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Journal of Virology, May 2003, p. 5275-5285, Vol. 77, No. 9
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.9.5275-5285.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Specific Cleavages by RNase H Facilitate Initiation of Plus-Strand RNA Synthesis by Moloney Murine Leukemia Virus

Sharon J. Schultz, Miaohua Zhang, and James J. Champoux*

Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington 98195-7242

Received 20 December 2002/ Accepted 8 February 2003

Successful generation, extension, and removal of the plus-strand primer is integral to reverse transcription. For Moloney murine leukemia virus, primer removal at the RNA/DNA junction leaves the 3' terminus of the plus-strand primer abutting the downstream plus-strand DNA, but this 3' terminus is not efficiently reutilized for another round of extension. The RNase H cleavage to create the plus-strand primer might similarly result in the 3' terminus of this primer abutting downstream RNA, yet efficient initiation must occur to synthesize the plus-strand DNA. We hypothesized that displacement synthesis, RNase H activity, or both must participate to initiate plus-strand DNA synthesis. Using model hybrid substrates and RNase H-deficient reverse transcriptases, we found that displacement synthesis alone did not efficiently extend the plus-strand primer at a nick with downstream RNA. However, specific cleavage sites for RNase H were identified in the sequence immediately following the 3' end of the plus-strand primer. During generation of the plus-strand primer, cleavage at these sites generated a gap. When representative gaps separated the 3' terminus of the plus-strand primer from downstream RNA, primer extension significantly improved. The contribution of RNase H to the initiation of plus-strand DNA synthesis was confirmed by comparing the effects of downstream RNA versus DNA on plus-strand primer extension by wild-type reverse transcriptase. These data suggest a model in which efficient initiation of plus-strand synthesis requires the generation of a gap immediately following the plus-strand primer 3' terminus.

* Corresponding author. Mailing address: Department of Microbiology, Box 357242, University of Washington, Seattle, WA 98195-7242. Phone: (206) 543-8574. Fax: (206) 543-8297. E-mail: champoux{at}u.washington.edu.

Journal of Virology, May 2003, p. 5275-5285, Vol. 77, No. 9
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.9.5275-5285.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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