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1 2 3 4 5 6 7 Crimean-Congo hemorrhagic fever replication interplays with regulation mechanisms of apoptosis Running title: CCHFV interplays with caspase induced apoptosis 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Helen Karlberg 1,2, Yee-Joo Tan 3,4,* and Ali Mirazimi 1,2,5* 1 Public Health Agency of Sweden, SE-171 82 Sweden Karolinska Institute, Stockholm, SE-171 77, Sweden 3 Department of Microbiology, Yong Loo Lin School of Medicine, National University Health System (NUHS), National University of Singapore, Singapore 4 Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore. 2 5 National Veterinary Institute, SE- 756 51 Uppsala Nr of Words Abstract: 223 Nr of Words MS: 4668 Nr of Figures: 6 * Corresponding authors: E-mails: [email protected] (A. Mirazimi) & [email protected] (Y.-J. Tan) 33 1 34 Abstract 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 Pathogenesis of viral hemorrhagic fevers (VHF) is associated with alteration of vascular barrier function and hemorrhage. To date, the specific mechanism behind this is unknown. Programmed cell death and regulation of apoptosis in response to viral infection is an important factor for host or virus survival but this has not been well-studied in the case of Crimean-Congo hemorrhagic fever virus (CCHFV). In this study, we demonstrated that CCHFV infection suppresses cleavage of poly (ADPribose) polymerase (PARP), triggered by staurosporine at early post infection. We also demonstrated that CCHFV infection suppresses activation of caspase-3 and caspase-9. Most interestingly, we found that CCHFV N can suppress induction of apoptosis by Bax and inhibit the release of cytochrome-c from the inner membrane of mitochondria to cytosol. However, CCHFV infection induces activation of Bid at late post infection, suggesting the activation of extrinsic apoptotic signaling. Consistently, supernatant from late post-infected cells stimulated was found to induce PARP cleavage, most probably through the TNF-α dead receptor pathway. In summary, we found that CCHFV has strategies to interplay with apoptosis pathways and thereby regulate caspase cascade. We suggest that CCHFV suppresses caspase activation at early stages of the CCHFV replication cycle, which perhaps benefits the establishment of infection. Furthermore, we suggest that the host cellular response at late post infection induces host cellular pro-apoptotic molecules through the death receptor pathway. 2 54 Introduction 55 56 57 58 59 60 61 62 Crimean-Congo hemorrhagic fever virus (CCHFV) is a member of the Nairovirus genus of the family Bunyaviridae. CCHFV is found worldwide (Asia, Africa Middle East, Europe) and causes severe disease in humans, with a reported mortality rate of up to 30%. CCHFV is transmitted through tick bites or through contact with viremic blood or tissues from patients or livestock. Viruses within this family encode for three single-stranded RNA segments with a negative sense. The large (L) segment encodes the RNA-dependent RNA polymerase, the medium (M) segment codes for the two mature structural glycoproteins (Gn and Gc) and the small (S) segment encodes the nucleocapsid protein (N) (Elliott, 1990; Ergonul et al., 2006). 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 The pathogenesis of viral hemorrhagic fevers (VHF) is associated with alteration of vascular barrier function and hemorrhage, most probably due to a combination of factors such as damage to the epithelium, consumption and degradation of clotting factors, and an overactive inflammatory response. The reduced capacity or dysfunction of endothelial cells observed in CCHF cases is believed to be due to virus-mediated host factors such as the pro-inflammatory response, to the virus infection itself, or a combination of both. Therefore, a hyperactive host response may be important in progression of the disease. Cell death might contribute to the increased permeability of the vascular system and bleeding disorders observed among CCHFV patients. Clinical studies indicate that high viremia is correlated with high cytokine levels and disease severity (Bray, 2005; Schnittler & Feldmann, 2003; Weber & Mirazimi, 2008). We have previously demonstrated that CCHF virus can induce cleavage of poly (ADP-ribose) polymerase (PARP), which is associated with apoptosis and has been