【病毒外文文献】2013 MERS-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin A or in

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1、 1 VIR/2013/052910 - REVISED VERSION1 2 MERS-coronavirus replication induces severe in vitro cytopathology and is strongly 3 inhibited by cyclosporin A or interferon-alpha treatment4 5 6 Adriaan H. de Wilde1, V. Stalin Raj2, Diede Oudshoorn1, Theo M. Bestebroer2, Stefan van 7 Nieuwkoop2, Ronald W. A

2、. L. Limpens3, Clara C. Posthuma1, Yvonne van der Meer1, Montserrat 8 Brcena3, Bart L. Haagmans2, Eric J. Snijder1*and Bernadette G. van den Hoogen2* 9 10 1Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical 11 Center, Leiden, The Netherlands12 2Viroscience L

3、ab, Erasmus MC, Rotterdam, The Netherlands13 3Section Electron Microscopy, Department of Molecular Cell Biology, Leiden University Medical 14 Center, Leiden, The Netherlands15 16 * Corresponding authors: Eric. J. Snijder (E.J.Snijderlumc.nl) and Bernadette G. van den Hoogen17 (b.vandenhoogenerasmusm

4、c.nl)18 19 Abstract word count: 23520 Text word count: 549921 Number of figures: 722 23 24 Running title: MERS-CoV replication and sensitivity to CsA and IFN-?25 26 Contents Category: Animal Viruses - Positive-strand RNA27 28 29 JGV Papers in Press. Published June 19, 2013 as doi:10.1099/vir.0.05291

5、0-0 2 SUMMARY30 31 Coronavirus (CoV) infections are commonly associated with respiratory and enteric disease in 32 humans and animals. The 2003 outbreak of severe acute respiratory syndrome (SARS) highlighted the 33 potentially lethal consequences of CoV-induced disease in humans. In 2012, a novel C

6、oV (Middle 34 East Respiratory Syndrome coronavirus; MERS-CoV) emerged, causing 49 human cases thus far, of 35 which 23 had a fatal outcome. In this study, we characterized MERS-CoV replication and cytotoxicity 36 in human and monkey cell lines. Electron microscopy of infected Vero cells revealed ex

7、tensive 37 membrane rearrangements, including the formation of double membrane vesicles and convoluted 38 membranes, which were previously implicated in the RNA synthesis of SARS-CoV and other CoVs. 39 Following infection, we observed rapidly increasing viral RNA synthesis and release of high titres

8、 of 40 infectious progeny, followed by pronounced cytopathology. These characteristics were used to 41 develop an assay for antiviral compound screening in 96-well format, which was used to identify42 cyclosporin A as an inhibitor of MERS-CoV replication in cell culture. Furthermore, MERS-CoV was 43

9、 found to be 50-100 times more sensitive to interferon-alpha (IFN-?-CoV, an 44 observation that may have important implications for the treatment of MERS-CoV-infected patients.45 MERS-CoV infection did not prevent the IFN-induced nuclear translocation of phosphorylated 46 STAT1, in contrast to infec

10、tion with SARS-CoV where this block inhibits the expression of antiviral 47 genes. These findings highlight relevant differences between these distantly related zoonotic CoVs in 48 terms of their interaction with and evasion of the cellular innate immune response. 49 50 3 INTRODUCTION51 52 In June 2

11、012, a previously unknown coronavirus was isolated from a 60 year-old Saudi-53 Arabian patient who died from acute respiratory distress syndrome and multiple organ failure (Zaki et 54 al., 2012). Subsequently, the novel virus was isolated from several additional residents and visitors of 55 the Arab

12、ian Peninsula suffering from similar respiratory symptoms. In retrospect, also a cluster of 56 respiratory infections in Jordan (April 2012) was linked to the same agent, although no convincing57 evidence for human-to-human transmission was obtained. This was clearly different for a cluster of 58 th

13、ree U.K. cases in early 2013, consisting of a patient who had travelled to Saudi Arabia and two 59 family members without recent travel history outside the U.K. In the past year, various names have 60 been used to refer to this newly identified CoV, including novel (beta)coronavirus (nCoV) and human

14、 61 coronavirus EMC (HCoV-EMC), but following a recent recommendation by the coronavirus study 62 group of ICTV and other experts (de Groot et al., 2013) we will use Middle East Respiratory 63 Syndrome coronavirus (MERS-CoV) throughout this paper. Up to May 2013, 49 confirmed MERS64 cases, including

15、 23fatalities, have been recorded65 (http:/www.who.int/csr/don/archive/disease/coronavirus_infections/en/).66 Coronavirus (CoV) infections are associated with respiratory and enteric disease in humans 67 and animals. Since the 1960s, two human CoVs (HCoVs OC43 and 229E) were known to cause mild 68 r

16、espiratory disease (Hamre & Procknow, 1966; McIntosh et al., 1967), but it was the 2003 outbreak of 69 severe acute respiratory syndrome (SARS; fatality rate 10%) that revealed the potentially lethal 70 consequences of CoV-induced disease in humans (Drosten et al., 2003; Ksiazek et al., 2003). Two 7

17、1 years later, bats were identified as the most likely animal reservoir for this zoonotic CoV (Lau et al.,72 2005; Li et al., 2005). Subsequently, a wide variety of bat-associated CoVswas discovered 73 (Vijaykrishna et al., 2007; Woo et al., 2007) and also two additional human CoVs (NL63 and HKU1; 7

