Mutational analysis of the HIV-1 Rev protein and its target sequence, the Rev responsive element

The human immunodeficiency virus type 1 (HIV-1) Rev protein is a positive posttranscriptional regulator of viral structural gene expression and essential for virus replication. Rev mediates its effects through interaction with an RNA target sequence, the Rev responsive element (RRE), present within the env mRNA. Previous studies have shown that the basic stretch of amino acids are required for Rev's ability to bind RNA, whereas residues present near the carboxy terminus are essential for full biological activity. Deletion mutagenesis was used to define the minimal domain required for RNA binding and function. We found that amino acids 8 through 67 confer full binding activity, whereas full biological activity requires the presence of residues 8 through 83. The minimal RNA binding sequence of HIV-1 Rev also interacts and functions with the HIV-2 and SIV RRE elements, indicating that the same domain is responsible for the biological activity with different, but related viruses. Mutational analysis of the RRE was also carried out in an effort to further define elements crucial for its function. Our findings indicate that interaction with Rev involves a stretch of three G nucleotides present at the base of a stem loop structure previously shown to be critical for Rev binding. These results suggest that the high degree of secondary structure of the RRE RNA may serve as a guide to bring Rev in contact with a primary nucleotide sequence required for stable protein-RNA association.