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J. Biol. Chem., Vol. 276, Issue 29, 27721-27730, July 20, 2001

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A Novel Interaction of tRNA^Lys,3 with the Feline Immunodeficiency Virus RNA Genome Governs Initiation of Minus Strand DNA Synthesis^*

Jennifer T. Miller, Bernard Ehresmann^§, Ulrich Hübscher^¶, and Stuart F. J. Le Grice

From the  HIV Drug Resistance Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702, the ^§ Institut de Biologie Moléculaire et Cellulaire du CNRS, rue Rene Decartes, 67084 Strasbourg Cedex, France, and the ^¶ Institute of Veterinary Biochemistry, University of Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zürich

Complementarity between nucleotides at the 5' terminus of tRNA^Lys,3 and the U5-IR loop of the feline immunodeficiency virus RNA genome suggests a novel intermolecular interaction controls initiation of minus strand synthesis in a manner analogous to other retroviral systems. Base pairing of this tRNA-viral RNA duplex was confirmed by nuclease mapping of the RNA genome containing full-length or 5'-deleted variants of tRNA^Lys,3 hybridized to the primer-binding site. A major pause in RNA-dependent DNA synthesis occurred 14 nucleotides ahead of the primer-binding site with natural and synthetic tRNA^Lys,3 primers, indicating it was not a consequence of tRNA base modifications. The majority of the paused complexes resulted in dissociation of the reverse transcriptase from the template/primer, as demonstrated by an assay limited to a single binding event. Hybridization of a tRNA mutant whose 5' nucleotides are deleted relieved pausing at this position and subsequently allowed high level DNA synthesis. Additional experiments with tRNA-DNA chimeric primers were used to localize the stage of minus strand synthesis at which the tRNA-viral RNA interaction was disrupted. Finally, replacing nucleotides of the feline immunodeficiency virus U5-IR loop with the (A)₄ sequence of its human immunodeficiency virus (HIV)-1 counterpart also relieved pausing, but did not induce pausing immediately downstream of the primer-binding site previously noted during initiation of HIV-1 DNA synthesis. These combined observations provide further evidence of cis-acting sequences immediately adjacent to the primer-binding site controlling initiation of minus strand DNA synthesis in retroviruses and retrotransposons.

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