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HIV-1 reverse transcription
The replication cycle of the human immunodeficiency
virus type 1 (HIV-1) and other retroviruses is characterized by
reverse transcription of the viral RNA genome into a double-stranded
DNA, which subsequently becomes integrated into the host cell genome.
This process is mediated by the virion-associated enzyme reverse
transcriptase (RT), and the cellular tRNAlys3 molecule
is used as a primer by HIV-1. The tRNA primer binds with its 3’-terminal
18 nucleotides to a complementary sequence in the viral genome,
the primer-binding site (PBS), which is located in the untranslated
leader region of the viral genome (FIG 1). Besides the interaction
between the PBS and the 3'-end of tRNAlys3, reverse transcription
was proposed to be stimulated by additional basepairing interactions
between other parts of the tRNA molecule and viral sequences flanking
the PBS site. In the HIV-1 RNA genome, the PBS is predicted to be
part of an extended RNA structure. This structure consists of a
small U5-PBS hairpin that contains part of the PBS sequence, and
a large stem region formed by sequences of the upstream U5 region
and the downstream leader region, the U5-leader stem (FIG 1). In
this project we study the role of the U5-PBS hairpin and U5-leader
stem in viral replication and reverse transcription.
Enlarged
view |
Fig 1. RNA secondary structure
model of the HIV-1 5’ untranslated leader region showing
the PBS (marked in gray), the U5-PBS hairpin and the U5-leader
stem.
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The U5-PBS hairpin
To study the role of the U5-PBS hairpin in viral replication we
introduced mutations that affect the stability of this RNA motif.
Stabilization or destabilization of the U5-PBS hairpin significantly
reduced virus replication. Forced evolution studies with the mutant
U5-PBS viruses, revealed that the thermodynamic stability of the
hairpin has to stay within narrow limits for efficient HIV-1 replication.
We also demonstrate that the U5-PBS hairpin is involved in the correct
placement of the tRNAlys3 primer onto the viral genome
by biochemical reverse transcription assays.
The U5-leader stem
Stabilization of the U5-leader stem was found
to affect the elongation of reverse transcription, causing the RT
enzyme to pause upon copying 7-8 bases into the extended basepaired
stem. The stabilizing mutations were also introduced into proviral
constructs for replication studies, demonstrating that the mutant
viruses have reduced replication capacity. Analysis of a revertant
virus demonstrated that opening of the stabilized U5-leader stem
can restore both virus replication and reverse transcription.
Two mutants containing large deletions in the U5-leader stem demonstrated
that sequences within the U5-leader stem are also important for
the initiation of reverse transcription. To accurately map these
motifs we mutated the stem by sequential 6 nt substitutions. Analysis
of these mutants in reverse transcription assays demonstrated that
the U5-leader stem contains a motif that interacts directly with
a complementary sequence in the tRNAlys3 molecule. The
tRNAlys3 primer was able to anneal onto RNA templates
lacking this motif, however the presence of this motif is required
to activate the primer and to initiate reverse transcription.
Relevant publications
Beerens N, Klaver B and Berkhout B. 2000.
A structured RNA motif is involved in correct placement of the tRNAlys3
primer onto the HIV-1 genome. Journal
of Virology 74, 2227-2238.
Beerens N and Berkhout B. 2000. In vitro studies on tRNA annealing
and reverse transcription with mutant HIV-1 RNA templates. Journal
of Biological Chemistry 275, 15474-15481.
Beerens N, Groot F and Berkhout B. 2000. Stabilization of the U5-leader
stem in the HIV-1 RNA genome affects initiation and elongation of
reverse transcription. Nucleic
Acids research 28, 4130-4137.
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