Reverse-transcriptase-associated RNaseH activity mediates template switching during reverse transcription in vitro.

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Retroviral genome reverse transcription involves reconstituting host cell PolII promoters.
• Template switching, utilizing direct repeats at RNA ends, is crucial for promoter assembly.
• Viral reverse transcriptase, with intrinsic RNaseH activity, likely plays a role in these steps.

Purpose of the Study:

• To investigate the specific role of reverse transcriptase's RNaseH activity in the initial template-switching event.
• To elucidate the mechanism by which RNaseH facilitates promoter reconstitution during reverse transcription.

Main Methods:

• Development of an in vitro system using primer extensions of synthetic RNAs.
• Utilizing wild-type and mutant reverse transcriptase (lacking RNaseH activity) in reactions with specific RNA substrates.
• Analysis of cDNA synthesis products to identify chimeric molecules and assess template switching efficiency.

Main Results:

• Wild-type reverse transcriptase facilitated template switching, producing chimeric cDNA from two RNA species.
• Template switching was undetectable when using a mutant reverse transcriptase lacking RNaseH activity.
• RNaseH activity is essential for cleaving the 5' RNA from the cDNA:RNA hybrid, enabling translocation.

Conclusions:

• The RNaseH activity of reverse transcriptase is indispensable for the initial template-switching event in retroviral reverse transcription.
• RNaseH enables promoter reconstitution by removing RNA templates, allowing cDNA to switch to a new RNA template.
• This finding clarifies a critical step in retroviral replication and genome processing.