FORCED EVOLUTION OF A REGULATORY RNA HELIX IN THE HIV-1 GENOME

The 5' and 3' end of the HIV-1 RNA genome forms a repeat (R) element t hat encodes a double stem-loop structure (the TAR and polyA hairpins). Phylogenetic analysis of the polyA hairpin in different human and sim ian immunodeficiency viruses suggests that the thermodynamic stability of the helix is fine-tuned. We demonstrated previously that mutant HI V-1 genomes with a stabilized or destabilized hairpin are severely rep lication-impaired. In this study, we found that the mutant with a dest abilized polyA hairpin structure is conditionally defective. Whereas r educed replication is measured in infections at the regular temperatur e (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 RN A structure is critical for function. An extensive analysis of reverta nt viruses was performed to further improve the understanding of the c ritical sequence and structural features of the element under scrutiny . The virus mutants with a stabilized or destabilized hairpin were use d as a starting point in multiple, independent selections for revertan t viruses with compensatory mutations. Both mutants reverted to hairpi ns with wild-type stability along various pathways by acquisition of c ompensatory mutations. We identified 19 different revertant HIV-1 form s 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.