A specific orientation of RNA secondary structures is required for initiation of reverse transcription.
The 5' end of avian retrovirus RNA near the primer-binding site (PBS) forms two secondary structures, the U5-inverted repeat (U5-IR) and the U5-leader stems, and contains a 7-nucleotide sequence that anneals to the T psi C loop of the tRNA(Trp) primer. Mutations that disrupt any of these base pair interactions cause defects in initiation of reverse transcription both in vivo and in vitro (D. Cobrinik, A. Aiyar, Z. Ge, M. Katzman, H. Huang, and J. Leis, J. Virol. 65:3864-3872, 1991; A. Aiyar, D. Cobrinik, Z. Ge, H.-J. Kung, and J. Leis, J. Virol. 66:2464-2472, 1992). We have now examined the effect of perturbing the non-base-paired intervening "spacer" sequences between these secondary-structure elements. Small deletions or insertions in these intervening sequences decreased initiation of reverse transcription in vitro. In contrast, base substitutions, which maintain the spacing distances between the structures, had no detectable effect. Additionally, a small deletion at the 3' end of the PBS caused a significant decrease in initiation of reverse transcription whereas substitution mutations again had no effect. Together, these results indicate that reverse transcriptase forms a complex in which the different structural elements are maintained in a specific orientation that is required for efficient initiation of reverse transcription. Specific sequence recognition of the duplex structures by reverse transcriptase is also required since mosaic RNAs that combine the human immunodeficiency virus type 1 PBS with avian sequences is not efficiently utilized for reverse transcription even though the primer used can anneal to the substituted PBS.
Aiyar
A
A
Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
Ge
Z
Z
Leis
J
J
D001358:Avian Sarcoma Viruses / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D002474:Cell-Free System; D004252:DNA Mutational Analysis; D015497:HIV-1 / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D014158:Transcription, Genetic
D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase