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Journal of Virology, January 2000, p. 600-610, Vol. 74, No. 2
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Mutations of the Kissing-Loop Dimerization Sequence Influence the
Site Specificity of Murine Leukemia Virus Recombination In
Vivo
Jacob Giehm
Mikkelsen,1
Anders H.
Lund,1
Mogens
Duch,1 and
Finn Skou
Pedersen1,2,*
Department of Molecular and Structural
Biology1 and Department of Medical
Microbiology and Immunology,2 University of
Aarhus, DK-8000 Aarhus, Denmark
Received 9 July 1999/Accepted 6 October 1999
The genetic information of retroviruses is retained within a
dimeric RNA genome held together by intermolecular RNA-RNA interactions near the 5' ends. Coencapsidation of retrovirus-derived RNA molecules allows frequent template switching of the virus-encoded reverse transcriptase during DNA synthesis in newly infected cells. We have
previously shown that template shifts within the 5' leader of murine
leukemia viruses occur preferentially within the kissing stem-loop
motif, a cis element crucial for in vitro RNA dimer formation. By use of a forced recombination approach based on single-cycle transfer of Akv murine leukemia virus-based vectors harboring defective primer binding site sequences, we now report that
modifications of the kissing-loop structure, ranging from a deletion of
the entire sequence to introduction of a single point mutation in the
loop motif, significantly disturb site specificity of recombination
within the highly structured 5' leader region. In addition, we find
that an intact kissing-loop sequence favors optimal RNA encapsidation
and vector transduction. Our data are consistent with the kissing-loop
dimerization model and suggest that a direct intermolecular RNA-RNA
interaction, here mediated by palindromic loop sequences within the
mature genomic RNA dimer, facilitates hotspot template switching during
retroviral cDNA synthesis in vivo.
*
Corresponding author. Mailing address: Department of
Molecular and Structural Biology, University of Aarhus, C. F. Moellers Allé, Bldg. 130, DK-8000 Aarhus, Denmark. Phone: 45 89422614. Fax: 45 86196500. E-mail: fsp{at}mbio.aau.dk.
Journal of Virology, January 2000, p. 600-610, Vol. 74, No. 2
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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