes (A) and serum virus titers (B) were determined at the indicated time points. Statistical 465 significance was calculated by analysis of variance (ANOVA) (* p < 0.05, ** p < 0.002, *** p < 466 0.0002, and **** p < 0.00001). 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