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Inhibitor of sarco/endoplasmic reticulum calcium-ATPase impairs paramyxovirus replication

Sarco/endoplasmic reticulum calcium-ATPase (SERCA) is a membrane bound cytosolic enzyme that is known to regulate the uptake of calcium into the sarco/endoplasmic reticulum. Herein, we demonstrate for the first time that SERCA can also regulate paramyxovirus [Peste des petits ruminants virus (PPRV) and Newcastle disease virus (NDV)] replication. Treatment of Vero cells with SERCA specific inhibitor (Thapsigargin) at a concentration that is nontoxic to the cells significantly reduced virus replication. Conversely, overexpression of SERCA rescued the inhibitory effect of Thapsigargin on virus replication. PPRV/NDV infection induced SERCA expression in Vero cells which could be blocked by Thapsigargin. With the help of time-of-addition and virus step-specific assays, it was observed that Thapsigargin specifically inhibits viral entry and subcellular localization of the viral proteins. Furthermore, NDV, but not PPRV acquired a significant resistance to Thapsigargin on long-term passage (P=70) in Vero cells. To the best of our knowledge, this is the first report describing virus supportive role of SERCA and a rare report suggesting that viruses may acquire resistance even in the presence of an inhibitor that targets a cellular factor. This study will contribute in understanding paramyxovirus replication and development of antiviral therapeutics using SERCA (host factor) as a candidate drug target.


ABSTRACT 21
Sarco/endoplasmic reticulum calcium-ATPase (SERCA) is a membrane bound 22 cytosolic enzyme that is known to regulate the uptake of calcium into the 23 sarco/endoplasmic reticulum. Herein, we demonstrate for the first time that SERCA can 24 also regulate paramyxovirus [Peste des petits ruminants virus (PPRV)  time-of-addition and virus step-specific assays, it was observed that Thapsigargin 31 specifically inhibits viral entry and subcellular localization of the viral proteins. 32 Furthermore, NDV, but not PPRV acquired a significant resistance to Thapsigargin on 33 long-term passage (P=70) in Vero cells. To the best of our knowledge, this is the first 34 report describing virus supportive role of SERCA and a rare report suggesting that 35 viruses may acquire resistance even in the presence of an inhibitor that targets a cellular 36 factor. This study will contribute in understanding paramyxovirus replication and 37

INTRODUCTION 43
The control strategies against pathogens have classically relied upon targeting 44 essential proteins of the pathogens (1). High mutation rate in viral genome allows the 45 virus to become resistant to antiviral drugs and preexisting immunity (2). Classically, 46 antiviral drugs have been developed by directly targeting viral proteins (3). Due to high 47 mutation rates, virus gets mutations at the druggable targets and becomes resistant. The 48 rise in incidence of drug resistance has prompted a shift in the development of novel 49 antiviral drugs (4). Viruses are obligate intracellular parasites that are highly dependent 50 on host. Host responses are equally important in determining actual outcome of the 51 diseases. Upon viral infection, numerous cellular factors are dysregulated (increased or 52 decreased expression); some of these host factors facilitate virus replication (proviral), 53 whereas others may have antiviral function (5). Proviral host factors may serve as 54 targets for development of novel antiviral therapeutics (1, 6, 7). 55 8 for 96 h. However, at higher concentrations (>2 µM), it was found to be toxic to the 162 cells. A non-cytotoxic concentration (0.5 µM) of Thapsigargin was used thereafter. 163 In order to determine the in vitro antiviral efficacy of Thapsigargin, we 164 measured the yield of infectious PPRV/NDV/BHV-1/BPXV in the presence of 0.5 µM 165 inhibitor or vehicle control (DMSO). Thapsigargin significantly inhibited 166 paramyxovirus viz; NDV (Fig. 1c), PPRV (Fig. 1d) replication. It also significantly 167 inhibited BHV-1 (Fig. 1e) but not BPXV (Fig. 1f) replication (DNA viruses), 168 suggesting its potent antiviral activity against paramyxoviruses and BHV-1 virus. 169 Furthermore, in order to determine whether the antiviral efficacy of 170 Thapsigargin against paramyxoviruses is partially due to direct inactivation of the cell 171 free virions, we incubated the infectious virions with either 0.5 µM or 5 µm 172 Thapsigargin for 1.5 h and subsequently tested the residual infectivity on Vero/MDBK 173 cells. Thapsigargin did not exhibit any virucidal effect on any of the prototype virus 174 tested (Fig. 1g) suggesting that the antiviral activity of Thapsigargin is due to the 175 inhibitory effect on virus replication in the target cells. 176 SERCA facilitates paramyxovirus replication. In order to further confirm the 177 role of SERCA in virus replication, HeLa/goat kidney cells were transfected with 178 plasmid expressing SERCA2 (pCR3-SERCA2), followed by viral infection. As 179 compared to control plasmid (empty vector)-transfected cells, overexpression of 180 SERCA2 not only facilitated NDV/PPRV replication, but also rescued the inhibitory 181 effect of Thapsigargin on virus replication suggesting that SERCA2 supports 182 paramyxovirus replication (Fig. 2a and 2b).
expression. PPRV infection of Vero cells resulted in enhanced SERCA2 expression. As 186 compared to mock-infected cells, a significant induction in SERCA2 expression was 187 observed at 3 hpi, which remained at the peak level between 24-72 hpi, before started 188 declining at 96 hpi (Fig. 3a, upper panel). However, the levels of house keeping 189 control gene (GAPDH) were similar at all the time points, suggesting that the enhanced 190 levels of SERCA2 expression were related to viral infection (Fig. 3a, lower panel). 191 Besides, we also observed that virus-induced SERCA2 expression could be blocked by 192 Thapsigargin treatment (Fig. 3b). 193 Time-of-addition assay. In order to ascertain the stage(s) of the viral life cycle 194 which can be impaired by Thapsigargin, we performed a time-of-addition assay (one-195 step growth curve), in which the inhibitor was applied at different time post-infection 196 and the virus released into the supernatant was quantified by plaque assay. The NDV 197 and PPRV varies in their length of replication cycle, ~10 h and ~24 h respectively, 198 therefore time-of-addition of inhibitor and time of virus harvest varied from virus to 199 virus. As shown in Fig. 4a, the magnitude of viral (NDV) inhibition gradually 200 decreased. The highest inhibition was observed when the inhibitor was applied 30 min 201 before infection. The inhibition levels progressively decreased from 1 hpi to 6 hpi. 202 Thapsigargin did not exhibit any inhibitory effect on virus replication if it was applied 203 at 10 hpi, a later time point in NDV life cycle when the virus is presumably undergoing 204 budding. Similar findings were observed with PPRV; highest inhibition in viral titers 205 was observed when the inhibitor was applied 30 min prior to infection, magnitude of 206 inhibition progressive decreased from 4 hpi to 24 hpi (Fig. 4b). The time-of-addition 207 assay, therefore suggested that Thapsigargin may inhibit multiple pre-budding steps of 208 paramyxovirus replication. 209

