The Production of Recombinant Infectious DI-Particles of a Murine Coronavirus in the Absence of Helper Virus

• Published in: Virology (New York, N.Y.) N.Y.), 1996-04, Vol.218 (1), p.52-60
• Main Authors: BOS, EVELYNE C.W., LUYTJES, WILLEM, MEULEN, HANS VAN DER, KOERTEN, HENK K., SPAAN, WILLY J.M.
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Coronavirus - genetics; Coronavirus - pathogenicity; Coronavirus - physiology; Defective Viruses - genetics; Defective Viruses - pathogenicity; Defective Viruses - physiology; DNA Probes; Helper Viruses - physiology; L Cells; Mice; Molecular Sequence Data; murine coronavirus; Regular; Viral Structural Proteins - physiology; Virus Assembly
• ISSN: 0042-6822; 1096-0341
• 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.