cited as one hallmark of apoptosis and caspase activation at late post infection (Karlberg et al., 2011). Rodrigues et al. subsequently showed that apoptosis is induced in human hepatoma cells at late stages of CCHFV infection (Rodrigues et al., 2012). Caspase-dependent apoptosis occurs following the activation of two main pathways initiated by both internal (intrinsic/mitochondria-dependent pathway) and external stimuli (extrinsic/death receptor-mediated pathway). Caspases, cysteine aspartate-specific proteases, are activated and amplify the apoptotic signaling pathways, leading to cell death. Effector caspases, including caspase-3, -6, and -7, and their activation are controlled by upstream initiator caspases such as caspase-8 and -9. Extrinsic and intrinsic pathways may function separately or mediate a crosstalk interconnection through cleavage of pro-apoptotic Bid, a member of the BCL-2 family of cell death regulators, which finally ends with cell death (Thornberry, 1998; Zimmermann et al., 2001). Pro-inflammatory host-derived mediators may contribute to receptor-mediated apoptosis in response to viral infection. External stimuli activate death receptors that include TNFR, Fas/CD95, and TRAIL, which function to induce apoptosis. Upon binding of their ligands, these receptors become activated and initiate activation of caspase-8, which in turn activates Bid. The intrinsic, or mitochondria, pathway is associated with the release of mitochondria proteins such as cytochrome-c, which activate downstream caspase activity through activation of caspase-9 from the intermembrane space into the cytoplasm (Thornberry, 1998; Zimmermann et al., 2001; Zimmermann & Green, 2001). Intracellular processes can result in loss of mitochondrial integrity via pro-apototic members of the BCL-2 superfamily members, for example Bid, Bak and Bax, which act directly on the outer mitochondrial membrane, facilitating secretion of mitochondria proteins (Zimmermann et al., 2001; Zimmermann & Green, 2001). 3 100 101 102 The aims of this study were to determine whether CCHFV regulates apoptosis pathways and to investigate the mechanism behind the regulatory properties of CCHFV, which interferes with the apoptotic signaling chain during the virus replication cycle. 103 4 104 105 106 107 Results CCHFV replication suppresses activation of caspase-3 and caspase-9. 108 109 110 111 112 113 114 115 116 117 We have previously demonstrated that CCHFV infection induces caspase-3 activation at late post infection in SW-13 (Karlberg et al., 2011). In order to investigate the events early in the CCHFV replication cycle, in the present study, mock or CCHFV infected SW-13 cells (MOI 1 at 24 h.p.i.) were treated with different concentrations of STS, a relatively non-selective protein kinase inhibitor, which is often used as a general method for inducing apoptosis. At 5 hours post treatment, detached cells were harvested and analyzed by Western blot. It was found that cleavage of PARP was clearly suppressed in the CCHFV-infected cells, compared with the mock-infected cells, in the 4µM STS treatment (Figure 1A-B). However, at the highest concentration of STS used, no inhibition of PARP cleavage within infected cells was observed. 118 119 120 121 122 123 124 125 126 To investigate whether CCHFV infection regulates caspase-3 activation during STS treatment, SW13 cells were infected and treated as described above (with 4uM STS) and analyzed for the presence of caspase-3 cleavage. It was found that the activation of caspase-3 in the CCHFV infected cells was significantly reduced compared with the mock-infected cells (Figure 2A-B). To further characterize the interaction of CCHFV replication and the apoptosis pathway, we analyzed these samples for STS-induced cleavage of caspase-9, which is upstream of caspase-3. The results showed that cleavage of caspase-9 was also suppressed during the early phase of CCHFV replication cycle in STS-treated infected cells, in contrast to mock infected cells (Figure 2C-D). 