A previously unknown coronavirus (CoV) is the aetiological agent causing severe acute respiratory syndrome (SARS), for which an effective antiviral treatment is urgently needed. To enable the rapid and biosafe identification of coronavirus replicase inhibitors, we have generated a non-cytopathic, selectable replicon RNA (based on human CoV 229E) that can be stably maintained in eukaryotic cells. Most importantly, the replicon RNA mediates reporter gene expression as a marker for coronavirus replication. We have used a replicon RNA-containing cell line to test the inhibitory effect of several compounds that are currently being assessed for SARS treatment. Amongst those, interferon-α displayed the strongest inhibitory activity. Our results demonstrate that coronavirus replicon cell lines provide a versatile and safe assay for the identification of coronavirus replicase inhibitors. Once this technology is adapted to SARS-CoV replicon RNAs, it will allow high throughput screening for SARS-CoV replicase inhibitors without the need to grow infectious SARS-CoV.
AlmazanF.,
GonzalezJ. M.,
PenzesZ.,
IzetaA.,
CalvoE.,
Plana-DuranJ.,
EnjuanesL.2000; Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome. Proc Natl Acad Sci U S A 97:5516–5521[CrossRef]
AnandK.,
PalmG. J.,
MestersJ. R.,
SiddellS. G.,
ZiebuhrJ.,
HilgenfeldR.2002; Structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain. EMBO J 21:3213–3224[CrossRef]
AnandK.,
ZiebuhrJ.,
WadhwaniP.,
MestersJ. R.,
HilgenfeldR.2003; Coronavirus main proteinase (3CLpro) structure: basis for design of anti-SARS drugs. Science 300:1763–1767[CrossRef]
BoothC. M.,
MatukasL. M.,
TomlinsonG. A.18 other authors2003; Clinical features and short-term outcomes of 144 patients with SARS in the greater Toronto area. JAMA 289:2801–2809[CrossRef]
CinatlJ.,
MorgensternB.,
BauerG.,
ChandraP.,
RabenauH.,
DoerrH. W.2003a; Glycyrrhizin, an active component of liquorice roots, and replication of SARS-associated coronavirus. Lancet 361:2045–2046[CrossRef]
CrottyS.,
CameronC. E.,
AndinoR.2001; RNA virus error catastrophe: direct molecular test by using ribavirin. Proc Natl Acad Sci U S A 98:6895–6900[CrossRef]
DonnellyM. L.,
HughesL. E.,
LukeG.,
MendozaH.,
ten DamE.,
GaniD.,
RyanM. D.2001; The ‘cleavage’ activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring ‘2A-like’ sequences. J Gen Virol 82:1027–1041
DonnellyC. A.,
GhaniA. C.,
LeungG. M.16 other authors2003; Epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in Hong Kong. Lancet 361:1761–1766[CrossRef]
DrostenC.,
GuntherS.,
PreiserW.23 other authors2003; Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med 348:1967–1976[CrossRef]
Elroy-SteinO.,
FuerstT. R.,
MossB.1989; Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5′ sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system. Proc Natl Acad Sci U S A 86:6126–6130[CrossRef]
FrolovI.,
HoffmanT. A.,
PragaiB. M.,
DrygaS. A.,
HuangH. V.,
SchlesingerS.,
RiceC. M.1996; Alphavirus-based expression vectors: strategies and applications. Proc Natl Acad Sci U S A 93:11371–11377[CrossRef]
GonzalezJ. M.,
Gomez-PuertasP.,
CavanaghD.,
GorbalenyaA. E.,
EnjuanesL.2003; A comparative sequence analysis to revise the current taxonomy of the family Coronaviridae. Arch Virol 148:2207–2235[CrossRef]
GuanY.,
ZhengB. J.,
HeY. Q.15 other authors2003; Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China. Science 302:276–278[CrossRef]
KerrS. M.,
SmithG. L.1991; Vaccinia virus DNA ligase is nonessential for virus replication: recovery of plasmids from virus-infected cells. Virology 180:625–632[CrossRef]
KsiazekT. G.,
ErdmanD.,
GoldsmithC. S.23 other authors2003; A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med 348:1953–1966[CrossRef]
KuikenT.,
FouchierR. A.,
SchuttenM.19 other authors2003; Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome. Lancet 362:263–270[CrossRef]
LaiM. M. C.,
HolmesK. V.2001; Coronaviridae : the viruses and their replication. In Fields Virology , 4th edn. pp 1163–1185 Edited by
KnipeD. M.,
HowleyP. M.
Philadelphia: Lippincott Williams & Wilkins;
LoM. K.,
TilgnerM.,
ShiP. Y.2003; Potential high-throughput assay for screening inhibitors of West Nile virus replication. J Virol 77:12901–12906[CrossRef]
MerchlinskyM.,
MossB.1992; Introduction of foreign DNA into the vaccinia virus genome by in vitro ligation: recombination-independent selectable cloning vectors. Virology 190:522–526[CrossRef]
PeirisJ. S.,
LaiS. T.,
PoonL. L. 13 other authors; 2003; Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 361:1319–1325[CrossRef]
RandallG.,
RiceC. M.2001; Hepatitis C virus cell culture replication systems: their potential use for the development of antiviral therapies. Curr Opin Infect Dis 14:743–747[CrossRef]
RotaP. A.,
ObersteM. S.,
MonroeS. S.32 other authors2003; Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science 300:1394–1399[CrossRef]
SnijderE. J.,
BredenbeekP. J.,
DobbeJ. C.7 other authors2003; Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage. J Mol Biol 331:991–1004[CrossRef]
SoL. K.,
LauA. C.,
YamL. Y.,
CheungT. M.,
PoonE.,
YungR. W.,
YuenK. Y.2003; Development of a standard treatment protocol for severe acute respiratory syndrome. Lancet 361:1615–1617[CrossRef]
TannerJ. A.,
WattR. M.,
ChaiY. B.,
LuL. Y.,
LinM. C.,
PeirisJ. S.,
PoonL. L.,
KungH. F.,
HuangJ. D.2003; The severe acute respiratory syndrome (SARS) coronavirus NTPase/helicase belongs to a distinct class of 5′ to 3′ viral helicases. J Biol Chem 278:39578–39582[CrossRef]
ThielV.,
RashtchianA.,
HeroldJ.,
SchusterD. M.,
GuanN.,
SiddellS. G.1997; Effective amplification of 20-kb DNA by reverse transcription PCR. Anal Biochem 252:62–70[CrossRef]
ThielV.,
HeroldJ.,
SchelleB.,
SiddellS. G.2001a; Infectious RNA transcribed in vitro from a cDNA copy of the human coronavirus genome cloned in vaccinia virus. J Gen Virol 82:1273–1281
ThielV.,
IvanovK. A.,
PuticsA.9 other authors2003a; Mechanisms and enzymes involved in SARS coronavirus genome expression. J Gen Virol 84:2305–2315[CrossRef]
YangH.,
YangM.,
DingY.12 other authors2003; The crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor. Proc Natl Acad Sci U S A 100:13190–13195[CrossRef]
YountB.,
CurtisK. M.,
BaricR. S.2000; Strategy for systematic assembly of large RNA and DNA genomes: transmissible gastroenteritis virus model. J Virol 74:10600–10611[CrossRef]
YountB.,
DenisonM. R.,
WeissS. R.,
BaricR. S.2002; Systematic assembly of a full-length infectious cDNA of mouse hepatitis virus strain A59. J Virol 76:11065–11078[CrossRef]
YountB.,
CurtisK. M.,
FritzE. A.,
HensleyL. E.,
JahrlingP. B.,
PrenticeE.,
DenisonM. R.,
GeisbertT. W.,
BaricR. S.2003; Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus. Proc Natl Acad Sci U S A 100:12995–13000[CrossRef]
ZiebuhrJ.,
SiddellS. G.2002; Nidovirales. In The Encyclopaedia of Life Sciences pp 190–198 Edited by
AtlasR.,
BynumW. F.,
CoxM.
London: Stockton Press;