18、4 (Fouchier et al., 2004; van der Hoek et al., 2004; Woo et al., 2005) were identified. Although the 75 general capacity of bat CoVs to switch hosts appears to be rather restricted (Muller et al., 2012), the 76 4 possibility of SARS-CoV re-emergence or zoonotic transfer of other animal CoVs has rema

19、ined a 77 public health concern over the past 10 years. 78 Coronaviruses are classified in four genera (alpha-, beta-, gamma- and deltacoronaviruses; (de 79 Groot et al., 2012) and our previous analysis of the MERS-CoV genome (van Boheemen et al., 2012)80 identified the newly emerging agent as a mem

20、ber of lineage C of the genus Betacoronavirus.81 Strikingly, as in the case of SARS-CoV, the closest known relatives of MERS-CoV are bat 82 coronaviruses, like HKU-4 and HKU-5(van Boheemen et al., 2012; Woo et al., 2007). The 83 evolutionary distance to SARS-CoV (lineage B) is considerable, a notion

21、 further supported by recent 84 comparative studies revealing important differences in receptor usage (Muller et al., 2012; Raj et al.,85 2013).86 Mammalian viruses have to cope with the host cells innate responses, including those87 triggered by activation of the type I interferon (IFN) pathway (re

22、viewed by (Randall & Goodbourn, 88 2008). Coronaviruses, including SARS-CoV, appear to have evolved a variety of mechanisms to 89 block or evade such antiviral responses (reviewed in (Perlman & Netland, 2009; Zhong et al., 2012).90 For example, it was postulated that the sensing of double-stranded (

23、ds) RNA replication intermediates91 by the innate immune system is inhibited by the elaborate virus-induced membrane structures with 92 which CoV RNA synthesis is associated (Knoops et al., 2008; Versteeg et al., 2007). Other evasion 93 mechanisms were attributed to protein functions that can be eit

24、her conserved across CoVs or specific 94 for certain CoV lineages. Proteins such as the nsp3 proteinase (Ratia et al., 2006), the nsp16 2-O-95 methyltransferase (Zust et al., 2011), and the products of SARS-CoV ORFs 3b, 6 and 7a (Frieman et 96 al., 2007; Hussain et al., 2008; Kopecky-Bromberg et al.

25、, 2006; Zhou et al., 2012) have all been97 described to prevent IFN induction/signalling. In particular, the SARS-CoV ORF6 protein is known to 98 inhibit IFN-induced JAK-STAT signalling by blocking the nuclear translocation of phosphorylated 99 STAT1 (p-STAT1), which contributes to the pathogenic po

26、tential of the virus in a mouse model (Sims100 et al., 2013). In spite of these immune evasion strategies, treatment with type I IFNs can inhibit CoV 101 replication in vitro (Garlinghouse et al., 1984; Haagmans et al., 2004; Paragas et al., 2005; Taguchi & 102 Siddell, 1985; Zheng et al., 2004) and

27、, for example, protected type I pneumocytes against SARS-CoV 103 infection in macaques (Haagmans et al., 2004).104 5 Clearly, well-characterized systems for MERS-CoV replication in cell culture will be 105 invaluable for future studies into basic virus properties and interactions with the host, incl

28、uding innate 106 immune responses. Therefore, we set out to characterize the replication of MERS-CoV in different 107 cell lines. Using this information, an assay to screen for antiviral compounds was developed, which 108 identified cyclosporin A (CsA) as an inhibitor of MERS-CoV replication. Our fi

29、rst screening 109 experiments also established that, compared to SARS-CoV, MERS-CoV replication is more sensitive 110 to type I interferon treatment.111 112 RESULTS113 114 Kinetics of MERS-CoV replication in Vero and Huh7 cells. Only a few laboratory studies on 115 MERS-CoV replication have been rep

30、orted thus far. Cells from a variety of mammalian hosts were 116 found to be susceptible and infection can induce pronounced cytopathology and cell death (Muller et 117 al., 2012; Zaki et al., 2012). Following entry, the CoV replicative cycle starts with the translation of 118 the positive-stranded

31、RNA genome into replicase polyproteins that are cleaved into 16 nsps 119 (Gorbalenya et al., 2006; van Boheemen et al., 2012). These direct both genome replication and the 120 synthesis of the subgenomic (sg) mRNAs required to express the structural and accessory proteins. To 121 investigate MERS-Co

32、V replication in more detail, we used Vero and Huh7 cells to analyse viral RNA 122 synthesis and progeny release in single-cycle infection experiments.123 Hybridisation analysis of the accumulation of viral RNA revealed the presence of genome 124 RNA and seven sg transcripts, with sizes closely matc

33、hing those previously predicted from the 125 positions of conserved transcription regulatory sequences (TRS) in the viral genome (van Boheemen126 et al., 2012) (Fig. 1a). The relative abundance of the various sg mRNAs is similar to what has been 127 observed for other CoVs, with the smallest species

34、 (encoding the N protein) being by far the most 128 abundant transcript (Fig. 1b). In both cell lines, viral mRNAs could be readily detected at 7 h p.i. and 129 reached maximum levels around 13 h p.i. (Fig. 1a). Viral RNA levels remained more or less constant 130 until 24 h p.i. in Vero cells, where

35、as the amount isolated from Huh7 cells declined due to the more 131 rapid development of cytopathology in this cell line between 13 and 24 h p.i. (see below). After the 132 6 peak of viral RNA accumulation had been reached, the titre of virus released from MERS-CoV-133 infected Vero cells steadily i

