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Journal of Virology, January 2006, p. 723-736, Vol. 80, No. 2
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.2.723-736.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Identification of 5' and 3' cis-Acting Elements of the Porcine Reproductive and Respiratory Syndrome Virus: Acquisition of Novel 5' AU-Rich Sequences Restored Replication of a 5'-Proximal 7-Nucleotide Deletion Mutant{dagger}

Yu-Jeong Choi,1,{ddagger} Sang-Im Yun,1,{ddagger} Shien-Young Kang,2 and Young-Min Lee1*

Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Korea,1 Research Institute of Veterinary Medicine, College of Veterinary Medicine, Chungbuk National University, Cheongju, Korea2

Received 23 June 2004/ Accepted 11 October 2005

We here demonstrate the successful engineering of the RNA genome of porcine reproductive and respiratory syndrome virus (PRRSV) by using an infectious cDNA as a bacterial artificial chromosome. Runoff transcription from this cDNA by SP6 polymerase resulted in capped synthetic RNAs bearing authentic 5' and 3' ends of the viral genome that had specific infectivities of >5 x 105 PFU/µg of RNA. The synthetic viruses recovered from the transfected cells were genotypically and phenotypically indistinguishable from the parental virus. Using our system, a series of genomic RNAs with nucleotide deletions in their 5' ends produced viruses with decreased or no infectivity. Various pseudorevertants were isolated, and acquisition of novel 5' sequences of various sizes, composed predominantly of A and U bases, restored their infectivities, providing a novel insight into functional elements of the 5' end of the PRRSV genome. In addition, our system was further engineered to generate a panel of self-replicating, self-limiting, luciferase-expressing PRRSV viral replicons bearing various deletions. Analysis of these replicons revealed the presence and location of a 3' cis-acting element in the genome that was required for replication. Moreover, we produced enhanced green fluorescent protein-expressing infectious viruses, which indicates that the PRRSV cDNA/viral replicon/recombinant virus can be developed as a vector for the expression of a variety of heterologous genes. Thus, our PRRSV reverse genetics system not only offers a means of directly investigating the molecular mechanisms of PRRSV replication and pathogenesis but also can be used to generate new heterologous gene expression vectors and genetically defined antiviral vaccines.

* Corresponding author. Mailing address: Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University, 12 Gaeshin-Dong, Heungduk-Ku, Cheongju, Korea. Phone: 82-43-261-2863 Fax: 82-43-272-1603. E-mail: ymlee{at}chungbuk.ac.kr.

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/.

{ddagger} These authors contributed equally to this work.

Journal of Virology, January 2006, p. 723-736, Vol. 80, No. 2
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.2.723-736.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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