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Journal of Virology, September 2003, p. 9790-9798, Vol. 77, No. 18
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.18.9790-9798.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Multigene RNA Vector Based on Coronavirus Transcription

Volker Thiel,1* Nadja Karl,1 Barbara Schelle,1 Petra Disterer,1 Ingo Klagge,1 and Stuart G. Siddell2

Institute of Virology and Immunology, University of Würzburg, Würzburg, Germany,1 Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, Bristol, United Kingdom2

Received 27 February 2003/ Accepted 19 June 2003

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.

* Corresponding author. Present address: Cantonal Hospital, St. Gallen Research Department, 9007 St. Gallen, Switzerland. Phone: 41-71-494 1074. Fax: 41-71-494 6321. E-mail: volker.thiel{at}kssg.ch.

Journal of Virology, September 2003, p. 9790-9798, Vol. 77, No. 18
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.18.9790-9798.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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