Nucleotide sequences and modifications that determine RIG-I/RNA binding and signaling activities

Area of Science:

• Immunology
• Virology
• Molecular Biology

Background:

• Cytoplasmic viral RNAs with 5' triphosphates (5'ppp) are recognized by RIG-I, triggering innate immune responses and interferon production.
• The hepatitis C virus (HCV) 3' untranslated region (UTR) is implicated in immune modulation, but its precise role and comparative activity remain under investigation.

Purpose of the Study:

• To compare the immune-stimulating activity of HCV 3' UTR RNA with other viral UTRs.
• To identify the specific regions and characteristics of HCV RNA responsible for activating RIG-I.
• To explore the potential of modified RNAs as modulators of RIG-I-mediated innate immunity.

Main Methods:

• In vitro transcription of various viral UTR RNAs, including HCV and flavivirus sequences.
• Assays to measure RIG-I activation and downstream signaling, including interferon-beta (IFN-beta) expression.
• Analysis of RNA structure-activity relationships, including the role of nucleotide sequence and modifications.

Main Results:

• HCV 3' UTR RNA exhibits significantly higher immune-stimulating activity compared to several flavivirus UTR RNAs.
• The poly(U/UC) region within the HCV 3' UTR is critical for robust RIG-I activation; its antisense poly(AG/A) sequence is also a potent activator.
• Modified uridine or pseudouridine ribonucleotides in the RNA abolish signaling activity, converting activators into competitive inhibitors of RIG-I.

Conclusions:

• The HCV 3' UTR, particularly its poly(U/UC) region and its antisense counterpart, is a potent activator of RIG-I-mediated innate immune signaling.
• RIG-I appears to recognize independent RNA domains, not solely dependent on proximity to the 5' triphosphate.
• RNA modifications can convert immune-activating sequences into inhibitors, presenting a novel strategy for modulating innate immune responses.