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Selection of peptides interfering with a ribosomal frameshift in the human immunodeficiency virus type 1

By Dominic Dulude, Gabriel Théberge-Julien, Léa Brakier-Gingras and Nikolaus Heveker

Abstract

The human immunodeficiency virus of type 1 (HIV-1) uses a programmed -1 ribosomal frameshift to produce the precursor of its enzymes, and changes in frameshift efficiency reduce replicative fitness of the virus. We used a fluorescent two-reporter system to screen for peptides that reduce HIV-1 frameshift in bacteria, knowing that the frameshift can be reproduced in Escherichia coli. Expression of one reporter, the green fluorescent protein (GFP), requires the HIV-1 frameshift, whereas the second reporter, the red fluorescent protein (RFP), is used to assess normal translation. A peptide library biased for RNA binding was inserted into the sequence of the protein thioredoxin and expressed in reporter-containing bacteria, which were then screened by fluorescence-activated cell sorting (FACS). We identified peptide sequences that reduce frameshift efficiency by over 50% without altering normal translation. The identified sequences are also active against different frameshift stimulatory signals, suggesting that they bind a target important for frameshifting in general, probably the ribosome. Successful transfer of active sequences to a different scaffold in a eukaryotic test system demonstrates that the anti-frameshift activity of the peptides is neither due to scaffold-dependent conformation nor effects of the scaffold protein itself on frameshifting. The method we describe identifies peptides that will provide useful tools to further study the mechanism of frameshift and may permit the development of lead compounds of therapeutic interest

Topics: Method
Publisher: Cold Spring Harbor Laboratory Press
OAI identifier: oai:pubmedcentral.nih.gov:2327360
Provided by: PubMed Central
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