Substrate requirements for secondary cleavage by HIV-1 reverse transcriptase RNase H

Area of Science:

• Molecular Biology
• Virology
• Enzymology

Background:

• Human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) possesses RNase H activity essential for degrading the viral RNA genome during DNA synthesis.
• Efficient cleavage of template RNA is critical for the transfer of minus-strand DNA, a key step in HIV-1 replication.
• The removal of the terminal RNA segment presents a challenge due to the formation of a blunt-ended RNA-DNA hybrid, which is resistant to in vitro cleavage.

Purpose of the Study:

• To investigate the influence of substrate structure on the secondary cleavage efficiency of HIV-1 RT.
• To determine factors that facilitate the cleavage of blunt-ended RNA-DNA hybrids by HIV-1 RT.

Main Methods:

• In vitro assays were performed using various RNA-DNA hybrid substrates with different extensions and structures at the 5' end.
• The cleavage efficiency of HIV-1 RT on these substrates was analyzed.
• The effect of the viral RNA cap structure and the NC protein on cleavage was evaluated.

Main Results:

• A short single-stranded DNA extension significantly stimulated secondary cleavage by HIV-1 RT.
• Annealing an RNA segment to the DNA extension further enhanced cleavage efficiency.
• Recessing the DNA from a blunt end by even one nucleotide prevented secondary cleavage, indicating sensitivity to substrate geometry. The viral RNA cap did not improve cleavage, but NC protein greatly facilitated secondary cleavage on blunt-ended substrates.

Conclusions:

• The structure of the substrate extending beyond the RNA 5' end is a critical determinant of HIV-1 RT secondary cleavage efficiency.
• Short DNA extensions and RNA annealing enhance cleavage, while precise blunt-end structures impede it.
• NC protein plays a vital role in overcoming substrate structural barriers, facilitating essential RNA cleavage for HIV-1 replication.