36、ncreased from 5 x 105to 5 x 107p.f.u. per ml (Fig. 1c). Interestingly, 134 the bulk of the viral progeny was released significantly earlier from Huh7 cells, although the final titre135 at 24 h p.i. was comparable to that obtained from Vero cells.136 137 Antisera raised against non-structural protein

37、s of other betacoronaviruses cross-react with 138 MERS-CoV proteins. Despite the relatively large evolutionary distance to better-characterized 139 CoVs, we tested a panel of antisera from our laboratory for cross-reactivity with MERS-CoV-infected 140 cells. In contrast to a polyclonal serum recogni

38、zing the SARS-CoV nucleocapsid (N) protein (data not 141 shown), antisera against various SARS-CoV nsps (nsp3, nsp5, nsp8; (Snijder et al., 2006) raised using 142 purified recombinant proteins as antigen, were found to strongly cross-react (Fig. 2a). In addition, 143 rabbit antisera raised against s

39、ynthetic peptides (23-mers) representing a small but conserved C-144 terminal part of SARS-CoV and MHV nsp4 strongly cross-reacted with MERS-CoV. Only small but 145 apparently immunogenic parts of these peptides (e.g., LYQPP) are absolutely conserved between 146 MHV and MERS-CoV nsp4 (Fig. 2b). Cons

40、ervation in other betacoronaviruses (data not shown) 147 suggests that antisera recognizing this nsp4 region may be used for immunodetection of additional 148 (newly emerging) CoVs.149 150 MERS-CoV replication structures. Subsequently, we employed a monoclonal antibody recognizing 151 dsRNA to local

41、ize intermediates in viral RNA synthesis (Knoops et al., 2008; Weber et al., 2006). In 152 various cell types, the immunolabelling signals for both replicase and dsRNA localized to the 153 perinuclear region (Fig. 2c), where the replication structures induced by other CoVs are known to 154 accumulat

42、e (Brockway et al., 2003; Gosert et al., 2002; Knoops et al., 2008; Snijder et al., 2006;155 Stertz et al., 2007; Ulasli et al., 2010).156 We next used electron microscopy (EM) to investigate the ultra-structural and potentially 157 cytopathic changes that MERS-CoV induces in infected cells, and foc

43、used on the membranous 158 replication structures that support MERS-CoV RNA synthesis. The preservation of such structures, 159 typically double-membrane vesicles (DMVs) and convoluted membranes (CMs), was previously 160 7 found to be significantly improved by using protocols that include cryo-fixat

44、ion and freeze-161 substitution (Knoops et al., 2008; Snijder et al., 2006). We now applied these advanced preservation 162 techniques, including newly developed protocols for high-pressure freezing (HPF), to MERS-CoV-163 infected Vero cells. Images of similarly prepared SARS-CoV-infected Vero E6 ce

45、lls are included for 164 comparison (Fig. 3f).165 Compared to mock-infected control cells (Fig. 3e), different degrees of distinct alterations166 were observed at 8 h p.i. Some cells contained relatively small DMV clusters (Fig. 3a,b; black 167 arrowheads, inset), whereas in others large numbers of

46、DMVs occupied extensive areas of the 168 perinuclear region (Fig. 3c,d), differences that likely reflect different stages in infection progression. 169 The diameter of MERS-CoV-induced DMVs ranged from 150 to 320 nm, comparable to what was170 previously measured for SARS-CoV-induced structures (Knoo

47、ps et al., 2008). An interesting171 morphological difference with our previous studies of SARS-CoV-infected cells was the presence of a 172 dense inner DMV core, which can be attributed to technical differences in sample preparation. In 173 terms of ultrastructural preservation, HPF is widely consid

48、ered superior to the previously used 174 plunge-freezing protocols. Also in the case of SARS-CoV (Fig. 3f) and the distantly related equine 175 arteritis virus (Knoops et al., 2012), a similar dense DMV core became apparent when HPF was 176 employed. Although DMV cores are known to contain dsRNA, th

49、e implications of these 177 ultrastructural observations remain unclear. Interestingly, CMs were always surrounded by DMV 178 clusters and were only observed in cells that appeared to be more advanced in infection (Fig. 3c,d; 179 white arrows, inset). This observation strengthens the notion that DMV

50、 formation precedes the180 development of CMs, as previously postulated for SARS-CoV (Knoops et al., 2008).181 182 MERS-CoV-induced cytophatology and cell death. In cell culture, many CoVs induce severe 183 cytopathic effect (CPE) and cell death. Infection with a number of CoVs can also induce exten

51、sive 184 syncytium formation, due to fusion activity of the viral spike protein at neutral pH (reviewed in 185 (Belouzard et al., 2012). MERS-CoV-induced cytopathology was monitored by light microscopy 186 following low-m.o.i. inoculation of monkey and human cells (Fig. 4). In line with previous 187

52、 observations (Zaki et al., 2012), Vero cells developed clear CPE at 2 days post infection (d p.i.) and 188 8 detached at 3 d p.i. (Fig. 4a). Similar observations were made for Calu3/2B4 cells (Fig. 4b). In 189 contrast, MERS-CoV-infected Vero E6 cells displayed only mild CPE starting at 3 d p.i and

