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 b e controlled by cis-acting sequence elements contained within the term inal MV genomic noncoding nucleotides. To validate these promoter and regulatory signal assignments, cDNAs were constructed allowing synthes is of RNAs corresponding to a MV genome in which all coding and interc istronic regions were replaced by the chloramphenicol acetyl transfera se (CAT) coding sequence. Transcript production by T7 polymerase start ing and ending precisely with the MV genome terminal residues was achi eved by fusing the T7 polymerase promoter and the hepatitis delta viru s genome ribozyme followed by tandem T7 polymerase termination sequenc es to the MV genomic 5' and 3' ends, respectively. Transfection of the se negative polarity transcripts, mimicking natural defective interfer ing RNAs of the internal deletion type, into MV-infected 293 cells gav e rise to CAT activity which could be serially transferred and massive ly amplified together with progeny helper virus in fresh cells. Transf er was blocked only by antibodies able to neutralize MV infectivity, i ndicating that the chimeric RNA not only was encapsidated, transcribed , and replicated, but also packaged into virions. Sequence analyses co nfirmed that both the expected chimeric antigenome and mRNA products w ere transcribed and replicated with fidelity during serial passage. Mi nor changes introduced in the transcription promoter markedly compromi sed function. This system now can be exploited to examine MV genomic c is-acting regulatory elements and extended to the development of full- length MV cDNAs. (C) 1995 academic Press, Inc.