Processing of the primer for plus strand DNA synthesis by human immunodeficiency virus 1 reverse transcriptase

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Reverse transcriptase (RT) from human immunodeficiency virus 1 (HIV-1) is crucial for viral replication.
• RT possesses multiple enzymatic activities, including RNA-dependent DNA polymerase and RNase H activity.
• Understanding primer processing is essential for developing antiviral therapies.

Purpose of the Study:

• To analyze the specific processing of RNA primers for (+) strand DNA synthesis by HIV-1 reverse transcriptase.
• To investigate the mechanism of primer selection, extension, and removal.
• To determine if these steps require coupling or can occur independently.

Main Methods:

• Construction of a 99-base pair RNA-DNA hybrid containing the viral polypurine tract and flanking sequences.
• Analysis of RNA cleavage, primer extension, and primer removal by HIV-1 reverse transcriptase.
• Utilizing nucleoside triphosphates and examining the role of p51 (a truncated RT form).

Main Results:

• HIV-1 reverse transcriptase's RNase H activity cleaves the RNA strand into multiple fragments.
• Only two specific primers are extended, with the major primer being the polypurine tract (minus the last adenosine).
• Reverse transcriptase releases the elongated primer intact via cleavage at the RNA-DNA junction, independent of DNA synthesis.

Conclusions:

• Primer processing, extension, and removal by HIV-1 reverse transcriptase are specific events.
• Specificity is maintained even when the three steps occur individually.
• The polypurine primer is selected and extended by both full-length RT and the p51 form, highlighting its importance in viral DNA synthesis.