Untying the FIV frameshifting pseudoknot structure by MS3D

Area of Science:

• Structural Biology
• Virology
• Biochemistry

Background:

• Ribosomal frameshifting is crucial for retroviral replication.
• Pseudoknots are key RNA structures involved in frameshifting.
• Feline immunodeficiency virus (FIV) utilizes a pseudoknot for frameshifting.

Purpose of the Study:

• To determine the three-dimensional (3D) structure of the FIV ribosomal frameshifting pseudoknot (PK).
• To validate the mass spectrometric three-dimensional (MS3D) approach for RNA structure determination.
• To provide a structural basis for understanding FIV replication mechanisms.

Main Methods:

• Mass spectrometric three-dimensional (MS3D) approach using solvent-accessibility probes and chemical crosslinkers.
• Electrospray ionization (ESI) Fourier transform mass spectrometry (FTMS) for nucleotide modification analysis.
• Computational modeling (MC-SYM, CNS) using protection maps and distance information.

Main Results:

• The MS3D approach successfully modeled the FIV-PK structure, confirming its pseudoknot nature.
• The FIV-PK structure shares similarities with mouse mammary tumor virus pseudoknot (VPK), differing from simian retrovirus type-1 pseudoknot.
• The model suggests a specific metal-binding site involving loop 1 and stem 2, explaining Mg(2+) effects.

Conclusions:

• The MS3D approach is effective for generating RNA 3D models and hypotheses in the absence of high-resolution structures.
• The determined FIV-PK structure provides insights into FIV replication and potential therapeutic targets.
• Structural features of FIV-PK, like the bent stems and unpaired U30, are critical for its function.