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 US-inverted repeat (US-IR) and the US-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-bas e-paired intervening ''spacer'' sequences between these secondary-stru cture elements. Small deletions or insertions in these intervening seq uences decreased initiation of reverse transcription in vitro. In cont rast, base substitutions, which maintain the spacing distances between the structures, had no detectable effect. Additionally, a small delet ion at the 3' end of the PBS caused a significant decrease in initiati on 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 mainta ined in a specific orientation that is required for efficient initiati on of reverse transcription. Specific sequence recognition of the dupl ex structures by reverse transcriptase is also required since mosaic R NAs that combine the human immunodeficiency virus type 1 PBS with avia n sequences is not efficiently utilized for reverse transcription even though the primer used can anneal to the substituted PBS.