Thapsigargin does not affect virus attachment, RNA synthesis and 210
budding. Virus step-specific assays viz; attachment, entry, RNA synthesis, subcellular 211 localization of viral proteins and budding were conducted as per the previously 212 described methods (24). Thapsigargin was not found to affect virus attachment, RNA 213 synthesis and budding (data not shown). we observed cytoplasmic foci in majority of Thapsigargin-treated cells (Fig. 6a and  228 6b). Contrary, in majority of the DMSO-treated cells, virions were found to be 229 localized at the cell surface ( Fig. 6a and 6b). Further, the cells with normal (cell 230 surface) or defective (cytoplasmic foci) localization were quantified. Thapsigargin 231 treatment showed defective localization in ~70% of the cells, as compared to DMSO 232 control wherein this proportion was 10-30 % (Fig. 6c and 6d), suggesting that the SERCA is required for proper localization of the viral proteins (cytoplasm to the cell 234

surface) in infected cells. 235
Selection of Thapsigargin-resistant viral mutants. Due to the high genetic 236 barrier to resistance, host-targeting agents provide an interesting perspective for novel 237 antiviral strategies, rather than the directly-acting agents. NDV, when passaged 238 sequentially in presence of a SERCA inhibitor (Thapsigargin, a host-targeting agent) 239 did not generate a completely resistant phenotype against Thapsigargin, even upon 70 240 passages in Vero cells (Fig. 7a). However, resistance began appearing at ~P25 and 241 significant resistance was observed at P35 (~100 fold-inhibition compared to ~10,000 242 fold inhibition at zero passage) after which it became stable without acquiring complete 243 resistance (Fig. 7a). Fluctuations in the overall viral titers and hence variation in fold 244 inhibition was observed (Fig. 7a), which might be due to the fact that the harvest time 245 (72-120 h) and MOI (0.01-0.001) used was not similar at each passage. Therefore, we 246 carried out an experiment wherein a similar MOI (MOI=0.1) and harvest time (24 h) 247 was used to evaluate the relative resistance in Thapsigargin-passaged and control-248 passaged viruses. As compared to P0 and P70-Control viruses, P70-Thapsigargin virus 249 exhibited significantly lower sensitivity to Thapsigargin, though a completely resistant 250 phenotype could not be observed (Fig. 7b). However, all PPRVs (P0, P70-Thapsigargin 251 and P70-Control) revealed similar sensitivity to Thapsigargin even at passage level 70 252 ( Fig. 7c), suggesting that PPRV is unlikely to develop Thapsigargin resistant mutants 253 upon long-term passage. Control-passaged viruses did not exhibit any significant 254 resistance against Thapsigargin even upon 70 passages ( Fig. 7b and 7c) suggesting that 255 resistance against Thapsigargin (NDV) is not a general phenomenon due to sequential 256 high passages but rather a specific event acquired in presence of Thapsigargin. 257

