The production of recombinant infectious DI-particles of a murine coronavirus in the absence of helper virus.
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.
Bos
E C
EC
Department of Virology, Leiden University, Leiden, 2300 AH, The Netherlands.
Luytjes
W
W
van der Meulen
H V
HV
Koerten
H K
HK
Spaan
W J
WJ
D000818:Animals; D001483:Base Sequence; D017934:Coronavirus / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D015342:DNA Probes; D003673:Defective Viruses / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D006378:Helper Viruses / Q000502:physiology; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D015678:Viral Structural Proteins / Q000502:physiology; D019065:Virus Assembly
D015342:DNA Probes; D015678:Viral Structural Proteins