53、 cell death 190 was not complete after six days (Fig. 4c). The development of CPE in Huh7 cells was strikingly faster 191 compared to the three other cell lines and, following extensive syncytium formation, cells detached 192 already around 17 h (Fig. 4d). Given the low m.o.i. used and the viral rep

54、lication kinetics (Fig. 1), the 193 syncytium formation in these only partially infected Huh7 cultures appeared to be a major factor in 194 CPE development. DPP4 expression on Vero and Huh7 cells (Raj et al., 2013) and expression levels 195 of DPP4 on Calu3/2B4 and Vero E6 cells correlated with susc

55、eptibility to MERS-CoV (data not 196 shown).197 198 Development of an assay to screen for compounds inhibiting MERS-CoV replication. The virus-199 induced CPE in Vero and Huh7 cells was used to develop a first assay to screen for compounds that 200 inhibit MERS-CoV replication in cell culture. Vero

56、cells were seeded in 96-well plates and infected at 201 an m.o.i. of 0.005 or 0.05 (Fig. 5a). After two and three days, CPE formation was monitored 202 microscopically and cytotoxicity was measured using a commercial cell viability assay. Moderate 203 CPE was observed on day 2, and by day 3 cell via

57、bility had dropped below 10% with both virus doses 204 used (Fig. 5a), indicating near-complete cell death. In MERS-CoV-infected Huh7 cells (Fig. 5b), 205 already after day 1, cell viability had dropped to 79% or 24% (after m.o.i. 0.005 or 0.05 infection, 206 respectively), which was in line with ou

58、r observations on rapid syncytium formation and CPE in this 207 particular cell line (Fig. 4d). One day later, CPE was complete for both virus doses used and cells had 208 detached (Fig. 5b). Based on this comparison, further experiments were done using an m.o.i. of 0.005209 and Huh7 and Vero cells

59、were incubated for two or three days, respectively, before measuring cell 210 viability.211 Previously, it was shown that replication of various CoVs, including SARS-CoV, can be 212 inhibited by the immunosuppressive drug CsA (de Wilde et al., 2011; Pfefferle et al., 2011).213 Therefore, while testi

60、ng whether the CPE-based assay described above could be used as an antiviral 214 screening method, we used CsA treatment to obtain a first proof of principle. Infected Vero cells were 215 given ?of CsA and were analysed at 3 d p.i. At the concentrations used, CsA did not 216 9 adversely affect the v

61、iability of mock-infected cells (Fig. 5c). ? ? ? ? ?ly217 inhibited CPE and left cell viability unchanged compared to mock-infected control cells. The 218 inhibitory effect of CsA was confirmed in Huh7 cells (Fig. 5d), which displayed reduced and lack of 219 ? ?!? ?#?$?#?%?&?#?#?#?!?&?220 immunofluo

62、rescence microscopy analysis of CsA-treated and high m.o.i.-infected Vero and Huh7 221 cells and by determining virus titres released into the medium. Both assays confirmed an almost 222 complete block of MERS-CoV-infection (data not shown). However, as previously reported for other 223 CoVs (de Wil

63、de et al., 2011), a small fraction of MERS-CoV-infected cells appeared to be refractive 224 to CsA treatment and supported a low level of MERS-CoV replication, even at high CsA 225 concentrations (data not shown). 226 227 Enhanced sensitivity of MERS-CoV to pegylated IFN-?-CoV. 228 Type I IFNs inhib

64、it CoV replication and can protect against infection in animal models (Haagmans et 229 al., 2004; Taguchi & Siddell, 1985). We therefore compared the effect of pegylated interferon-?230 (PEG-IFN) treatment on MERS-CoV and SARS-CoV replication in vitro. Vero cells were given 231 PEG-IFN 4 h before lo

65、w-m.o.i. infection, together with the inoculum or 4 h after infection. At 2 d p.i. 232 CPE was scored microscopically.233 Treatment with PEG-IFN profoundly inhibited both MERS-CoV- and SARS-CoV-induced 234 CPE and RNA levels in a dose-dependent manner (Fig. 6). At 2 d p.i., SARS-CoV-induced CPE was

66、235 reduced for all time points of PEG-IFN addition when using a dose of at least 30 ng/ml PEG-IFN 236 (Fig. 6a), whereas MERS-CoV-induced CPE already decreased using a dose of 1 ng/ml (Fig. 6b). For 237 SARS-CoV, only pre-treatment with 1000 ng/ml PEG-IFN completely prevented CPE. For MERS-238 CoV,

67、 complete inhibition of CPE was observed at much lower concentrations, specifically 3, 10 or 30239 ng/ml when the drug was added to the cells before, during or after infection, respectively. Although 240 decreased CPE was also observed in SARS-CoV-infected cultures treated with 30 ng/ml PEG-IFN, 241

68、 only a 30-fold reduction of viral RNA was detected in their medium at 2 d p.i. (Fig. 6c). For 242 comparison, treatment of MERS-CoV-infected cells with the same PEG-IFN dose completely blocked 243 10 CPE and reduced viral RNA levels in the medium 600- to 2,000-fold, depending on the timing of 244 P

69、EG-IFN addition (Fig. 6d).245 Our data revealed that in the same cell line MERS-CoV infection is 50-100 times more 246 sensitive to PEG-IFN treatment than SARS-CoV infection. This difference may be explained by 247 important lineage-specific genetic differences between these two zoonotic betacoronav

