Analysis of Binding Elements in the Human Immunodeficiency Virus Type 1 Genomic RNA and Nucleocapsid Protein

Abstract

We previously used RNA gel mobility shift assays to demonstrate specific binding of the HIV-1 gag precursor polyprotein and nucleocapsid (NC) protein to HIV-1 RNA and to map the binding elements in each species by mutagenesis. Here we report finer mapping of binding elements in the HIV-1 genomic RNA and NC protein, performed by analyzing the binding behavior of fragments of each species in the gel shift assay. With regard to the RNA, the strongest binding activity resided in a 120-nucleotide segment flanking the gag start codon, containing three potential stem-loop structures. Binding analysis of various combinations of these three potential stem-loop structures and their flanking sequences revealed that no one element could bind to the gag polyprotein or NC protein as well as the entire 120-nucleotide segment. Mutational analysis of the NC protein showed that two nonoverlapping regions exhibited specific binding for HIV-1 RNA. Each region includes a Cys-His box, though each box could not bind to HIV-1 RNA on its own. In contrast, a construct lacking both boxes exhibited primarily nonspecific RNA-binding activity.