Analysis of Protein and RNA Interactions of the HIV-1 rev Protein
The HIV-1 Rev protein enters the nucleus via it’s nuclear localization sequence/RNA binding domain and interacts with a ≈234 nt region of viral RNA, termed the Rev Response Element (RRE), which is located in the env region of unspliced and singly-spliced HIV-1 mRNA. The Rev nuclear export sequence (NES) then mediates translocation of the viral transcript to the cytoplasm, allowing for translation of viral structural genes and active virion formation. We report identification of two yeast nucleopore proteins, NUP49 and NUP100, that have been found to interact with Rev in vivo using the yeast two-hybrid system. NUP49 and NUP100 contain GLFG repeats in their amino termini, but their carboxyl termini diverge. We have overexpressed and purified six-His tagged versions of these nucleoporins and have demonstrated by in vitro assays that their interaction with Rev in the two-hybrid system must be indirect. Additional two-hybrid experiments have shown that Rev’s NES and the GLFG domains of NUP49 and NUP100 are both required for their interaction. Based on these experiments we propose that Rev-NUP interactions are important in Rev-mediated export of HIV-1 mRNA’s, but these interactions are most likely mediated by another factor. In vivo expression of the primary binding site of the RRE, known as the Rev Binding Element (RBE), has previously been shown to inhibit HIV-1 replication in vivo. The specific interaction of such molecules with Rev is necessary for their usefulness as therapeutics. We have found that RBE aptamers evolved by SELEX interact specifically with a Rev RNA binding domain peptide. In addition the linking of a ribozyme to an aptamer did not affect its specific interaction with Rev protein. RBE aptamers and RBE aptamer-ribozymes have the potential to function as anti-viral therapeutics. In addition an RBE aptamer-ribozyme may enhance the ribozymes function by facilitating colocalization with target HIV-1 mRNA. We also report the use of novel templates for in vitro transcription. An RBE aptamer transcribed from these templates was efficiently terminated by propane diol units, and gave rise to a functional molecule in vitro. These templates have the potential to be used in vivo for screening optimal anti-HIV RNA therapeutics.
Microbiology, Molecular Genetics, HIV-1; Genes, env; Gene Products, rev; RNA-Binding Proteins; Acquired Immundodeficiency Syndrome
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