127 CCHFV infection suppresses release of cytochrome-c in apoptotic cells 128 129 130 131 132 133 134 135 In order to investigate whether CCHFV infection interferes or interplays with release of cytochrome-c from inner mitochondria membrane in apoptotic cells caused by STS, mock infected and CCHFV-infected cells (24 h p.i.) were not treated or treated with STS. Attached and detached cells were harvested at 5 h post treatment, and the cytosol and mitochondria fractions were collected separately ((Gogvadze et al., 2003)) and analyzed for estimated release of cytochrome-c by WB. It was found that release of cytochrome-c into the cytosol was decreased in infected cells compared with mock-infected cells, for both attached and detached cells (Figure 3). 136 137 138 139 140 141 142 143 144 145 146 147 148 CCHFV N protein suppresses activation of apoptosis by Bax Release of proteins, such as cytochrome-c, from mitochondria inter-membrane space to cytosol is controlled by BCL-2 pro-apoptotic members such as Bax. To investigate whether the CCHFV N protein is involved or contributes to regulation of mitochondria membrane permabilisation and downstream caspase activation, we established an in vitro system (see Materials & Methods section). The SW13 cells were transfected with a plasmid coding for CCHFV ORF N (myc-N), with different concentrations and a plasmid coding for Bax (flagBax), a potent inducer of apoptosis that acts via the mitochondria. Induction of apoptosis in the presence and absence of CCHFV N was then analyzed by measuring the activation of caspase-3. The results showed that overexpression of Bax (with a flag epitope at the N terminus) in SW13 cells induced a high level of apoptosis, as determined by the activation of caspase-3 and cleavage of endogenous PARP, when compared with mock transfected cells (Figure 4). However, the level of apoptosis induced by Bax was significantly reduced when 5 149 150 151 152 153 Bax was co-expressed with different amounts of recombinant N protein. These results suggest that expression of N in the early phase of CCHFV infection may suppress the apoptotic processes that are coordinated by Bax or downstream of Bax and cause the CCHFV-infected cells to become resistant to apoptosis. 154 155 Pro-inflammatory cytokines released during CCHFV infection can induce PARP cleavage 156 157 158 159 160 161 162 163 164 165 166 167 As demonstrated above, we have evidence that at 24 h p.i., CCHFV has the ability to suppress caspase-3 activation through inhibition of release of cytochrome-c by interfering with Bax This finding seems to contradict our previous observation that CCHFV infection induces apoptosis at late post infection (Karlberg et al., 2011). To investigate whether CCHFV infection initiates apoptotic signaling pathways through the extrinsic pathway at late post infection, SW13 were mock or CCHFV-infected (MOI 1). At 24 and 48 h p.i., cells were harvested and analyzed for activation and cleavage of Bid (truncated Bid, tBid), which is a pro-apoptotic member of the BCL-2 family, by WB. External stimuli such as proinflammatory factors (TNF-α) activate the extrinsic or death receptor signaling pathways, which in turn activate caspase-8, leading to activation of Bid. Our results clearly demonstrated that CCHFV infection at late post infection led to cleavage of Bid to tBid, in attached and detached cells (Figure 5). 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 It has been demonstrated, by us and others (Karlberg et al., 2011; Rodrigues et al., 2012), that CCHFV infection induces secretion of TNF-α and other pro-inflammatory factors at late post infection through activation of dendritic cells and macrophages. In the present study we examined whether supernatant of infected SW13 releases pro-inflammatory factors, such as TNF-α, during infection, using SW13 cells which were mock infected or CCHFV-infected (MOI 1) and harvested at 24, 48, and 72 h.p.i. Analysis of supernatants for the presence of TNF-α showed that infected cells secreted TNF-α at late post infection (data not shown). However, to confirm whether the induction of cleavage of PARP at late post infection is due to an extrinsic pathway, all supernatants, mock and CCHFV infected, were UV-inactivated in order to inactivate the virus (which may be present in the supernatant) and then transferred to seeded new SW13 cells for 24 hours. The presence of CCHFV N, as an indicator of inactivation with UV and cleavage of PARP, was determined by Western blot for all lysates (data not shown). The results showed clearly that activation and cleavage of PARP was induced in cells where supernatant from infected cells collected at either 48 or 72 h.p.i. was added (Figure 6A-B). In contrast, no PARP cleavage was observed for cells incubated with supernatant from mock infected or UV-inactivated virus infected cells. 