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Requirements for minus-strand transfer catalyzed by Rous sarcoma virus reverse transcriptase
S Werner1, K Vogel-Bachmayr, B Hollinderbäumer
1Abteilung Physikalische Biochemie, Max-Planck-Institut für Molekulare Physiologie, 44227 Dortmund, Germany.
Journal of Virology
|October 3, 2001
Summary
Rous sarcoma virus (RSV) reverse transcriptases require RNase H activity for minus-strand transfer. Human immunodeficiency virus type 1 nucleocapsid protein enhances this strand transfer process.
Area of Science:
- Molecular Biology
- Virology
- Enzymology
Background:
- Rous sarcoma virus (RSV) reverse transcriptase exists in three forms: alphabeta, alpha, and beta.
- Minus-strand DNA synthesis is a critical step in retroviral replication.
Purpose of the Study:
- To investigate the mechanism of minus-strand transfer in RSV reverse transcriptase.
- To determine the role of RNase H activity and different enzyme subunits in strand transfer.
Main Methods:
- Utilized model primer-template substrates mimicking RSV RNA termini.
- Assessed minus-strand transfer efficiency with various RSV reverse transcriptase forms.
- Investigated the effect of RNase H deficiency and human immunodeficiency virus type 1 nucleocapsid protein.
Main Results:
- RSV reverse transcriptase RNase H activity is essential for efficient minus-strand transfer.
- RNase H-deficient enzymes showed significantly reduced strand transfer (<2%).
- The alpha homodimer performed strand transfer comparably to alphabeta and alphaPol heterodimers, while beta and Pol activities were reduced.
- Human immunodeficiency virus type 1 nucleocapsid protein increased minus-strand transfer activity two- to fivefold.
Conclusions:
- RNase H function is indispensable for RSV minus-strand transfer.
- The alpha subunit plays a key role in mediating strand transfer.
- Nucleocapsid proteins can modulate retroviral reverse transcriptase activity.