The conversion of the single-stranded RNA genome into
double-stranded DNA by virus-coded reverse transcriptase (RT)
is an essential step of the retrovirus life cycle. In human
immunodeficiency virus type 1 (HIV-1), RT uses the cellular
tRNA3Lys to initiate
the (−) strand DNA synthesis. Placement of the primer
tRNA3Lys involves
binding of its 3′-terminal 18 nt to a complementary
region of genomic RNA termed PBS. However, the PBS sequence
is not the unique determinant of primer usage and additional
contacts are important. This placement is believed to be achieved
in vivo by the nucleocapsid domain of Gag or by the mature protein
NCp. Up to now, structural information essentially arose from
heat-annealed primer-template complexes (Isel et al., J
Mol Biol, 1995, 247:236–250; Isel et al.,
EMBO J, 1999, 18:1038–1048). Here, we
investigated the formation of the primer–template complex
mediated by NCp and compared structural and functional properties
of heat- and NCp-annealed complexes. We showed that both heat-
and NCp-mediated procedures allow comparable high yields of
annealing. Then, we investigated structural features of both
kinds of complexes by enzymatic probing, and we compared their
relative efficiency in (−) strong stop DNA synthesis.
We did not find any significant differences between these
complexes, suggesting that information derived from the
heat-annealed complex can be transposed to the NCp-mediated
complex and most likely to complexes formed in vivo.