184 185 186 187 188 189 190 In order to characterize the process in more detail, supernatants collected at 48 h.p.i. from infected cells were mock treated or treated with neutralizing TNF-α antibodies (5ug/mL) for 1 h at 37oC and transferred to seeded SW13. At 24 h post treatment, detached cells were harvested and analyzed with Western blot. The analysis showed that antibodies against TNF-α significantly suppressed cleavage of PARP (Figure 6C), indicating that TNF-α secreted from infected cells is a contributor to induction of the extrinsic apoptotic pathway during the late phase of infection. 191 6 192 193 Discussion 194 195 196 197 A characteristic of CCHF and other VHF is loss of epithelium cell function, in particular function of endothelial cells, which leads to changes in vascular permeability and dysfunction and imbalanced fluid distribution between the intra- and extra-vascular tissue space, causing coagulation disorders, hemorrhage, and multi organ failure (Peters & Zaki, 2002). 198 199 200 201 202 203 204 205 206 207 Recently, two animal models with defective interferon response, IFNAR- and STAT1- mice, which are killed by/susceptible to CCHFV infection, have been used to study the pathogenesis of the disease (Bente et al., 2010; Bereczky et al., 2010). Infected mice developed leukopenia, thrombocytopenia, and elevated levels of liver enzymes. The highest levels of CCHF vRNA were detected mainly in liver and spleen. These organs were clearly affected, with visible symptoms such as necrosis of the liver and massive lymphocyte depletion of the spleen. Infected mice also had elevated levels of pro-inflammatory cytokine (TNF-α, IL 6, IL10) in serum/blood samples (Bente et al., 2010; Bereczky et al., 2010). An over-stimulated host protective response might be one contributing factor to coagulation disturbances, with hemorrhage and the overall vascular system affected. 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 In previous studies, we and others revealed that caspase-dependent apoptotic pathways are induced in different human cell types late during the CCHFV replication cycle (Fraisier et al., 2014; Karlberg et al., 2011; Rodrigues et al., 2012). Activation of executor caspase-3 then leads to cleavage of the viral structural protein, N. We found that caspase activation does not favor progeny viral production and therefore suggested that it is induced by the host cell as a protective response, rather than induced by the virus infection (Karlberg et al., 2011). Viruses have different strategies to interfere with cell death pathways during infection to secure efficient progeny viral production by expressing viral proteins (encode gene products) or down-/up-regulate cellular pro- and anti-apoptotic proteins, which interfere with apoptotic signaling pathways. Rift Valley fever virus, a member of the Bunyaviridae, encodes NSm protein with anti-apoptotic functions by inhibiting caspase-3 activity and upstream initator caspases (Won et al., 2007). Another member of the Bunyaviridae, La Crosse virus, induces increased levels of cellular BCL-2 during infection, both in vivo and in vitro, which prolongs cell survival and seems to be important in decreasing apoptosis and preventing tissue damage (Pekosz et al., 1996). 223 224 225 226 227 228 In the present study, where SW13 cells were used as a model system, we demonstrated that apoptotic signaling pathways are regulated early during the CCHFV replication cycle. By challenging CCHFV-infected cells at 24 h p.i. with STS, we were able to show that activation both upstream and on executor caspase level (caspase-9 and caspase-3) was suppressed. In line with these results, we also showed that activation and cleavage of PARP, a marker of apoptosis, are suppressed. 