Multigene RNA vector based on coronavirus transcription.
Coronavirus genomes are the largest known autonomously replicating RNAs with a size of ca. 30 kb. They are of positive polarity and are translated to produce the viral proteins needed for the assembly of an active replicase-transcriptase complex. In addition to replicating the genomic RNA, a key feature of this complex is a unique transcription process that results in the synthesis of a nested set of six to eight subgenomic mRNAs. These subgenomic mRNAs are produced in constant but nonequimolar amounts and, in general, each is translated to produce a single protein. To take advantage of these features, we have developed a multigene expression vector based on human coronavirus 229E. We have constructed a prototype RNA vector containing the 5' and 3' ends of the human coronavirus genome, the entire human coronavirus replicase gene, and three reporter genes (i.e., the chloramphenicol acetyltransferase [CAT] gene, the firefly luciferase [LUC] gene, and the green fluorescent protein [GFP] gene). Each reporter gene is located downstream of a human coronavirus transcription-associated sequence, which is required for the synthesis of individual subgenomic mRNAs. The transfection of vector RNA and human coronavirus nucleocapsid protein mRNA into BHK-21 cells resulted in the expression of the CAT, LUC, and GFP reporter proteins. Sequence analysis confirmed the synthesis of coronavirus-specific mRNAs encoding CAT, LUC, and GFP. In addition, we have shown that human coronavirus-based vector RNA can be packaged into virus-like particles that, in turn, can be used to transduce immature and mature human dendritic cells. In summary, we describe a new class of eukaryotic, multigene expression vectors that are based on the human coronavirus 229E and have the ability to transduce human dendritic cells.
Thiel
Volker
V
Institute of Virology and Immunology, University of Würzburg, Würzburg, Germany. volker.thiel@kssg.ch
Karl
Nadja
N
Schelle
Barbara
B
Disterer
Petra
P
Klagge
Ingo
I
Siddell
Stuart G
SG
D000818:Animals; D017934:Coronavirus / Q000235:genetics*; D006224:Cricetinae; D003713:Dendritic Cells / Q000276:immunology; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007167:Immunotherapy; D005810:Multigene Family; D012367:RNA, Viral / Q000235:genetics*; D014158:Transcription, Genetic; D014161:Transduction, Genetic; D014162:Transfection; D019065:Virus Assembly
D012367:RNA, Viral