Repression of the Internal Ribosome Entry Site-dependent Translation of Hepatitis C Virus by an Engineered PUF Protein

Abstract

Background: Pumilio/fem-3 mRNA binding factor (PUF) proteins can bind RNA in a sequence-specific manner. The deciphered RNA-recognition code of these proteins has enabled researchers to design engineered PUF proteins, capable of binding to any desired target in order to modify its ultimate fate. In this study, a modified Homo sapiens Pumilio 1-homology domain (HsPUM1-HD) was engineered to bind to the internal ribosome entry site (IRES) of hepatitis C virus (HCV) genome to potentially inhibit viral translation. Methods: Based on the RNA-recognition code, required modifications were applied to HsPUM1-HD in order to change its natural recognition sequence to a sequence in the stem-loop III of HCV IRES. RNA protein pull-down assay was performed to assess the sequence specificity of the modified HsPUM1-HD (mHsPUM1-HD). Translational inhibitory effect of mHsPUM1-HD was evaluated in a dual-luciferase reporter assay. Results: The mHsPUM1-HD was found to bind to its cognate RNA in a sequence-specific manner, as a biotinylated target RNA captured mHsPUM1-HD through binding to streptavidin magnetic beads. This protein also reduced HCV IRES-dependent firefly luciferase translation by 40% in HEK293 cells. Conclusions: The present study is the first report of an engineered HsPUM1-HD with potential anti-HCV activity. These findings suggest that PUM-HDs can be engineered to target desired RNAs of infectious agents in order to specifically interrupt protein translation, as an essential step of their life cycle.