Three substitutions are sufficient to alter the specificity of RRE IIB toward wild-type Rev or Rev-R35G:N40V -

Abstract

The binding of human immune-deficiency virus type 1 (HIV-1) Rev arginine-rich motif (ARM) to stem-loop IIB of Rev-Response Element (RRE) is necessary for viral replication. Despite the essential role of Asn40 in recognition, Rev-R35G:N40V binds RRE IIB well when fused to lambda N protein. Neutral theories of molecular evolution claim that genotypes are sufficiently mutable that new phenotypes occur when genotypes diffusing along neutral mutational paths of one phenotype intersect those of distinct phenotypes. How well neutral theories describe protein-RNA interactions is unclear. The questions raised by Rev-R35G:N40V are: 1) How mutable is the Rev-RRE IIB interaction? and 2) How neutral are evolutionary paths between these distinct specificities? To examine the mutagenic potential of RRE IIB, ten RRE IIB libraries, each completely randomized in a separate over-lapping sub-region, were screened with RNA-binding domain of wild-type Rev and Rev-R35G:N40V using a reporter system based on bacteriophage lambda N-nut antitermination. Flanking regions of RRE IIB are mutable, and the internal loop of RRE IIB allows compensatory mutations that widen the groove. That Rev-R35G:N40V accepts a different panel of RRE variants is consistent with the two ARMs binding RRE IIB using distinct recognition strategies. Many RRE variants obtained showed relaxed specificity with both Rev and Rev-R35G:N40V. From RRE IIB, one substitution was sufficient to confer specificity to wild-type Rev, and three substitutions were sufficient to confer specificity to Rev-R35G:N40V. Retroviruses have high mutation rates and undergo rapid genetic drift; neutral theories are plausible means of generating the diversity of extant arginine-rich motif-RNA recognition strategies.