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Journal of Virology, March 2000, p. 2227-2238, Vol. 74, No. 5
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
A Structured RNA Motif Is Involved in Correct
Placement of the tRNA3Lys Primer onto the Human
Immunodeficiency Virus Genome
Nancy
Beerens,
Bep
Klaver, and
Ben
Berkhout*
Department of Human Retrovirology, Academic
Medical Center, University of Amsterdam, Amsterdam, The Netherlands
Received 3 September 1999/Accepted 22 November 1999
Human immunodeficiency virus type 1 (HIV-1) reverse transcription
is primed by the cellular tRNA3Lys molecule that binds
with its 3'-terminal 18 nucleotides to the fully complementary
primer-binding site (PBS) on the viral RNA genome. Besides this
complementarity, annealing of the primer may be stimulated by
additional base-pairing interactions between other parts of the 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
sequence is involved in base pairing to form a small stem-loop
structure, termed the U5-PBS hairpin. This hairpin may be involved in
the process of reverse transcription. To study the role of the U5-PBS
hairpin in the viral replication cycle, we introduced mutations in the
U5 region that affect the stability of this structured RNA motif.
Stabilization and destabilization of the hairpin significantly
inhibited virus replication. Upon prolonged culturing 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
apparently 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 virions 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 stable. These results suggest
that the U5-PBS hairpin is involved in correct placement of the tRNA
primer on the viral genome. The analysis of virus mutants and
revertants and the RNA structure probing experiments presented in this
study are consistent with the existence of the U5-PBS hairpin as
predicted in the RNA secondary structure model.
*
Corresponding author. Mailing address: Department of
Human Retrovirology, Academical Medical Center, University of
Amsterdam, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands. Phone:
31-20-5664822. Fax: 31-20-6916531. E-mail:
B.Berkhout{at}AMC.UVA.NL.
Journal of Virology, March 2000, p. 2227-2238, Vol. 74, No. 5
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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