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Journal of Virology, February 2002, p. 1521-1526, Vol. 76, No. 3
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.3.1521-1526.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Kissing Interaction between 3' Noncoding and Coding Sequences Is Essential for Porcine Arterivirus RNA Replication

M. H. Verheije,1* R. C. L. Olsthoorn,2 M. V. Kroese,1 P. J. M. Rottier,3 and J. J. M. Meulenberg1,{dagger}

Department of Infectious Diseases and Food Chain Quality, Institute for Animal Science and Health, Lelystad,1 Institute of Molecular Plant Sciences, Gorlaeus Laboratories, Leiden University, Leiden,2 Virology Division, Department of Infectious Diseases and Immunology, Veterinary Faculty, Utrecht University, Utrecht, The Netherlands3

Received 12 June 2001/ Accepted 24 October 2001

We used an infectious cDNA clone of Porcine reproductive and respiratory syndrome virus (PRRSV) to investigate the presence of essential replication elements in the region of the genome encoding the structural proteins. Deletion analysis showed that a stretch of 34 nucleotides (14653 to 14686) within ORF7, which encodes the nucleocapsid protein, is essential for RNA replication. Strand-specific reverse transcription-PCR analysis of viral RNA isolated from transfected BHK-21 cells revealed that this region is required for negative-strand genomic RNA synthesis. The 34-nucleotide stretch is highly conserved among PRRSV isolates and folds into a putative hairpin. A 7-base sequence within the loop of this structure was suggested to base-pair with a sequence present in the loop of a hairpin located in the 3' noncoding region, resulting in a kissing interaction. Mutational analyses confirmed that this kissing interaction is required for RNA replication.

* Corresponding author. Mailing address: Virology Division, Department of Infectious Diseases and Immunology, Veterinary Faculty, Utrecht University, Yalelaan 1, NL-3584 CL Utrecht, The Netherlands. Phone: 31 30 253 5094. Fax: 31 30 253 6723. E-mail: h.verheije{at}vet.uu.nl.

{dagger} Present address: Amsterdam Molecular Therapeutics, Amsterdam, The Netherlands.

Journal of Virology, February 2002, p. 1521-1526, Vol. 76, No. 3
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.3.1521-1526.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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