229 230 231 232 233 234 235 236 Opening of the mitochondrial permeability transition (MPT) pore and loss of membrane potential leads to release of pro-apoptotic proteins such as cytochrome-c from the intermembrane space into the cytosol of cells undergoing apoptosis (Zimmermann et al., 2001). The released cytochrome-c and Apaf-1 form a complex known as the apoptosome, leading to caspase-9 activation, which further activates downstream effector caspases (Zimmermann et al., 2001). In the present study, we showed that secretion of cytochrome-c from the mitochondria into the cytosol was decreased in STS-treated CCHFV-infected cells, but not in STS-treated mock infected cells. This indicates that the release of mitochondria 7 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 proteins is regulated and caspase activation downstream of mitochondria level, including caspase-3, is suppressed or delayed during the early phase of CCHFV infection. It has previously been demonstrated that hepatitis C virus (HCV) encodes proteins with antiapoptotic activity, which interfere with apoptosis signaling pathways by different mechanisms. HCV NS2 interferes with caspase-dependent induced apoptosis by counteracting cytochrome-c release from mitochondria, which contributes to viral persistence by interfering with host cell defense (Erdtmann et al., 2003). In the mitochondrial pathway, pro-apoptotic members of the BCL-2 super family are associated with the mitochondria and release of cytochrome-c. Bax is one of the key pro-apoptotic (BCL-2 member) molecules, an inactive monomer, and is normally found in the cytosol or loosely bound to the mitochondria membrane. Upon activation by apoptotic stimuli, Bax changes confirmation and forms oligomers, which are then integrated by formation of membrane pores that facilitate release of mitochondrial proteins such as cytochrome-c, which leads to downstream caspase activation (Antonsson et al., 2000; Wolter et al., 1997). By using a recombinant expression system, we showed that the overexpression of viral structural nucleocapsid (N) could inhibit Bax-induced apoptosis. These results indicate that the high level of CCHFV N expressed in infected cells has the ability to protect against apoptotic stimuli by acting at the level of Bax or downstream of it. Hence N could be one of the major factors controlling cell death in the early phase of infection. It has recently been demonstrated that rubella virus (RV) capsid protein interferes with import of Bax into mitochondria and thereby prevents mitochondria membrane permabilisation and promotes cell survival, and that adenovirus (ADV) induces expression of anti-apoptotic protein E1B 19K (vBcl-2), which inhibits the TNF-α-mediated death signaling pathway, including release of mitochondria proteins such as cytochrome-c by directly interacting with Bax (Ilkow et al., 2011; Sundararajan et al., 2001). It has also been reported that Hantaan virus (HTNV) nucleocapsid protein modulates apoptosis pathways through NFƙB (Ontiveros et al., 2010). 263 264 265 266 267 268 269 270 271 272 As discussed above, we have evidence that at 24 h p.i., CCHFV infection is able to inhibit caspase-3 activation through regulation of cytochrome-c release by interplaying or interfering with Bax. This finding can be interpreted as contradicting our previous finding that CCHFV infection induces apoptosis at late post infection (Karlberg et al., 2011). External stimuli through the death receptor pathway might initially induce caspase activation, triggered by the viral infection, and often results in crosstalk between the extrinsic and intrinsic cell death pathways through the pro-apoptotic member Bid, thereby facilitating induction of the mitochondrial pathway (Luo et al., 1998; Perez & White, 2000). Transmissible gastroenteritis virus infection induces both the FasL and mitochondria pathways through interconnection with Bid and upregulation of Bid (Ontiveros et al., 2010). 273 274 275 276 277 278 279 280 281 282 283 284 In the present study, we clearly demonstrated that Bid is active at late post infection and also that supernatant of CCHFV-infected cells contains the pro-inflammatory factors. These findings together indicate that CCHFV at late post infection most probably induces apoptosis through induction of pro-inflammatory factors, which in turn activates the extrinsic apoptotic pathway. This assumption is in line with the data in Figure 6A and B, which show that UVinactivated supernatants collected from CCHFV-infected cells at 48 and 72 h p.i. can induce cleavage of PARP in fresh cells from as early as 24 h post-treatment. Since all the supernatants collected were UV-inactivated before being transferred to the fresh cells, apoptosis could not have been induced by virus replication. Instead, it is most likely caused by pro-inflammatory mediators released into