Dynamics of the HIV-1 reverse transcription complex during initiation of DNA synthesis.
Initiation of human immunodeficiency virus-1 (HIV-1) reverse transcription requires formation of a complex containing the viral RNA (vRNA), tRNA(3)(Lys) and reverse transcriptase (RT). The vRNA and the primer tRNA(3)(Lys) form several intermolecular interactions in addition to annealing of the primer 3' end to the primer binding site (PBS). These interactions are crucial for the efficiency and the specificity of the initiation of reverse transcription. However, as they are located upstream of the PBS, they must unwind as DNA synthesis proceeds. Here, the dynamics of the complex during initiation of reverse transcription was followed by enzymatic probing. Our data revealed reciprocal effects of the tertiary structure of the vRNA.tRNA(3)(Lys) complex and reverse transcriptase (RT) at a distance from the polymerization site. The structure of the initiation complex allowed RT to interact with the template strand up to 20 nucleotides upstream from the polymerization site. Conversely, nucleotide addition by RT modified the tertiary structure of the complex at 10-14 nucleotides from the catalytic site. The viral sequences became exposed at the surface of the complex as they dissociated from the tRNA following primer extension. However, the counterpart tRNA sequences became buried inside the complex. Surprisingly, they became exposed when mutations prevented the intermolecular interactions in the initial complex, indicating that the fate of the tRNA depended on the tertiary structure of the initial complex.
Lanchy
J M
JM
UPR 9002 du CNRS, IBMC, 67084 Strasbourg cedex, France.
Isel
C
C
Keith
G
G
Le Grice
S F
SF
Ehresmann
C
C
Ehresmann
B
B
Marquet
R
R
D000926:Anticodon; D001483:Base Sequence; D004261:DNA Replication; D015497:HIV-1; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D012357:RNA, Transfer, Lys / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D013698:Templates, Genetic
D000926:Anticodon; D012357:RNA, Transfer, Lys; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase