Rescue of measles viruses from cloned DNA

EMBO J. 1995 Dec 1;14(23):5773-84.

Abstract

A system has been established allowing the rescue of replicating measles viruses (MVs) from cloned DNA. On one hand, plasmids were constructed from which MV antigenomic RNAs with the correct termini are transcribed by phage T7 RNA polymerase. On the other hand, helper cells derived from the human embryonic kidney 293 cell line were generated constitutively expressing T7 RNA polymerase together with MV nucleocapsid protein and phosphoprotein. Simultaneous transfection of the helper cells with the MV antigenomic plasmid and with a plasmid encoding the MV polymerase under direction of a T7 promoter led to formation of syncytia from which MVs were easily recovered. A genetic tag comprising three nucleotide changes was present in the progeny virus. As a first application of reverse genetics, a segment of 504 nucleotides from the 5' non-coding region of the fusion gene was deleted, leading to an MV variant whose replication behaviour in Vero cells was indistinguishable from that of the laboratory Edmonston B strain. Since no helper virus is involved, this system, in principle, should be applicable to the rescue of any member of the large virus order Mononegavirales, i.e. viruses with a nonsegmented negative-strand RNA genome.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • Cell Line
  • Cloning, Molecular
  • DNA-Directed RNA Polymerases / metabolism
  • Gene Expression Regulation / genetics
  • Genes, Reporter / genetics
  • Giant Cells
  • Humans
  • Kidney
  • Measles virus / genetics*
  • Measles virus / isolation & purification*
  • Measles virus / physiology
  • Molecular Sequence Data
  • Plasmids / genetics
  • Polymerase Chain Reaction
  • RNA, Antisense / genetics
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Sequence Analysis
  • Transcription, Genetic / genetics
  • Transfection / genetics
  • Viral Proteins / genetics
  • Viral Proteins / metabolism
  • Virus Replication

Substances

  • RNA, Antisense
  • RNA, Messenger
  • Viral Proteins
  • bacteriophage T7 RNA polymerase
  • DNA-Directed RNA Polymerases

Associated data

  • GENBANK/K01711
  • GENBANK/Z66517