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Journal of Virology, May 2001, p. 4922-4928, Vol. 75, No. 10
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.10.4922-4928.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Transfer of Primer Binding Site-Mutated Simian Immunodeficiency Virus Vectors by Genetically Engineered Artificial and Hybrid tRNA-Like Primers

Anette Chemnitz Hansen,¹ Thomas Grunwald,² Anders Henrik Lund,^1, Alexander Schmitz,¹ Mogens Duch,¹ Klaus Überla,² and Finn Skou Pedersen^1,3,*

Department of Molecular and Structural Biology¹ and Department of Medical Microbiology and Immunology,³ Aarhus University, DK-8000 Aarhus, Denmark, and Institute of Virology, Leipzig University, D-04103 Leipzig, Germany²

Received 19 October 2000/Accepted 14 February 2001

Simian immunodeficiency viruses (SIV) harbor primer binding sites (PBS) matching tRNA or tRNA. To study determinants of primer usage in SIV, a SIVmac239-based vector was impaired by mutating the PBS to a sequence (PBS-X2) with no match to any tRNA. By cotransfection of a synthetic gene encoding a tRNA^Pro-like RNA with a match to PBS-X2, the activity of this vector could be restored to a transduction efficiency slightly lower than that of the wild-type vector. A vector with a PBS matching tRNA^Pro was functional at a level slightly below that of the wild-type vector, but higher transduction efficiency could be obtained by cotransfection of a gene for an engineered tRNA^Pro-tRNA hybrid with a match to PBS-Pro. The importance of tRNA backbone identity was further analyzed by complementing the PBS-X2 vector with a gene for a matching x2 primer with a tRNA backbone, which led to three- to fourfold-higher titers than those observed for the x2 primer with the tRNA^Pro backbone. In summary, our results demonstrate flexibility in PBS and primer usage for SIVmac239, with PBS-primer complementarity being the major determinant, in analogy with previous findings for murine leukemia viruses and human immunodeficiency virus type 1.

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^* Corresponding author. Mailing address: Department of Molecular and Structural Biology, Aarhus University, C.F. Moellers Allé, Bldg. 130, DK-8000 Aarhus, Denmark. Phone: 45 89423188. Fax: 45 86196500. E-mail: fsp{at}mbio.aau.dk.

Present address: Division of Molecular Carcinogenesis, The Netherlands Cancer Institute, NL-1066CX Amsterdam, The Netherlands.

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Journal of Virology, May 2001, p. 4922-4928, Vol. 75, No. 10
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.10.4922-4928.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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