N. Beerens et al., A structured RNA motif is involved in correct placement of the tRNA(3)(Lys) primer onto the human immunodeficiency virus genome, J VIROLOGY, 74(5), 2000, pp. 2227-2238
Human immunodeficiency virus type 1 (HIV-1) reverse transcription is primed
by the cellular tRNA(3)(Lys) molecule that binds with its 3'-terminal 18 n
ucleotides to the fully complementary primer-binding site (PBS) on the vira
l RNA genome. Besides this complementarity, annealing of the primer may be
stimulated by additional base-pairing interactions between other parts of t
he tRNA molecule and viral sequences flanking the PBS, According to the RNA
secondary structure model of the HIV-1 leader region, part of the PBS sequ
ence is involved in base pairing to form a small stem-loop structure, terme
d the U5-PBS hairpin, This hairpin may be involved in the process of revers
e transcription. To study the role of the U5-PBS hairpin in the viral repli
cation cycle, we introduced mutations in the U5 region that affect the stab
ility of this structured RNA motif, Stabilization and destabilization of th
e hairpin significantly inhibited virus replication, Upon prolonged culturi
ng of the virus mutant with the stabilized hairpin, revertant viruses were
obtained with additional mutations that restore the thermodynamic stability
of the U5-PBS hairpin. The thermodynamic stability of the U5-PBS hairpin a
pparently has to stay within narrow limits for efficient HIV-1 replication,
Transient transfection experiments demonstrated that transcription of the
proviral genomes, translation of the viral mRNAs, and assembly of the virio
ns with a normal RNA content is not affected by the mutations within the U5
-PBS hairpin. We show that stabilization of the hairpin reduced the amount
of tRNA primer that is annealed to the PBS. Destabilization of the hairpin
did not affect tRNA annealing,: but the viral RNA-tRNA complex was less sta
ble. These results suggest that the U5-PBS hairpin is involved incorrect pl
acement of the tRNA primer on the viral genome. The analysis of virus mutan
ts and revertants and the RNA structure probing experiments presented in th
is study are consistent with the existence of the:U5-PBS hairpin as predict
ed in the RNA secondary structure model.