70、iruses in terms 248 of accessory protein genes encoded in the 3 part of the genome (Snijder et al., 2003; van Boheemen249 et al., 2012). In particular, MERS-CoV does not encode a homolog of the SARS-CoV ORF6 protein, 250 which was reported to block the IFN-induced nuclear translocation of phosphoryl

71、ated transcription 251 factor STAT1. As nuclear translocation of p-STAT1 is essential for transcriptional activation of 252 downstream antiviral genes, the ORF6 protein makes SARS-CoV less sensitive to treatment with type 253 I IFN (Frieman et al., 2007; Sims et al., 2013). IFN-induced translocation

72、 of p-STAT1 was readily 254 observed in IFN-treated mock-infected Vero cells (Fig. 7a-d), but not in IFN-treated SARS-CoV-255 infected cells (Fig. 7e,f). In contrast, in MERS-CoV-infected and IFN-treated cultures the 256 translocation of p-STAT1 was detected (Fig. 7g,h). Together with the data on IF

73、N sensitivity (Fig. 5), 257 these observations highlight important differences between SARS-CoV and MERS-CoV in terms of 258 their interaction with the IFN signalling pathways. 259 260 11 261 DISCUSSION262 263 Following the 2003 SARS epidemic, global CoV hunting efforts identified a wealth of previo

74、usly 264 unknown family members, in particular in bat species from several continents (de Groot et al., 2012).265 Moreover, at least three of the four current established human CoVs (NL63, 229E, and OC43) were 266 postulated to have originated from zoonotic reservoirs (Huynh et al., 2012; Pfefferle

75、et al., 2009;267 Vijgen et al., 2005). Recently, about a decade after the SARS outbreak, MERS-CoV was identified as 268 the next zoonotic CoV (Zaki et al., 2012) and appears to be highly pathogenic to humans: of the 49269 cases confirmed thus far, 23had a fatal outcome 270 (http:/www.who.int/csr/don

76、/archive/disease/coronavirus_infections/en/).Whether zoonotic CoVs271 cause transient epidemics or establish a long-lasting relationship with the human host, an in-depth 272 understanding of virus-host interactions will be required to develop effective countermeasures. In this 273 study, we defined

77、several basic but important parameters of MERS-CoV replication in cell culture 274 (Figs. 1-4). Among the tools for MERS-CoV research developed are immunoassays based on cross-275 reacting antisera raised against other betacoronaviruses (Fig. 2) and a CPE-based assay that can be 276 used to screen f

78、or antiviral effects (Figs. 5-6).277 Following the development of a high-throughput screening method for antiviral effects, proof 278 of principle was obtained using CsA, a recently discovered inhibitor of CoV replication (de Wilde et 279 al., 2011; Pfefferle et al., 2011). This drug affects the fun

79、ction of several members of the cellular 280 cyclophilin (Cyp) family and appears to block functional interactions between viral proteins and one 281 or multiple cyclophilin family members (Nagy et al., 2011). Low-micromolar CsA concentrations 282 blocked MERS-CoV-induced CPE i? (? ?!? )? ?#? *? ? ?

80、!? ? ?$? ?#?%?&? as283 previously observed for other CoVs (de Wilde et al., 2011; Pfefferle et al., 2011). As in those 284 previous studies (de Wilde et al., 2011), a small fraction of the cells somehow remained susceptible to 285 MERS-CoV infection, even at high CsA concentrations. Thus, virus repl

81、ication could not be 286 completely eliminated, which may ultimately lead to the development of CsA resistance in cell 287 culture. In conclusion, these experiments established that monitoring MERS-CoV-induced CPE can 288 12 be a valuable and rapid tool in screening for the potential antiviral activ

82、ity of e.g. small-molecule 289 compounds or FDA-approved drugs like PEG-IFN.290 Type I IFN induction, a hallmark of the early innate immune response, is counteracted by 291 different CoV-encoded proteins. Despite these evasion strategies, IFN can be detected in sera of CoV-292 infected mice and huma

83、ns (Cameron et al., 2012; Garlinghouse et al., 1984; Taguchi & Siddell, 293 1985), and CoV-infected plasmacytoid DCs have been identified as a source of high IFN-?%?#?294 (Cervantes-Barragan et al., 2007; Roth-Cross et al., 2007). The SARS-CoV ORF6 protein, however, 295 (partially) disrupts the down

84、stream IFN-induced signalling in infected cells by inhibiting the nuclear 296 translocation of p-STAT1, a critical component of both the IFN-?!?+,-.?#?/?/?&#?297 (Frieman et al., 2007). Although contributions from additional immune evasion mechanisms are 298 likely, the lack of a SARS-CoV ORF6 homol

85、og (van Boheemen et al., 2012) may be a major factor in 299 the higher sensitivity of MERS-CoV to PEG-IFN treatment, as observed in this study and other recent 300 work (Kindler et al., 2013). This was further substantiated by the finding that nuclear translocation of 301 p-STAT1 is not blocked in M

86、ERS-CoV-infected cells (Fig. 7), which indicates that MERS-CoV has 302 not evolved an alternative strategy to achieve the same goal. MHV has been shown to be relatively 303 insensitive to IFN pre-treatment, however also this virus does not block activation and translocation of 304 p-STAT1 but instea

87、d inhibits the induction of a subset of ISGs by IFN-?01 (Rose et al., 2010). Future305 studies may elucidate whether MERS-CoV has evolved alternative strategies to cope with the hosts306 IFN response. In addition, it will be important to test whether MERS-CoV is attenuated in vivo as a 307 result of

