RNA degradation and primer selection by Moloney murine leukemia virus reverse transcriptase contribute to the accuracy of plus strand initiation.
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.
Kelleher
C D
CD
Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington 98195, USA.
Champoux
J J
JJ
D004926:Escherichia coli / Q000378:metabolism; D008979:Moloney murine leukemia virus / Q000201:enzymology* / Q000235:genetics*; D019590:Nucleocapsid Proteins / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D016914:Ribonuclease H / Q000378:metabolism*; D013696:Temperature; D020079:Terminal Repeat Sequences; D014158:Transcription, Genetic
D019590:Nucleocapsid Proteins; C034477:RNA primers; D012367:RNA, Viral; D012313:RNA; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H