Reverse transcription of a retroviral RNA genome is a critical part of its life cycle and
needs mechanisms for appropriate timing and regulation. Our study set out to confirm
and explain a prior observation (unpublished results) that reverse transcription
initiation (RTI) of the HIV-1 LAI leader RNA varies among 3’-end truncated
transcripts. We confirmed this observation and observed that the RTI of shorter
transcripts (3’-end positions 202, 248 & 256) increased with transcript length.
However, an opposite pattern was observed for longer transcripts (3’-end positions
263, 270, 281, 290 & 368) of which the RTI decreased with transcript length. As the
HIV-1 leader RNA can adopt two different conformations, the rodlike LDI and the
branched BMH structure, we investigated if the conformational state of the transcripts
influence RTI efficiency. RTI of mutants that either fold the LDI or the BMH structure
was not affected, thereby excluding the possibility of RTI regulation by the LDI/BMH
status. We next set out to determine whether the availability of the primer activation
sequence (PAS) is responsible for the observed variation in reverse transcription
initiation. This sequence has been reported to be crucial for efficient RTI through
interaction with a complementary sequence in the tRNA primer. We performed primer
extension assays on transcripts that were RTI impaired due to their mutations in the
PAS sequence, and stimulated in RTI as the PAS sequence was exposed by
mutating the complementary sequence in the PBS stem. The combined results show
that the observed decrease in RTI for wt transcripts is due to an occlusion of the PAS
motif. However, calculating the availability of the PAS sequence, expressed in
ΔΔGPAS values, with the secondary structure prediction program Mfold showed that
the availability of this sequence is equal in all 3’-end truncated transcripts. Therefore,
the primer extension data combined with the calculated ΔΔGPAS values suggests that
the regulation of reverse transcription initiation is complicated by the existence of a
tertiary interaction. Furthermore, Goldschmidt et al. questioned the existence of the
PAS interaction since their results show that the observed differences in reverse
transcription efficiencies of tRNA primed extensions are equal for an RNA
oligonucleotide (lacking the antiPAS sequence) primed extensions. However, we
show here that the observed down-regulation of RTI of the PAS mutants and upregulation of a mutant that exposes the PAS motif is only observed when a complete
tRNA or a half tRNA (containing the antiPAS sequence) is used and not when an
RNA oligonucleotide lacking the antiPAS sequence is used for RTI. This underscores
the importance of the PAS motif for the regulation of reverse transcription initiation.