88、 the relative high IFN sensitivity.308 PEG-IFN is a registered drug used for the treatment of chronic hepatitis B and C infections in 309 humans (Bergman et al., 2011). Several CoVs, including SARS-CoV, were shown to be sensitive to 310 both type I IFN treatment in vitro and PEG-IFN treatment in viv

89、o (Haagmans et al., 2004; Paragas et 311 al., 2005; Zheng et al., 2004), and in this study we established a relatively high sensitivity for MERS-312 CoV. For example, in cynomolgus macaques plasma levels of 1-5 ng/ml were reached (Haagmans et 313 al., 2004), a dose which in this study significantly

90、reduced MERS-CoV replication in vitro. The 314 sensitivity of MERS-CoV to exogenous IFN suggests that administration of recombinant IFN merits 315 13 further evaluation as a therapeutic intervention strategy if new infections with the novel virus would 316 occur.317 318 MATERIAL AND METHODS319 320 C

91、ells culture and virus infection. Vero cells (ATCC: CCL-81) were cultured in Eagles minimal 321 essential medium (EMEM; Lonza) with 8% fetal calf serum (FCS; PAA) and antibiotics. Huh7 cells 322 were grown in Dulbeccos Modified Eagle Medium (DMEM; Lonza) containing 8% FCS, 2 mM L-323 Glutamine (PAA)

92、, non-essential amino acids (PAA), and antibiotics. Vero E6 and Calu3/2B4 cells 324 were cultured as previously described (Snijder et al., 2006; Yoshikawa et al., 2010). Infection of 325 Vero, Vero E6, Huh7, and Calu3/2B4 cells with MERS-CoV (strain EMC/2012; (van Boheemen et 326 al., 2012; Zaki et

93、al., 2012) at high multiplicity of infection (m.o.i. 5) was done in PBS containing 50 327 ?/0?2?-!?3?!?45?,?+?#?!?#?*?6?7?7 ?8?MERS-CoV or 328 SARS-CoV (strain HKU-39849; (Zeng et al., 2003) were done directly in EMEM containing 2% 329 FCS. Virus titrations by plaque assay were performed as describe

94、d before (van den Worm et al.,330 2012). All work with live MERS-CoV and SARS-CoV was performed inside biosafety cabinets in 331 biosafety level 3 facilities at Leiden University Medical Center or Erasmus Medical Center.332 333 Antibodies and drugs. Rabbit antisera recognizing the SARS-CoV replicase

95、 subunits nsp3, nsp4, 334 nsp5 and nsp8 have been described previously (Snijder et al., 2006; van Hemert et al., 2008b). Rabbit 335 antisera recognizing the SARS-CoV nucleocapsid (N) protein and MHV nsp4 were raised as 336 described (Snijder et al., 1994). Antigens were a full-length recombinant SAR

96、S-CoV N protein 337 (purified from E. coli) and a synthetic peptide representing the 23 C-terminal residues of MHV nsp4, 338 respectively. p-STAT1 was detected with Alexa Fluor 488-labelled mouse-anti-STAT1 (pY701) (BD 339 Biosciences) and FITC-labelled anti-mouse-IgG was used to enhance the green f

97、luorescence. Virus 340 infection was detected using the above-mentioned anti-nsp3 sera and Alexa Fluor 594-labeled anti-341 rabbit IgG.342 14 Cyclosporin A (CsA; Sigma) was dissolved in DMSO and a 10-mM stock was stored in aliquots for 343 single use at -20C. Peg-interferon alfa-2b (PEG-IFN; Pegintr

98、on, Merck, USA) was prepared 344 according to the manufacturers instruction as a 100 g/ml stock stored at 4C.345 346 Immunofluorescence microscopy.Cellsweregrown on coverslipsandfixed with 3% 347 paraformaldehyde in PBS or with 4% formaldehyde and 70% ethanol (p-STAT1 experiments),348 permeabilized

99、with 0.1% Triton X-100, and processed for immunofluorescence microscopy as349 described previously (van der Meer et al., 1998). Specimens were examined with a Zeiss Axioskop 2 350 fluorescence microscope with an Axiocam HRc camera and Zeiss Axiovision 4.4 software or with a 351 confocal microscope (

100、Zeiss, LSM 700) (p-STAT1 experiments). 352 353 Electron microscopy. Vero cells were grown on sapphire discs and fixed at 8 h p.i. for 30 min at 354 room temperature with 3% paraformaldehyde and 0.25% glutaraldehyde in 0.1 M PHEM buffer pH 355 6.9 60 mM piperazide-1,4-bis (2-ethanesulfonic acid), 25

101、mM HEPES, 2mM MgCl2, 10mM EGTA 356 containing 50% diluted Eagles minimal essential medium and 1% FCS. Cells were stored in fixative 357 at 4C for 72 h and then high-pressure frozen using a Leica EM PACT2. Freeze-substitution was 358 performed in an automated system (Leica AFS2) using as freeze-subst

102、itution medium acetone 359 containing 1% OsO4, 0.5% uranyl acetate and 10% H2O. First, the samples were maintained at -90C 360 for 6 h in this medium and then slowly warmed to -20C within 14 h, kept at -20C for 1 h, warmed to 361 0C at a 5C/h rate and left at 0C for 1 h before letting the samples re

103、ach room temperature. After 362 washing with acetone, the samples were gradually infiltrated with epoxy resin LX-112 and 363 polymerized at 60C. The samples were cut into thin sections (100 nm) and counterstained with 364 uranyl acetate and lead citrate. Imaging was performed in an FEI Tecnai12 TWIN

104、 electron 365 microscope operating at 120 kV and equipped with an Eagle 4k cooled slow-scan charge-coupled 366 device (CCD) camera (FEI company). The images were acquired using binning mode 2.367 368 Intracellular viral RNA analysis. Isolation of intracellular viral RNA was described previously (van

105、 369 Kasteren et al., 2013). After drying of the gel, viral mRNAs were detected by hybridization with a 370 15 32P-labeled oligonucleotide probe (5-GCAAATCATCTAATTAGCCTAATC-3) complementary to 371 the 3end of all MERS-CoV mRNAs. Equal loading was verified in a second hybridization using a 372 32P-la

106、beled oligonucleotide probe (5-GTAACCCGTTGAACCCCATT-3)recognizing 18S 373 ribosomal RNA (van Hemert et al., 2008a). ImageQuant TL (GE Healthcare) software was used for 374 quantification.375 376 Real-time reverse transcription-polymerase chain reaction (RT-PCR). ?-?8?477?culture 377 medium of CoV-in

107、fected cells was isolated with the MagnaPure LC total nucleic acid isolation kit 378 *?!?!?77?RT-PCR conditions for quantifying MERS-CoV and SARS-CoV RNA379 and amplification parameters were described previously (Kuiken et al., 2003; Raj et al., 2013). 380 Dilutions of viral RNA isolated from MERS-C

108、oV or SARS-CoV virus stocks with a known virus titre381 were used to produce a standard curve. 382 383 Development of a screening assay for antiviral compounds. Huh7 or Vero cells were seeded in 96-384 well plates at a density of 104or 2x104cells per well, respectively. After overnight growth, cells

109、 were 385 infected with an m.o.i. of 0.005 or 0.05. One to three days after incubation, differences in cell viability 386 caused by virus-induced CPE or by compound-specific side effects were analysed using the CellTiter 387 96 AQueousNon-Radioactive Cell Proliferation Assay (Promega), according to

110、the manufacturers 388 instructions. Absorbance (A490) was measured using a Berthold Mithras LB 940 96-well plate reader. 389 Infected cells were given CsA or DMSO (solvent control) prior to infection (m.o.i 0.005). Cytotoxic 390 effects caused by CsA treatment alone were monitored in parallel plates

111、 containing mock-infected 391 cells. 392 393 IFN sensitivity and p-STAT1 translocation experiments. One day prior to infection, Vero cells 394 were plated at a density of 104cells per well in a 96-well plate format. At -4, 0 and 4 h p.i., cells were 395 incubated with 0 to 1000 ng/ml PEG-IFN in 250

112、l. At t=0 h, all wells were washed with PBS and 396 infected with MERS-CoV or SARS-CoV (100 TCID50per 100 l medium). Those cultures receiving 397 treatment from t=-4 or t=0 were infected in the presence of the indicated concentration PEG-IFN. 398 16 After 1 h, 150 l medium was added to the cultures

113、of t=-4 or t=0 cultures, and 100 l medium was 399 added to the untreated cultures, which at 4 h p.i. received 50 l medium supplemented with PEG-IFN 400 to reach a final concentration of 0 to 1000 ng/ml PEG-IFN. At 48 h p.i., RNA was isolated from 50 l 401 cell culture supernatant and quantified usin

114、g virus-specific real time RT-PCR assays (see above). 402 Furthermore at 48 h p.i., CPE was scored microscopically as either (0) none, (1) mild, (2) moderate, 403 (3) severe or (4) complete.404 For p-STAT1 nuclear translocation experiments, Vero cells were infected with MERS-CoV405 or SARS-CoV (m.o.

115、i. 1). At 8 h p.i., cells were treated with 1000 ng/ml PEG-IFN for 30 min and 406 fixed with 4% formaldehyde and 70% ethanol and subsequently stained for presence of viral antigen 407 and p-STAT1 translocation.408 409 410 17 ACKNOWLEDGEMENTS 411 412 We are grateful to Ron Fouchier, Chris Lauber and

116、Alexander Gorbalenya for helpful discussions, and 413 we thank Dennis Ninaber and Corrine Beugeling for technical assistance. This research was supported 414 in part by TOP grant 700.57.301 from the Council for Chemical Sciences (CW) and MEERVOUD415 grant 836.10.003 from the Council for Earth and Li

117、fe Sciences (ALW) of the Netherlands 416 Organization for Scientific Research (NWO) and the EU-FP7-Health project SILVER (Grant 417 #260644). 418 419 18 FIGURE LEGENDS420 421 Fig 1. Kinetics of MERS-CoV replication in Vero and Huh7 cells. Vero and Huh7 cells were 422 infected with MERS-CoV (m.o.i. 5

118、). (a) Hybridization analysis of viral mRNAs isolated from MERS-423 CoV-infected cells using an oligonucleotide recognizing the viral genome and all sg mRNAs. 424 Additional minor bands of 3 and 4 kb were observed (*) and may represent additional viral mRNA 425 species that remain to be studied in m

119、ore detail. However, the corresponding positions in the ORF4a/b 426 and ORF5 coding regions do not contain a canonical core TRS sequence (AACGAA; (van Boheemen427 et al., 2012) that might provide a direct explanation for their synthesis. (b) Analysis of the relative 428 molarities of viral genome an

