Polypyrimidine-Tract-Binding Protein Isoforms Differentially Regulate the Hepatitis C Virus Internal Ribosome Entry...

Area of Science:

• Virology
• Molecular Biology
• Biochemistry

Background:

• Hepatitis C virus (HCV) mRNA translation initiation relies on an internal ribosome entry site (IRES).
• The HCV IRES spans the 5' untranslated region (5'UTR) and part of the core coding sequence.
• Polypyrimidine-tract-binding protein (PTB) is an RNA-binding protein with four RNA recognition motifs (RRMs).

Purpose of the Study:

• To investigate the role of PTB isoforms (PTB1, PTB2, PTB4) in stimulating HCV IRES activity.
• To determine the cell-type specificity of PTB isoform function in HCV translation.
• To elucidate the contribution of PTB's RNA recognition motifs (RRMs) to HCV IRES stimulation.

Main Methods:

• Expression of PTB isoforms (PTB1, PTB2, PTB4) and their RRM mutants in HuH-7 and HEK293T cells.
• Assays measuring HCV IRES activity in the presence of different PTB isoforms and mutants.
• Site-directed mutagenesis of PTB RRMs to assess RNA-binding disruption.

Main Results:

• PTB1 and PTB4, but not PTB2, significantly stimulated HCV IRES activity in both cell types.
• PTB1 showed stronger stimulation than PTB4 in HuH-7 cells, while both isoforms had similar effects in HEK293T cells.
• Mutations disrupting PTB RRM RNA-binding capacity abrogated or reduced HCV IRES stimulation, with cell-type specific differences observed for PTB1 and PTB4.

Conclusions:

• PTB1 and PTB4 function as IRES trans-acting factors for the HCV IRES.
• The stimulatory effect of PTB1 and PTB4 on HCV IRES activity is cell-type specific.
• Specific PTB RRM domains are critical for mediating HCV IRES stimulation in a manner dependent on the cellular context.