The native structure of the human immunodeficiency virus type 1 RNA genome is required for the first strand transfer of reverse transcription.
Retroviral particles contain two genomic RNAs of approximately 9 kb that are linked in a noncovalent manner. In vitro studies with purified transcripts have identified particular RNA motifs that contribute to the RNA-dimerization reaction, but the situation may be more complex within virion particles. In this study, we tested whether the primer-binding site (PBS) of the human immunodeficiency virus type 1 (HIV-1) RNA genome and the associated tRNA(Lys3) primer play a role in the process of RNA dimerization. Deletion of the PBS motif did not preclude the formation of RNA dimers within virus particles, indicating that this motif and the tRNA primer do not participate in the interactions that control RNA packaging and dimerization. Genome dimerization has been proposed to play a role in particular steps of the reverse transcription mechanism. To test this, reverse transcription was performed with the native RNA dimer and the heat-denatured template. These two template forms yielded equivalent levels of minus-strand strong-stop cDNA product, which is an early intermediate of reverse transcription. However, melting of the RNA dimer precluded the next step of reverse transcription, in which the minus-strand strong-stop cDNA is translocated from the 5' repeat element to the 3' repeat element. The results suggest that the conformation of the dimeric RNA genome facilitates the first strand-transfer reaction of the reverse transcription mechanism.
Berkhout
B
B
Academic Medical Center, University of Amsterdam, Meibergdreef 15, Amsterdam, The Netherlands. b.berkhout@mc.uva.nl
Das
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van Wamel
J L
JL
D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D019281:Dimerization; D016679:Genome, Viral; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D009690:Nucleic Acid Conformation; D012313:RNA; D012357:RNA, Transfer, Lys / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
C034477:RNA primers; D012357:RNA, Transfer, Lys; D012367:RNA, Viral; D012313:RNA