RNA secondary structure and binding sites for gag gene products in the 5' packaging signal of human immunodeficiency virus type 1
| Autor: | Tristram G. Parslow, Jared L. Clever, Christopher M. Sassetti |
|---|---|
| Rok vydání: | 1995 |
| Předmět: |
Base Sequence
RNase P Sequence analysis Molecular Sequence Data Immunology Gene Products gag RNA Biology Group-specific antigen Microbiology Molecular biology Nucleic acid secondary structure Virology Insect Science Transfer RNA HIV-1 Nucleic Acid Conformation RNA Viral Binding site Nucleic acid structure Research Article DNA Primers Protein Binding |
| Zdroj: | Journal of Virology. 69:2101-2109 |
| ISSN: | 1098-5514 0022-538X |
| DOI: | 10.1128/jvi.69.4.2101-2109.1995 |
| Popis: | The selective encapsidation of retroviral RNA requires sequences in the Gag protein, as well as a cis-acting RNA packaging signal (psi site) near the 5' end of the genomic transcript. Gag protein of human immunodeficiency virus type 1 (HIV-1) has recently been found to bind specifically to the HIV-1 psi element in vitro. Here we report studies aimed at mapping features within the genetically defined psi locus that are required for binding of HIV-1 Gag or of its processed nucleocapsid derivative. The full-length HIV-1 Gag (p55) and nucleocapsid (p15) sequences were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. In a gel shift assay containing excess competitor tRNA, affinity-purified GST-p15 and GST-p55 proteins bound to a 206-nucleotide psi RNA element spanning the major splice donor and gag start codons but did not bind to antisense psi transcripts. Quantitative filter-binding assays revealed that both GST-p55 and GST-p15 bound to this RNA sequence with identical affinities (apparent Kd congruent to 5 x 10(-8) M), indicating that all major determinants of psi binding affinity reside within the nucleocapsid portion of Gag. Chemical and RNase accessibility mapping, coupled with computerized sequence analysis, suggested a model for psi RNA structure comprising four independent stem-loops. Filter-binding studies revealed that RNAs corresponding to three of these hypothetical stem-loops can each function as a independent Gag binding site and that each is bound with approximately fourfold-lower apparent affinity than the full-length psi locus. Interaction of Gag with these regions is likely to play a major role in directing HIV-1 RNA encapsidation in vivo. |
| Databáze: | OpenAIRE |
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