Nucleic Acids Research, Vol 25, Issue 5 940-947, Copyright © 1997 by Oxford University Press
B Berkhout, B Klaver and AT Das
The 5'and 3'end of the HIV-1 RNA genome forms a repeat (R) element that
encodes a double stem-loop structure (the TAR and polyA hairpins).
Phylogenetic analysis of the polyA hairpin in different human and simian
immunodeficiency viruses suggests that the thermodynamic stability of the
helix is fine-tuned. We demonstrated previously that mutant HIV-1 genomes
with a stabilized or destabilized hairpin are severely
replication-impaired. In this study, we found that the mutant with a
destabilized polyA hairpin structure is conditionally defective. Whereas
reduced replication is measured in infections at the regular temperature
(37 degrees C), this mutant is more fit than the wild-type virus at reduced
temperature (33 degrees C). This observation of a temperature-dependent
replication defect underscores that the stability of this RNA structure is
critical for function. An extensive analysis of revertant viruses was
performed to further improve the understanding of the critical sequence and
structural features of the element under scrutiny. The virus mutants with a
stabilized or destabilized hairpin were used as a starting point in
multiple, independent selections for revertant viruses with compensatory
mutations. Both mutants reverted to hairpins with wild-type stability along
various pathways by acquisition of compensatory mutations. We identified 19
different revertant HIV-1 forms with improved replication characteristics,
providing a first look at some of the peaks in the total sequence landscape
that are compatible with virus replication. These experiments also
highlight some general principles of RNA structure building.
ARTICLES
Forced evolution of a regulatory RNA helix in the HIV-1 genome
Academic Medical Center, University of Amsterdam, Department of Human Retrovirology, PO Box 22700, 1100 DE Amsterdam, The Netherlands. b.berkhout@amc.uva.nl
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