Construction of a severe acute respiratory syndrome coronavirus infectious cDNA clone and a replicon to study coronavirus RNA synthesis.
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.
Almazán
Fernando
F
Department of Molecular and Cell Biology, Centro Nacional de Biotecnología, CSIC, Darwin 3, Campus Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
Dediego
Marta L
ML
Galán
Carmen
C
Escors
David
D
Alvarez
Enrique
E
Ortego
Javier
J
Sola
Isabel
I
Zuñiga
Sonia
S
Alonso
Sara
S
Moreno
Jose L
JL
Nogales
Aitor
A
Capiscol
Carmen
C
Enjuanes
Luis
L
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
D018076:DNA, Complementary; D004279:DNA, Viral; D012367:RNA, Viral