DISCUSSION 258
High mutation rate in RNA viruses induces resistance to antiviral drugs and 259 preexisting immunity. The rise in incidence of drug resistance has prompted a shift 260 towards the development of novel antiviral drugs. As compared to the viral genome, 261 genetic variability of the host is quite low and therefore host-targeting agents are 262 considered to impose a higher genetic barrier to generation of resistant viruses (24, 27-263 29). Thus, a potentially better approach for development of novel antiviral therapeutics 264 would be to target host factors required for viral replication. Targeting host factors 265 could have a significant impact on multiple virus genotypes (strain/serotype) and 266 provide broad spectrum inhibition against different families of viruses which might use 267 the same cellular pathway(s) for replication (24,(30)(31)(32)(33). This novel approach has led to 268 the development of some promising compounds for treatment of HCV and HIV (34, 269 35). In this study, we have shown that targeting SERCA (a Ca2+ ATPase) by a small 270 molecule chemical inhibitor-Thapsigargin can block paramyxovirus replication at the 271 level of viral entry and localization of viral proteins in infected cell. We were able to 272 verify the specific requirement of SERCA in paramyxovirus replication; 273 overexpression of SERCA2 rescued the inhibitory effect of Thapsigargin. Therefore, 274 SERCA may present as a novel target for antiviral drug development. When we were 275 writing this manuscript, Hoffmann and coworkers identified that SPCA1 [a secretary 276 pathway calcium (Ca 2+ ) transporter that facilitates Ca +2 and Mn 2+ uptake into the trans-It is generally believed that viruses do not acquire resistance against host-281 targeting antiviral agents (1,31,36,37). However, in a recent study (38), Schaar and 282 colleagues identified Coxsackievirus B3 (CVB3) mutants that replicate efficiently in 283 the presence of several potent antiviral drugs known to inhibit phosphatidylinositol-4-284 kinase IIIα (PI4KIIIα), a key cellular factor for CVB3 replication. The authors observed 285 that a single point mutation in the viral 3A protein confers resistance and the drug 286 resistant escape mutants of CVB3 can replicate in cells with low PI4KIIIα. 287 Additionally, cyclosporine A (CsA) resistant hepatitis C virus (HCV) mutant has also 288 been identified (39, 40). In our study, resistance acquired by NDV against SERCA 289 inhibitor adds another example to a short list of viruses which can acquire resistance to 290 host-targeting antiviral agents. To the best of our knowledge, this is the first 291 documented example where a paramyxovirus significantly bypassing its dependency on 292 a cellular factor that is targeted by a small molecule inhibitor. While not yet 293 understood, one possible mechanism underlying acquisition of drug resistance is due to 294 change in host factor requirement (41). For example, under selection pressure in 295 CLDN1 (tight junction protein claudin-1 which serves as an entry factor for HCV)-296 knock-out cells, CLDN1-dependent HCV evolved to use alternate host factors viz; 297 CLDN6 or CLDN9 (41). Alternatively, resistant viruses may simply have enhanced 298 affinity for its natural substrate, thereby allowing the virus to propagate despite 299 reduction in concentration of the cellular factors (42). Though a complete Thapsigargin 300 resistant NDV phenotype could not be achieved even up to passage level 70, it is a 301 matter of further study how NDV became less dependent on SERCA (under the 302 selection pressure of Thapsigargin). Additional studies are also required to determine 303 which viral proteins(s) have acquired mutations upon prolonged passage in presence of SERCA inhibitor and how these mutations confer resistance against Thapsigargin. In 305 immunofluorescence assay, we could not observe a perfect co-localization of SERCA 306 and viral proteins. The co-immunoprecipitation assay to analyze the interaction 307 between SERCA/virus was unsuccessful, therefore, in this study, we could not 308 determine any direct interaction between SERCA and viral proteins. 309 To conclude, we have provided strong evidence for SERCA as a crucial host 310 factor in facilitating optimal paramyxoviral replication, thus validating this as a 311 candidate drug target for the development of antiviral therapeutics.

Western blot analysis. (b) Thapsigargin inhibits NDV-induced SERCA2 expression in 481
Vero cells: Vero cells were infected with NDV at MOI 5 for 1 h followed by washing 482 with PBS and addition of fresh medium containing Thapsigargin or vehicle control. 483 Cell lysates were prepared at 16 hpi to probe SERCA2 and GAPDH by Western blot 484 analysis. 485   Thapsigargin inhibits NDV-induced SERCA2 expression in Vero cells: Vero cells were infected with NDV at MOI 5 for 1 h followed by washing with PBS and addition of fresh medium containing Thapsigargin or vehicle control. Cell lysates were prepared at 16 hpi to probe SERCA2 and GAPDH by Western blot analysis.  Thapsigargin-free medium for 1h at 4 o C to permit attachment, followed by washing and addition of fresh medium containing Thapsigargin or vehicle-control. Entry was allowed to proceed at 37 o C for 1h after which the cells were washed again with PBS to remove any extracellular viruses and incubated with cell culture medium without any inhibitor.
The progeny virus particles released in the cell culture supernatants in the treated and untreated cells were titrated by plaque assay. Error bars indicate SD. Pair-wise statistical comparisons were performed using Student's t test (** = P<0.01,*** = P<0.001).