Mutational characterization of HIV-1 Rev R35G-N40V and selection of altered-apecificity mutants -

Abstract

The Rev protein of the human immunodeficiency virus-type 1 (HIV-1) binds stem-loop region IIB of its target RNA, the Rev Response Element (RRE IIB), via its arginine-rich motif (ARM). This ARM-RNA interaction is an essential step in the virus’s lifecycle, for it mediates the nuclear export of viral transcripts. Interestingly, the recognition of RRE IIB is maintained upon mutation of Rev Asn40 to valine simultaneously with the mutation of Arg35 to glycine. The absence of the important arginine at position 35 and the critical asparagine at position 40 means that the Rev ARM double mutant, R35G-N40V, is employing a distinct recognition strategy to recognize RRE IIB. Biophysical data of both interactions and mutational analysis of RRE IIB align with this finding, however no structural model of the R35G-N40V-RRE interaction is available. Using a plasmid-based, bacteriophage lambda, N-nut antitermination reporter system, 15 N-fusion libraries of individual Rev R35G-N40V residues were individually screened for active mutants in bacteria. This scanning mutagenesis identified 8 R35G-N40V positions that behave differently than their counterpart in wild-type Rev. Further screening of both wild-type Rev and Rev R35G-N40V libraries against a panel of mutant RREs identified sequences with compensatory mutations that rescue recognition of incompatible RREs, providing insight to the novel recognition strategy adopted by Rev R35G-N40V. Remarkably, screens also identified several mutants of both ARMs that do not bind RRE IIB yet have affinities for specific RRE variants. These results characterize the distinct recognition strategy by which Rev R35G-N40V binds RRE IIB, and these results will help interpret an anticipated structural model. The recovery of mutants with enhanced specificities will inform mechanisms by which RNA-protein recognition can evolve, and these mutants demonstrate a speciation network between at least two distinct recognition strategies.