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Journal of Virology, January 2002, p. 88-95, Vol. 76, No. 1
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.1.88-95.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

RNase H Activity Is Required for High-Frequency Repeat Deletion during Moloney Murine Leukemia Virus Replication

Jennifer L. Brincat, Julie K. Pfeiffer, and Alice Telesnitsky*

Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0620

Received 11 July 2001/ Accepted 26 September 2001

It has been postulated that retroviral recombination, like strong stop template switching, requires the RNase H activity of reverse transcriptase. To address this hypothesis, Moloney murine leukemia virus-based vectors, which were designed to test the recombination-related property of direct repeat deletion, were encapsidated in virions engineered to contain phenotypic mixtures of wild-type and RNase H catalytic site point mutant reverse transcriptase. Integrated provirus titers per milliliter were determined for these phenotypically mixed virions, and vector proviruses were screened to determine what percentage contained repeat deletions. The results revealed a steady decline in direct repeat deletion frequency that correlated with decreases in functional RNase H, with greater than fourfold decreases in repeat deletion frequency observed when 95% of virion reverse transcriptase was RNase H defective. Parallel experiments were performed to address effects of molar excesses of RNase H relative to functional DNA polymerase. These experiments demonstrated that increasing the stoichiometry of RNase H relative to the amount of functional DNA polymerase had minimal effects on direct repeat deletion frequency. DNA synthesis was error prone when directed principally by RNase H mutant reverse transcriptase, suggesting a role for RNase H catalytic integrity in the fidelity of intracellular reverse transcription.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Michigan Medical School, 1150 W. Medical Ctr. Dr., Rm. 5641, Ann Arbor, MI 48109-0620. Phone: (734) 936-6466. Fax: (734) 764-3562. E-mail:ateles{at}umich.edu.

Journal of Virology, January 2002, p. 88-95, Vol. 76, No. 1
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.1.88-95.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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