Rescue of Synthetic Measles Virus Minireplicons

Measles Genomic Termini Direct Efficient Expression and Propagation of a Reporter Gene

Mohinderjit S. Sidhu, John Chan, Karin Kaelin, Pius Spielhofer, Frank Radecke, Henriette Schneider, Malthi Masurekar, Peter C. Dowling, Martin A. Billeter, Stephen A. Udem

Research output: Contribution to journalArticle

106 Citations (Scopus)

Abstract

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. Transfaction 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 arid extended to the development of full-length MV cDNAs.

Original languageEnglish (US)
Pages (from-to)800-807
Number of pages8
JournalVirology
Volume208
Issue number2
DOIs
StatePublished - Apr 20 1995
Externally publishedYes

Fingerprint

Measles virus
Measles
Reporter Genes
Genome
RNA
Chloramphenicol
Transferases
Complementary DNA
Helper Viruses
Hepatitis Delta Virus
Serial Passage
Catalytic RNA
Messenger RNA
Intergenic DNA
Virion
Sequence Analysis
Nucleotides
Antibodies

ASJC Scopus subject areas

  • Virology
  • Infectious Diseases

Cite this

Rescue of Synthetic Measles Virus Minireplicons : Measles Genomic Termini Direct Efficient Expression and Propagation of a Reporter Gene. / Sidhu, Mohinderjit S.; Chan, John; Kaelin, Karin; Spielhofer, Pius; Radecke, Frank; Schneider, Henriette; Masurekar, Malthi; Dowling, Peter C.; Billeter, Martin A.; Udem, Stephen A.

In: Virology, Vol. 208, No. 2, 20.04.1995, p. 800-807.

Research output: Contribution to journalArticle

Sidhu, MS, Chan, J, Kaelin, K, Spielhofer, P, Radecke, F, Schneider, H, Masurekar, M, Dowling, PC, Billeter, MA & Udem, SA 1995, 'Rescue of Synthetic Measles Virus Minireplicons: Measles Genomic Termini Direct Efficient Expression and Propagation of a Reporter Gene', Virology, vol. 208, no. 2, pp. 800-807. https://doi.org/10.1006/viro.1995.1215
Sidhu, Mohinderjit S. ; Chan, John ; Kaelin, Karin ; Spielhofer, Pius ; Radecke, Frank ; Schneider, Henriette ; Masurekar, Malthi ; Dowling, Peter C. ; Billeter, Martin A. ; Udem, Stephen A. / Rescue of Synthetic Measles Virus Minireplicons : Measles Genomic Termini Direct Efficient Expression and Propagation of a Reporter Gene. In: Virology. 1995 ; Vol. 208, No. 2. pp. 800-807.
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