Specific interactions between HIV-1 nucleocapsid protein and the TAR element

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Retroviral reverse transcription involves minus-strand strong-stop DNA (ss-cDNA) transfer to the genomic RNA's 3' end, a process dependent on repeat (R) sequences.
• Human immunodeficiency virus type 1 (HIV-1) R sequences can mediate DNA strand transfer even in ectopic positions, with R sequences and nucleocapsid protein (NC) being key determinants.

Purpose of the Study:

• To investigate the specific interactions between HIV-1 NC and RNA.
• To understand the influence of NC on R sequence folding.
• To elucidate the roles of NC zinc fingers and basic domains in NC/RNA interactions during ss-cDNA transfer.

Main Methods:

• Studied secondary structures of R sequences in natural (5'/3' ends) and ectopic internal positions.
• Utilized NC mutants to probe the roles of zinc fingers and basic domains.
• Employed chemical/enzymatic probings and gel retardation assays on viral RNA/NC complexes under conditions supporting ss-cDNA transfer.

Main Results:

• NC specifically binds the TAR apical loop in all four genetic contexts without significantly altering TAR hairpin or R region folding.
• NC binding to the TAR apical loop requires its zinc fingers.
• Efficient annealing of cTAR DNA to the 3' R depends on sequence complementarity between TAR and cTAR terminal loops.

Conclusions:

• The TAR apical loop serves as the initiation site for annealing during minus-strand transfer.
• HIV-1 NC chaperones the annealing reaction at the TAR apical loop, a process dependent on NC's zinc fingers.
• These findings clarify the mechanism of NC-mediated DNA strand transfer in HIV-1 reverse transcription.