MMTV RNA packaging requires an extended long-range interaction for productive Gag binding to packaging signals

Area of Science:

• * Virology
• * Molecular Biology
• * RNA Structure and Function

Background:

• * Retroviral genomic RNA (gRNA) packaging into viral particles depends on specific recognition of packaging signals (Psi) by the Gag precursor.
• * The Psi element possesses a complex secondary structure maintained by long-range interactions (LRIs), but their precise role in packaging remains unclear.
• * The sufficiency of Gag-Psi binding for packaging and the contribution of LRIs require further investigation.

Purpose of the Study:

• * To investigate the role of long-range interactions (LRIs) in the secondary structure of the mouse mammary tumor virus (MMTV) packaging signal (Psi).
• * To determine if Gag binding to Psi alone is sufficient for efficient gRNA packaging.
• * To elucidate the structural requirements for efficient retroviral gRNA packaging.

Main Methods:

• * Site-directed mutagenesis to disrupt proposed LRIs within the MMTV Psi element.
• * Chemical probing (hSHAPE) to assess gRNA secondary structure.
• * Filter-binding assays to quantify Gag-Psi binding affinity.
• * Footprinting experiments to map Gag binding sites on Psi.

Main Results:

• * Identification of an extended LRI within the MMTV Psi element.
• * hSHAPE analysis confirmed that maintaining a wild-type-like Psi structure is critical for efficient gRNA packaging.
• * Most Psi mutants retained significant binding to Pr77Gag, indicating Gag binding alone is insufficient for packaging.
• * Efficient packaging requires Pr77Gag binding to two specific sites within Psi, not just general binding.

Conclusions:

• * The 3D structure of the Psi/Pr77Gag complex, not just binding affinity, is key for regulating viral particle assembly.
• * Specific binding interactions within Psi, alongside structural integrity, are crucial for selective gRNA packaging.
• * This mechanism ensures efficient discrimination against non-specific viral and cellular RNAs.