A novel interaction of tRNA(Lys,3) with the feline immunodeficiency virus RNA genome governs initiation of minus strand DNA synthesis.
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.
Miller
J T
JT
HIV Drug Resistance Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702, USA.
Ehresmann
B
B
Hübscher
U
U
Le Grice
S F
SF
D001483:Base Sequence; D004261:DNA Replication / Q000235:genetics*; D016679:Genome, Viral; D016089:Immunodeficiency Virus, Feline / Q000235:genetics*; D009690:Nucleic Acid Conformation; D012357:RNA, Transfer, Lys / Q000737:chemistry / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*
D012357:RNA, Transfer, Lys; D012367:RNA, Viral