Rescue of synthetic measles virus minireplicons: measles genomic termini direct efficient expression and propagation of a reporter gene.
Measles virus (MV) mRNA transcription and replication are thought to be controlled by cis-acting sequence elements contained within the terminal MV genomic noncoding nucleotides. To validate these promoter and regulatory signal assignments, cDNAs were constructed allowing synthesis of RNAs corresponding to a MV genome in which all coding and intercistronic regions were replaced by the chloramphenicol acetyl transferase (CAT) coding sequence. Transcript production by T7 polymerase starting and ending precisely with the MV genome terminal residues was achieved by fusing the T7 polymerase promoter and the hepatitis delta virus genome ribozyme followed by tandem T7 polymerase termination sequences to the MV genomic 5' and 3' ends, respectively. Transfection of these negative polarity transcripts, mimicking natural defective interfering RNAs of the internal deletion type, into MV-infected 293 cells gave rise to CAT activity which could be serially transferred and massively amplified together with progeny helper virus in fresh cells. Transfer was blocked only by antibodies able to neutralize MV infectivity, indicating that the chimeric RNA not only was encapsidated, transcribed, and replicated, but also packaged into virions. Sequence analyses confirmed that both the expected chimeric antigenome and mRNA products were transcribed and replicated with fidelity during serial passage. Minor changes introduced in the transcription promoter markedly compromised function. This system now can be exploited to examine MV genomic cis-acting regulatory elements and extended to the development of full-length MV cDNAs.
Sidhu
M S
MS
Department of Neurosciences, New Jersey Medical School, Newark 07103, USA.
Chan
J
J
Kaelin
K
K
Spielhofer
P
P
Radecke
F
F
Schneider
H
H
Masurekar
M
M
Dowling
P C
PC
Billeter
M A
MA
Udem
S A
SA
D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D016679:Genome, Viral; D006378:Helper Viruses / Q000502:physiology; D008459:Measles virus / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012093:Replicon / Q000235:genetics*; D012692:Serial Passage; D014158:Transcription, Genetic / Q000235:genetics*; D014162:Transfection; D014779:Virus Replication / Q000235:genetics
D012333:RNA, Messenger; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase