title: Construction of a severe acute respiratory syndrome coronavirus infectious cDNA clone and a replicon to study coronavirus RNA synthesis.
abstract: The engineering of a full-length infectious cDNA clone and a functional replicon of the severe acute respiratory syndrome coronavirus (SARS-CoV) Urbani strain as bacterial artificial chromosomes (BACs) is described in this study. In this system, the viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and further amplified in the cytoplasm by the viral replicase. Both the infectious clone and the replicon were fully stable in Escherichia coli. Using the SARS-CoV replicon, we have shown that the recently described RNA-processing enzymes exoribonuclease, endoribonuclease, and 2'-O-ribose methyltransferase were essential for efficient coronavirus RNA synthesis. The SARS reverse genetic system developed as a BAC constitutes a useful tool for the study of fundamental viral processes and also for developing genetically defined vaccines.
authors.affiliation: Department of Molecular and Cell Biology, Centro Nacional de Biotecnología, CSIC, Darwin 3, Campus Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
authors.forename: Fernando
authors.initials: F
authors.lastname: Almazán
authors.forename: Marta L
authors.initials: ML
authors.lastname: Dediego
authors.forename: Carmen
authors.initials: C
authors.lastname: Galán
authors.forename: David
authors.initials: D
authors.lastname: Escors
authors.forename: Enrique
authors.initials: E
authors.lastname: Alvarez
authors.forename: Javier
authors.initials: J
authors.lastname: Ortego
authors.forename: Isabel
authors.initials: I
authors.lastname: Sola
authors.forename: Sonia
authors.initials: S
authors.lastname: Zuñiga
authors.forename: Sara
authors.initials: S
authors.lastname: Alonso
authors.forename: Jose L
authors.initials: JL
authors.lastname: Moreno
authors.forename: Aitor
authors.initials: A
authors.lastname: Nogales
authors.forename: Carmen
authors.initials: C
authors.lastname: Capiscol
authors.forename: Luis
authors.initials: L
authors.lastname: Enjuanes
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D012093:Replicon; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics* / Q000378:metabolism*; D014709:Vero Cells
chemical_list: D018076:DNA, Complementary; D004279:DNA, Viral; D012367:RNA, Viral