Utilizing fowlpox virus recombinants to generate defective RNAs of the coronavirus infectious bronchitis virus

J Gen Virol. 2000 Dec;81(Pt 12):2855-2865. doi: 10.1099/0022-1317-81-12-2855.

Abstract

Coronavirus defective RNAs (D-RNAs) have been used as RNA vectors for the expression of heterologous genes and as vehicles for reverse genetics by modifying coronavirus genomes by targetted recombination. D-RNAs based on the avian coronavirus infectious bronchitis virus (IBV) D-RNA CD-61 have been rescued (replicated and packaged into virions) in a helper virus-dependent manner following electroporation of in vitro-generated T7 transcripts into IBV-infected cells. In order to increase the efficiency of rescue of IBV D-RNAs, cDNAs based on CD-61, under the control of a T7 promoter, were integrated into the fowlpox virus (FPV) genome. The 3'-UTR of the D-RNAs was flanked by a hepatitis delta antigenomic ribozyme and T7 terminator sequence to generate suitable 3' ends for rescue by helper IBV. Cells were co-infected simultaneously with IBV, the recombinant FPV (rFPV) containing the D-RNA sequence and a second rFPV expressing T7 RNA polymerase for the initial expression of the D-RNA transcript, subsequently rescued by helper IBV. Rescue of rFPV-derived CD-61 occurred earlier and with higher efficiency than demonstrated previously for electroporation of in vitro T7-generated RNA transcripts in avian cells. Rescue of CD-61 was also demonstrated for the first time in mammalian cells. The rescue of rFPV-derived CD-61 by M41 helper IBV resulted in leader switching, in which the Beaudette-type leader sequence on CD-61 was replaced with the M41 leader sequence, confirming that helper IBV virus replicated the rFPV-derived D-RNA. An rFPV-derived D-RNA containing the luciferase gene under the control of an IBV transcription-associated sequence was also rescued and expressed luciferase on serial passage.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bacteriophage T7 / genetics
  • Base Sequence
  • Cell Line
  • Chickens
  • Chlorocebus aethiops
  • DNA, Recombinant / genetics*
  • DNA, Viral / genetics*
  • Defective Viruses / genetics*
  • Defective Viruses / physiology
  • Fowlpox virus / genetics*
  • Fowlpox virus / physiology
  • Gene Expression Regulation, Viral
  • Genes, Reporter / genetics
  • Genes, Viral / genetics
  • Genetic Complementation Test
  • Genetic Vectors / genetics
  • Genetic Vectors / physiology
  • Helper Viruses / genetics
  • Helper Viruses / physiology
  • Infectious bronchitis virus / genetics*
  • Infectious bronchitis virus / physiology
  • Kidney / cytology
  • Kidney / virology
  • Molecular Sequence Data
  • Promoter Regions, Genetic / genetics
  • RNA, Catalytic / genetics
  • RNA, Catalytic / metabolism
  • RNA, Messenger / analysis
  • RNA, Messenger / biosynthesis
  • RNA, Messenger / genetics
  • RNA, Viral / analysis
  • RNA, Viral / biosynthesis*
  • RNA, Viral / genetics
  • Terminator Regions, Genetic / genetics
  • Vero Cells
  • Virus Assembly

Substances

  • DNA, Recombinant
  • DNA, Viral
  • RNA, Catalytic
  • RNA, Messenger
  • RNA, Viral