Structural requirement for the two-step dimerization of human immunodeficiency virus type 1 genome

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• RNA dimerization of the human immunodeficiency virus type 1 (HIV-1) genome is vital for viral replication.
• The dimerization initiation site (DIS) forms stem-loop structures, facilitating dimer formation.
• HIV-1 RNA fragments exhibit two dimer types: loose and tight, with tight dimers formed upon addition of nucleocapsid protein NCp7.

Purpose of the Study:

• To elucidate the biochemical mechanism of the two-step RNA dimerization process mediated by the HIV-1 DIS.
• To determine the specific RNA sequences and structural elements essential for the observed two-step dimerization.

Main Methods:

• Chemical synthesis of a 39-mer RNA encompassing the entire DIS sequence.
• Chemical synthesis of a 23-mer RNA covering the DIS self-complementary loop and flanking stem.
• Electrophoretic dimerization assays to analyze dimer formation kinetics and structures.
• Mutational analysis involving deletion of the bulge region from the 39-mer RNA.

Main Results:

• The 39-mer RNA successfully replicated the two-step dimerization process (loose to tight dimer conversion).
• The 23-mer RNA, lacking the stem bulge, formed tight dimers immediately.
• Deletion of the bulge from the 39-mer RNA abolished NCp7-assisted tight dimer formation.
• The entire DIS sequence was found to be necessary and sufficient for the two-step dimerization.

Conclusions:

• The complete DIS sequence is essential for the regulated, two-step RNA dimerization observed in HIV-1.
• The bulge region within the DIS stem plays a critical role in regulating stem stability.
• The bulge region acts as a key determinant guiding the DIS towards the specific two-step dimerization pathway.