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

Abstract

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.

Contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity

“…It has been shown for several coronaviruses that coexpression of S, M, and E results in the formation of virus-like particles that bud from infected cells (19,(26)(27)(28). Also, whereas M expressed alone was retained in the Golgi network, coexpression of M and E resulted in expression at more distal cell membranes (19,20,27,29).…”

Role of Mouse Hepatitis Coronavirus Envelope Protein Transmembrane Domain

“…Expression of E and M alone is sufficient for virus-like particle (VLP) assembly. [1][2][3] E protein containing vesicles are released from cells when E is expressed alone. 4 E protein may also be involved in determining the virus budding site at the endoplasmic reticulum-Golgi intermediate compartment membrane (ERGIC).…”

Retargeting of Coronavirus by Substitution of the Spike Glycoprotein Ectodomain: Crossing the Host Cell Species Barrier

“…Furthermore, the coexpression system was used to show that S protein is also dispensable in the assembly process; only the M and E proteins are required for VLP formation (4,60). This observation accorded well with earlier studies that noted the release of spikeless, noninfectious virions from mouse hepatitis virus (MHV)-infected cells treated with the glycosylation inhibitor tunicamycin (21, 49).The VLP assembly system has provided a valuable avenue to begin exploring the roles of individual proteins in coronavirus morphogenesis (2,4,5,7,8,60), leading to conclusions that, in some cases, have been complemented and extended by the construction of viral mutants (7,14). One of many critical questions to be resolved is the nature of the apparently passive and optional participation of S protein in the budding process.…”

“…The VLP assembly system has provided a valuable avenue to begin exploring the roles of individual proteins in coronavirus morphogenesis (2,4,5,7,8,60), leading to conclusions that, in some cases, have been complemented and extended by the construction of viral mutants (7,14). One of many critical questions to be resolved is the nature of the apparently passive and optional participation of S protein in the budding process.…”