the medium as a cellular protective response to infection. This suggests in turn that caspase activation is triggered by external stimuli as a secondary effect. In the present study, we also demonstrated that neutralizing TNF-α 8 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 antibodies reduced the cleavage of PARP triggered by supernatant from infected SW13 cells at 48 h p.i. (Fig 6C). However, the neutralization was not complete, most probably because we could not neutralize all TNF-α or because other molecules are also involved in triggering the extrinsic pathway. Hepatitis C virus induces apoptosis by an indirect immunologically mediated mechanism and viral core protein enhances this process through TNF signaling pathways (Zhu et al., 1998). Cytokines and chemokines regulate the host immune response, but when present in high concentrations might contribute to toxic effects. Dysregulation of the cytokine and chemokine response is anticipated to cause capillary fragility, leading to leakage of erythrocytes and plasma through the vascular endothelium and resulting in dangerous hypotension, which is a prominent feature of VHF (Geisbert & Jahrling, 2004). Previous studies have shown that key molecules in CCHF progression seem to be cytokines such as IL 6, IL 8, and TNF-alpha. In an animal model study, mice lacking STAT-1 had elevated levels of IL-6, IL-10, and TNFalpha concentrations, confirming findings in some severe human cases (Bente et al., 2010; Ergonul et al., 2006; Papa et al., 2006). 300 301 302 303 304 305 306 307 308 In summary, this study showed that CCHFV has strategies to interplay with apoptotic molecular signaling pathways and thereby regulatory properties. It also showed that CCHFV N possesses the function of suppressing mitochondria permeability through Bax, which in turn has an influence on downstream executor caspase activity. This delays or suppresses induced caspase activation during the early phase of the CCHFV replication cycle. This event perhaps works to the benefit of the virus, helping or allowing it to establish infection. In later phases of CCHFV infection, we suggest that the host cellular response may induce the death receptor pathway, leading to activation of Bid. However, this hypothesis should be followed up in future experiments. 309 310 9 311 312 Methods 313 Cells, antibodies, chemicals and virus 314 315 316 317 318 319 320 321 322 323 324 325 326 SW13 cells (human adrenal cortex adeno carcinoma cells) were maintained in Leibovitz’s medium (L15) supplemented with 2% fetal bovine serum and antibiotics (10 U/mL penicillin and 10 µg/mL streptomycin). The antibodies used in this study included a rabbit polyclonal anti-CCHFV nucleocapsid antibody (Andersson et al., 2004). Anti-myc monoclonal (Santa Cruz Biotechnology, Santa Cruz, CA), anti-actin monoclonal, anti-flag polyclonal (Sigma, St. Louis, MO), anti-PARP polyclonal, anti-caspase 3, 9 monoclonal (Cell Signaling Technology, Beverly, MA), anti-cytochrome c and anti-TNF-α antibodies, recombinant TNF-α (rTNF-α) (BD Pharmingen), and staurosporine (STS) (Cell Signaling Technology) were used according to the manufacturer’s instructions. Horseradish peroxidase (HRP)-conjugated antibodies (Pierce, Rockford, IL or Bio-Rad, Hercules, CA) were used, again according to the manufacturer’s instructions. The Nigerian CCHFV Ibar10200-strain, originally isolated in Nigeria, was used in the experiments and all handling of live virus was performed in a BSL-4 facility. 327 328 329 UV-inactivation Harvested cell supernatant was centrifuged at 13000 rpm for 10 minutes and irradiated at 254 nm (UVG-54) in order to inactivate viral particles. 330 In vitro infection model (challenging SW13 cells) 331 332 333 334 335 SW13 cells were seeded in 6-well plates and then infected with CCHFV (MOI=1). After 24 hours post infection (h.p.i.), cells were challenged with 4, 8, 16, or 32 µM STS to induce apoptosis. The cells were then further incubated for 5 hours, harvested (detached and attached cells separately), and analyzed with Western blot analysis for activation of caspase-3, caspase-9 and induced cleavage of PARP. 336 337 338 339 340 SW13 cells were infected with CCHFV, mock infected or treated with UV-inactivated virus. At different times post infection (24, 48 and 72 h p.i.), supernatants were harvested and UVinactivated. These supernatants were then transferred to new SW13 cells seeded in 6-well plates. At 24 h p.i., the cells was collected and analyzed for cleavage of PARP in the absence of replicating virus. 