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 RNArecognition 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.

Engineered Puf Proteins: New Flexible Toolkits to Target the Replication of RNA Viruses

“…Any obstacle to such an interaction can decrease viral polyprotein translation. This is in agreement with our previous results of a luciferase reporter assay that showed more than 30% decrease in IRES-dependent translation of Firefly luciferase in the presence of mPUM protein targeted for stem-loop IIIb [ 8 ]. This was also in line with the results of our viability assay in Huh7.5 clone 5 cells, where the expression of mPUM protein in clone 5 cells in a medium supplemented with G-418 resulted in more than 70% reduction in cell viability.…”

“…In order to check the effect of mPUM protein on IRES-dependent translation, a dual luciferase reporter assay was performed. For this, the psiCHECK2 plasmid (Promega Corporation) was modified to harbor HCV IRES between its two luciferase genes, as described previously [ 8 ]. Transcription from this modified plasmid (psiCHECK2-IRES) results in a bicistronic mRNA, in which the translation of Renilla luciferase is cap-dependent, while the translation of Firefly luciferase is IRES-dependent.…”

“…In our previous study, sequence-specific binding of a modified PUM-HD protein to HCV IRES was assessed by a RNA-protein pull-down assay as well as a dual-luciferase reporter assay in HEK-293 cells [ 8 ]. In this study, the efficiency of the modified PUM-HD protein to repress virus replication was investigated in human hepatoma cell line (Huh7.5) infected with genotype 2a HCV JFH-1 strain.…”