The production of recombinant infectious DI-particles of a murine coronavirus in the absence of helper virus

Virology. 1996 Apr 1;218(1):52-60. doi: 10.1006/viro.1996.0165.


We have studied the production and release of infectious DI-particles in vaccinia-T7-polymerase recombinant virus-infected L cells that were transfected with five different plasmids expressing the synthetic DI RNA MIDI-HD and the four structural proteins (M, N, S, and E) of the murine coronavirus MHV-A59. The DI cDNA contains the hepatitis delta ribozyme sequences to generate in the transfected cells a defined 3' end. In EM studies of transfected cells virus-like particles (VLP) were observed in vesicles. Release of the particles into the medium was studied by immunoprecipitations of proteins released into the culture supernatant. Particle release was independent of S or N, but required M and E. Coexpression of E and M was sufficient for particle release. Coexpression of the structural proteins and the MIDI-HD RNA resulted in the production and release of infectious DI-particles. Infectivity of the DI-particles was determined by adding helper virus MHV-A59 to the medium containing the VLPs and using this mixture to infect new L cells. Intracellular RNA of several subsequent undiluted passages was isolated to detect the MIDI-HD RNA. Passage of the MIDI-HD RNA was dependent on the expression of the structural proteins of MHV-A59 in the transfected cells. In the absence of either E or M, MIDI-HD RNA could not be passaged to fresh L cells. We have thus developed a system in which we can produce coronavirus-like particles and an assay to test their infectivity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Coronavirus / genetics
  • Coronavirus / pathogenicity
  • Coronavirus / physiology*
  • DNA Probes
  • Defective Viruses / genetics
  • Defective Viruses / pathogenicity
  • Defective Viruses / physiology*
  • Helper Viruses / physiology
  • L Cells
  • Mice
  • Molecular Sequence Data
  • Viral Structural Proteins / physiology
  • Virus Assembly*


  • DNA Probes
  • Viral Structural Proteins