341 Cytosolic and mitochondrial cytochrome-c analysis 342 343 344 345 346 347 348 349 350 In order to analyze the release of cytochrome-c from mitochondria, SW13 cells were seeded in 6-well plates and either infected with CCHFV (MOI=1) or not infected. At 24 h p.i., the cells were incubated with apoptotic stimuli (0.1 µM STS) overnight 37°C, and attached and detached cells were harvested separately, centrifuged for 5 min at 5000 rpm, washed with icecold PBS, and centrifuged a second time. The pellets were resuspended in a solution containing 0.15 M KCl, 5mM Tris, 1mM MgCl2, and 0.01% digitonin, incubated on ice for 20 min, and thereafter centrifuged at 10 min at 13000 rpm. Cytosol and mitochondria fractions (Gogvadze et al., 2003) of mock and infected cells, detached and attached, were harvested and analyzed using Western blot with anti-cytochrome-c antibodies. 10 351 352 353 354 355 356 357 358 359 360 361 362 363 Transient transfections and CaspACE fluorometric assay Transient transfections of SW13 cells were performed using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Expression plasmids for N with N-terminal myc-tag and Bax with N-terminal flag-tag were previously constructed (Karlberg et al., 2011; Mohd-Ismail et al., 2009).Approximately 24 h after transfection, the cells were harvested by scraping them into the medium, spun down in a bench-top centrifuge, and washed twice with cold PBS. The cell pellets were then resuspended in RIPA buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 0.5% NP40, 0.5% deoxycholic acid, 0.005% SDS, and 1 mM phenylmethylsulfonyl fluoride) and subjected to freeze-thawing five times before being centrifuged at 13000 rpm to remove cellular debris. The cell lysate was then used for Western blot analysis and the activation of caspase-3 was quantified using the CaspACE fluorometric assay system from Promega Corporation (Madison, WI). 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 SDS-PAGE and Western blot analysis Samples were resuspended in reducing sample buffer (10mM Tris-HCl, 0.5% SDS, 10% glycerol; 2% -mercaptpethenol, bromophenol blue), boiled for 5 minutes, and separated by SDS-PAGE using pre-cast gels from Life technologies (Europe). Electrophoresis was carried out at 200V and proteins were transferred to nitrocellulose membranes using a transfer buffer containing 25 mM Tris, 192mM glycine, and 20% methanol at 100 V for 1 hour. Membranes were blocked in 5% non-fat dry milk overnight at 4C. After washing in PBS containing 0.01% Tween (PBST), the membranes were incubated with primary antibody for 1 hour at room temperature or overnight at 4C. The membranes were then washed with PBST before addition of secondary antibody. After incubation at room temperature for 1 hour, the membranes were washed in PBST. Proteins were detected with ECL Plus Western Blotting Detection Reagents (Amersham Pharmacia, Buckinghamshire, UK) according to the manufacturer’s instructions. The intensity of bands were analysed within the linear range of the detector. Percent of cleaved product is analyzed compared with total density of product (Cleaved and uncleaved) in each well. Mean of three independent experiments are analyzed (see statistical analysis). 380 Measurement of pro-inflammatory mediators 381 382 383 384 Supernatant from infected SW13 cells (MOI 1) were collected at 24, 48, and 72 h p.i., centrifuged at 13000 rpm 10 min, and assayed for different cytokines and chemokines according to the manufacturer’s instructions (Qiagen Mix-N-Match Multi-Analyte ElisaArray kit). 385 Neutralizing assay 386 387 388 389 390 391 SW13 were infected with CCHFV, mock infected, or treated with UV-inactivated virus. Supernatants were harvested 48 h p.i., centrifuged at 13000 rpm for 10 min, and UVinactivated. The supernatants and rTNF-α suspension (2ng/mL) were pre-incubated separately with or without neutralizing antibody against TNF-α (5 μg/mL) for 1 h at 37C and subsequently transferred to seeded SW13 and incubated for 24 hours. All material was analyzed with Western blot for PARP activation. 