Exchange of the basic domain of human immunodeficiency virus type 1 Rev for a polyarginine stretch expands the RNA binding specificity, and a minimalarginine cluster is required for optimal RRE RNA binding affinity, nuclearaccumulation, and trans-activation

The Rev regulatory protein of human immunodeficiency virus (HIV) facilitate s the nuclear export of unspliced and partially spliced HIV RNAs. Using a R ev:MS2 phage coat protein fusion that could be targeted to bind and activat e the Rev-responsive element (RRE) RNA or heterologous MS2 phage operator R NA, we analyzed the role(s) of the arginine-rich RNA binding domain in RNA binding and transactivation. The arginine-rich domain could be functionally replaced by a stretch of nine arginines. However, polyarginine substitutio ns expanded the RNA binding specificity of the resultant mutant Rev protein . Polyarginine insertions in place of residues 24 to 60 that excised the RN A binding and oligomerization domains of Rev preserved the activation for M S2 RNA, but not for the RRE. A nine-arginine insertion outside of the natur al context of the Rev nuclear localization signal domain was incompatible w ith activation of either RNA target. Insertions of fewer than eight arginin es impaired RRE activation. Interrupted lysine clusters and disruption of t he arginine stretch with lysine or neutral residues resulted in a similar p henotype. Some of these mutants,vith a null phenotype for RRE activated the heterologous MS2 RNA target. Under steady-state conditions, mutants that p reserved the Rev response for RRE RNA localized to the nuclei; those with p oor or no Rev response accumulated mostly in the cytoplasm. Many of the cyt oplasmically resident derivatives became nuclear when leptomycin B (LMB) tr eatment inhibited nuclear export of nuclear export signal-containing protei ns. Mutants that had a null activation potential for either RNA target were particularly resistant to LMB treatment. Abbreviated nuclear residence tim es and differences in RRE binding affinity may have compromised their activ ation potential for RRE. High-affinity binding to MS2 RNA through the intac t coat protein was sufficient to overcome the short nuclear residence times and to facilitate MS2 activation by some derivatives.