120、d each of the sg mRNAs (% of total viral mRNA). mRNA sizes were 429 calculated on the basis of the TRS positions in the viral genome sequence (van Boheemen et al.,430 2012). Phosphorimager quantification was performed on the gel lanes with the RNA samples isolated 431 from Vero cells at 10, 13 and 2

121、4 h p.i. (Fig. 1a; lanes 3, 4, and 5; avg SD). (c) Release of infectious 432 MERS-CoV progeny into the medium of infected Vero or Huh7 cells at the indicated time points, as 433 determined by plaque assay (avg SD; n=4). 434 435 Fig 2. Selected rabbit antisera raised against SARS-CoV and MHV nsps cro

122、ss-react with 436 MERS-CoV proteins. (a) MERS-CoV-infected Vero cells (m.o.i. 5) were fixed at 8 h p.i. For 437 immunofluorescence microscopy, cells were double-labelled with a mouse monoclonal antibody 438 recognizing dsRNA (bottom row) and rabbit antisera raised against SARS-CoV nsp3, nsp4, nsp5 o

123、r439 nsp8, ?)(?#?9?*?:?$?47?*b) Sequence comparison of the C-terminal domain of 440 nsp4 of SARS-CoV (isolate Frankfurt 1), MERS-CoV (strain EMC/2012) and MHV (strain A59). The 441 SARS-CoV and MHV sequences corresponds to the synthetic peptides used to raise rabbit anti-nsp4 442 sera. Residues cons

124、erved in all three viruses are highlighted in yellow, whereas residues conserved in 443 two out of three are highlighted in grey. Amino acid numbers refer to the full-length pp1a sequence.444 (c) Monolayers of Vero, Vero E6, Huh7 and Calu3/2B4 cells were infected with MERS-CoV (m.o.i. 445 5) and dou

125、ble-labelled fo?!#?-?*/?!?#?*?!?:?$?97?446 19 447 Fig.3. Membrane structures induced by MERS-CoV infection. Electron micrographs of thin 448 sections (100 nm) of (a-d) MERS-CoV-infected Vero cells at 8 h p.i. (a) Low magnification images of 449 a cell containing a small cluster of double-membrane ve

126、sicles, enlarged in (b). Some DMVs are 450 indicated by black arrowheads and the inset displays a close-up of the boxed DMV in (b). (c) 451 Extensive membrane alterations in the perinuclear region. The boxed area in (c) is displayed at higher 452 magnification in (d), where CMs (white arrows, inset)

127、 embedded in clusters of DMVs (black 453 arrowheads) can be observed. For comparison, (e) shows the unaltered cytoplasm of a mock-infected 454 cell and (f) contains SARS-CoV-induced DMV (black arrowheads) as observed after HPF and freeze 455 substitution. N, nucleus; m, mitochondria. Scale bars, 2 m

128、 (a,c,e), 500 nm (b,d,f).456 457 Fig 4. MERS-CoV infection induces severe cytopathology in monkey and human cell lines. 458 Monolayers of Vero (a), Calu3/2B4 (b), Vero E6 (c) and Huh7 (d) cells infected with MERS-CoV459 (m.o.i. 0.05) and analysed by light microscope at ?!?!?#?:?$?77?460 461 Fig. 5.

129、Development of an assay to screen for compounds inhibiting MERS-CoV replication.462 (a,c) Vero and (b,d) Huh7 cells in a 96-well plate format were infected at an m.o.i. of 0.005 or 0.05.463 Mock-infected cells were used as reference for unchanged cell viability (their relative viability was set 464

130、at 100%). (a) Infected Vero cells were incubated for 2 or 3 days and (b) Huh7 cells were incubated for 465 1 or 2 days. (c) Vero cells were infected with MERS-CoV (m.o.i. 0.005) in the presence of 3 M or 9 466 M CsA, or 0.09% DMSO as solvent control. (d) Huh7 cells were infected with MERS-CoV (m.o.i

131、. 467 0.005) in the presence of 3.75 to 15 M CsA, or 0.15% DMSO. (c,d) Graphs show the results of a 468 representative experiment (avg SD; n=4). All experiments were repeated at least twice.469 470 Fig. 6. Sensitivity of MERS-CoV and SARS-CoV to PEG-IFN. Vero cells were incubated with 0 to 471 1000

132、ng/ml PEG-IFN at t=-4, t=0, at t=4 h p.i. Cells were infected with 100 TCID50virus per well.472 (a,b) At 2 d p.i. cells were examined for CPE. Effect of PEG-IFN treatment on CPE induced by (a) 473 20 SARS-CoV or (b) MERS-CoV. CPE was scored as either (0) none, (1) mild, (2) moderate, (3) severe 474

133、or (4) complete. (c,d) Viral genomes in the culture medium of virus-infected cells were determined by 475 RT-PCR. Influence of PEG-IFN treatment on the viral RNA load (genome equivalents (gen. eq.) per 476 ml) in the supernatants of cells infected with (c) SARS-CoV or (d) MERS-CoV.477 478 Fig. 7. IF

134、N?-STAT1 in MERS-CoV-infected Vero cells. 479 Confocal immunofluorescence microscopy of uninfected Vero cells (a-d) and Vero cells infected 480 (m.o.i. 1) with SARS-CoV (e,f) or MERS-CoV (g,h). At 8 h p.i. cells were (a, b) left untreated or (c-481 h) treated with 1000 ng/ml PEG-IFN for 30 minutes,

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