Abstract
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.
Highlights
Cis-acting sequences throughout the RNA genome are important mediators of several events during replication of retroviruses and retrotransposons, including transcription [1, 2], translation [3], nuclear transport [4, 5], and genome packaging [6, 7]
Feline immunodeficiency virus (FIV) RT shares a heterodimeric organization of its p66 and p51 subunits with the HIV-1 enzyme and likewise exploits tRNALys,3 as its replication primer, the absence of an A-rich U5-IR loop upstream of the primer-binding site (PBS) suggests base pairing with the tRNA anticodon loop is unlikely
Further inspection of feline immunodeficiency virus (FIV) U5-IR loop sequences indicated extensive homology with nucleotides at the extreme 5Ј-end of tRNALys,3. These tRNA nucleotides would make up in part the acceptor stem in free tRNALys,3, they might be available for pairing upon its hybridization to the FIV PBS, an event mediated through the 18 3Ј terminal tRNA nucleotides
Summary
Vol 276, No 29, Issue of July 20, pp. 27721–27730, 2001 Printed in U.S.A. A Novel Interaction of tRNALys, with the Feline Immunodeficiency Virus RNA Genome Governs Initiation of Minus Strand DNA Synthesis*. Complementarity between nucleotides at the 5 terminus of tRNALys, 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 tRNALys, hybridized to the primer-binding site. We have evaluated the initiation program of FIV by a variety of approaches, directed at both the tRNA primer and viral RNA genome The first of these involved nuclease mapping of the 5Ј end of the FIV genome to which full-length tRNALys, and a variant lacking nucleotides constituting part of the D stem-loop and the entire 5Ј acceptor stem terminus was hybridized. Initiation of reverse transcription in many retroviruses and retrotransposons can be viewed as a complex, multistep process where productive elongation arises through “escape” synthesis from an abortive initiation complex, in many respects analogous to the initiation program exhibited by prokaryotic RNA polymerase
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