392 Densitometric and Statistical analysis 393 394 Densitometric analysis was done on the western blot results using quantity software to determine the intensity of each band. Each band (cleavage products and uncleaved product) 11 395 396 397 398 399 were correlated to the total amount of each proteins PARP, Caspase3, etc), which then be used for statistical analysis. 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After 5 hours post treatment, detached cells were harvested and analyzed with Western blot. (A) Percent of cleaved PARP compared with total density of PARP. Mean of three independent experiments. Error bars = S.D. Significance level: p<0.001 (***), p<0.01 (**). NS: Non-Significance. (B) Cleavage of PARP in treated cells with 4 or 32 µM STS in one representative gel, as analyzed in Figure 1A. 492 493 494 495 496 497 498 499 Figure 2. CCHFV infection inhibits activation of caspase-3 and caspase-9 induced by STS. SW13 cells were mock or CCHFV infected (MOI=1). At 24 h p.i., detached cells were treated with 4 uM STS. After 5 hours post treatment, detached cells were harvested and analyzed with Western blot. (A) Percent of cleaved caspase-3 relative to total density of caspase-3. Mean of three independent experiments. Error bars = S.D. (B) Cleavage of caspase-3 in one representative gel, as analyzed in Figure 2A. (C) Percent of cleaved caspase-9 relative to total amount of caspase-9. Mean of three independent experiments. Error bars = S.D. (D) Cleavage of caspase-9 in one representative gel, as analyzed in Figure 2C. Significance level: p<0.0005 500 501 502 503 504 505 506 507 Figure 3. Release of cytochrome-c is suppressed in STS-treated CCHF virus-infected cells. SW13 cells were mock or CCHFV infected (MOI=1). At 24 h p.i., cells were treated with 4 STS uM for 5 hours. Attached and detached cells was separated and fractionated into cytosolic and mitochondrial fractions. Release of cytochrome-c from mitochondria to the cytosol following CCHFV infection was analyzed by Western blot with anti-cytochrome c antibody. (A) Percent of cytochrome-c released to the cytosol. Mean of three independent experiments. Error bars = S.D. Significance level: p<0.0005 (***). (B) A representative gel, as analyzed in Figure 2A. Mitochondrial fraction (a and c) and cytosolic fraction (b and d). 508 509 510 511 512 513 514 515 516 517 518 519 520 521 Figure 4. CCHFV N protein suppresses activation of apoptosis by Bax. (A) A CaspACE fluorometric assay system from Promega Corporation (Madison, WI) was used to measure the activation of caspase-3 in SW13 cells that were mock transfected or transfected with CCHFV N only, or Bax in the absence or presence of CCHFV N. DNAs used in each of the transfections are indicated in micrograms. In each transfection, the total amount of DNA was normalized to 1.8 µg with the addition of empty vector if necessary. All experiments were performed in triplicate, and mean with standard deviations are plotted. (B) Western blot analysis also was performed to determine the cleavage of endogenous PARP (top). Similarly, the expression levels of the different proteins were determined using anti-flag or anti-myc antibodies (middle). The amounts of total cell lysates loaded were verified by measuring the levels of endogenous actin (bottom). Significance level: p<0.05 (*). 522 523 524 525 526 527 528 529 530 531 Figure 6. CCHFV infection induces apoptosis through induction of pro-inflammatory response in cells. SW13 cells were CCHFV (a), UV-treated CCHFV (b) or Mock infected (c) (MOI=1). At 24, 48, and 72 h p.i., the supernatant was collected and treated with UV. The UV-treated supernatant was then transferred to new freshly seeded SW13. After 24 h, cells were harvested and analyzed by Western blot. (A) Percent of cleaved PARP compared with the total amount of PARP. Mean of three independent experiments. (B) Cleavage of PARP in one representative gel, as analyzed in Figure 6A. (C) SW13 cells were incubated with rTNF-α or supernatant from 48 h p.i. infected cells, with or without anti TNF-α. After 24 h, cells were harvested and analyzed by Western blot. Percent of cleaved PARP compared with the total density of PARP. Mean of three independent experiments. Significance levels: p < 0.0005 (***), p < 0.005 (**), p < 0.05 (*) Figure 5. CCHFV infection induces activation of Bid. SW13 cells were mock or CCHFV infected (MOI=1). At 24 and 48 h p.i., attached and detached cells were harvested and analyzed for presence of activation of Bid (tBid). 16