Modulation of Viral Programmed Ribosomal Frameshifting and Stop Codon Readthrough by the Host Restriction Factor...

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Interferon-stimulated genes play a role in antiviral defense.
• Programmed ribosomal frameshifting is a crucial mechanism for viral polyprotein synthesis.
• Shiftless (SHFL) is an interferon-stimulated gene product implicated in restricting human immunodeficiency virus (HIV) replication.

Purpose of the Study:

• To investigate the mechanism by which SHFL restricts viral replication.
• To determine if SHFL affects programmed ribosomal frameshifting at various viral signals.
• To elucidate the role of SHFL's RNA-binding activity in its function.

Main Methods:

• Bacterial expression and purification of SHFL protein.
• In vitro assays to measure programmed ribosomal frameshifting efficiency.
• Size-exclusion chromatography to assess protein-ribosome interactions.
• Electrophoretic mobility shift assays (EMSAs) and mutational analysis to study RNA binding.

Main Results:

• Purified SHFL inhibits in vitro programmed ribosomal frameshifting at diverse sites, including coronavirus (IBV, SARS-CoV, SARS-CoV-2) and cardiovirus (EMCV, TMEV) signals.
• SHFL reduces stop-codon readthrough efficiency at the murine leukemia virus (MLV) gag/pol signal.
• SHFL binds to mammalian ribosomes in vitro.
• SHFL exhibits strong RNA binding activity essential for frameshifting inhibition, though without specific recognition of stimulatory RNA structures.

Conclusions:

• SHFL broadly inhibits viral programmed ribosomal frameshifting through direct RNA binding.
• SHFL's interaction with ribosomes and RNA is key to its antiviral activity.
• SHFL represents a potential target for antiviral therapeutic strategies.