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J Biol Chem, Vol. 275, Issue 17, 13061-13070, April 28, 2000
From the Department of Microbiology, School of Medicine, University
of Washington, Seattle, Washington 98195
During reverse transcription, plus strand DNA
synthesis is initiated at a purine-rich RNA primer generated by the
RNase H activity of reverse transcriptase (RT). Specific initiation of plus strand synthesis from this polypurine tract (PPT) RNA is essential
for the subsequent integration of the linear viral DNA product. Based
on current models, it is predicted that priming from sites upstream of
the PPT may be tolerated by the virus, whereas efficient extension from
RNA primers located downstream from the PPT is predicted to generate
dead-end products. By using hybrid duplex substrates derived from the
Moloney murine leukemia virus long terminal repeat, we investigated the
extent to which RNase H degrades the viral RNA during time course
cleavage assays, and we tested the capacity of the polymerase activity
of RT to use the resulting cleavage products as primers. We find that
the majority of the RNA fragments generated by RNase H are 2-25
nucleotides in length, and only following extensive degradation are
most fragments reduced to 10 nucleotides or smaller. Although extensive
RNA degradation by RNase H likely eliminates many potential RNA
primers, based on thermostability predictions it appears that some RNA
fragments remain stably annealed to the DNA template. RNA primers
generated by RNase H within the long terminal repeat sequence are found to have the capacity to initiate DNA synthesis by RT; however, the
priming efficiency is significantly less than that observed with the
PPT primer. We find that Moloney murine leukemia virus nucleocapsid
protein reduces RNase H degradation and slightly alters the cleavage
specificity of RT; however, nucleocapsid protein does not appear to
enhance PPT primer utilization or suppress extension from non-PPT RNA primers.
RNA Degradation and Primer Selection by Moloney Murine Leukemia
Virus Reverse Transcriptase Contribute to the Accuracy of Plus
Strand Initiation*
*
This work was supported by Grant R37 CA51605 from the
National Institutes of Health.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Microbiology,
Box 357242, University of Washington, Seattle, WA 98195-7242. Tel.:
206-543-8574; Fax: 206-543-8297; E-mail:
champoux@u.washington.edu.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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