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Journal of Virology, April 2000, p. 3156-3165, Vol. 74, No. 7
Department of Virology, Center for Infectious
Diseases, Leiden University Medical Center, Leiden, The Netherlands
Received 13 October 1999/Accepted 5 January 2000
Equine arteritis virus (EAV), the type member of the
family Arteriviridae, is a single-stranded RNA virus with a
positive-stranded genome of approximately 13 kb. EAV uses a
discontinuous transcription mechanism to produce a nested set of six
subgenomic mRNAs from which its structural genes are expressed.
We have generated the first documented arterivirus defective
interfering (DI) RNAs by serial undiluted passaging of a wild-type EAV
stock in BHK-21 cells. A cDNA copy of the smallest DI RNA (5.6 kb) was
cloned. Upon transfection into EAV-infected BHK-21 cells, transcripts derived from this clone (pEDI) were replicated and packaged. Sequencing of pEDI revealed that the DI RNA was composed of three segments of the
EAV genome (nucleotides 1 to 1057, 1388 to 1684, and 8530 to 12704)
which were fused in frame with respect to the replicase reading frame.
Remarkably, this DI RNA has retained all of the sequences encoding the
structural proteins. By insertion of the chloramphenicol
acetyltransferase reporter gene in the DI RNA genome, we were able to
delimitate the sequences required for replication/DI-based
transcription and packaging of EAV DI RNAs and to reduce the maximal
size of a replication-competent EAV DI RNA to approximately 3 kb.
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Isolation and Characterization of an Arterivirus
Defective Interfering RNA Genome
*
Corresponding author. Mailing address: Department of
Virology, Leiden University Medical Center, LUMC P4-26, PO Box
9600, 2300 RC Leiden, The Netherlands. Phone: 31 71 5261657. Fax:
31 71 5266761. E-mail: e.j.snijder{at}lumc.nl.
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