Abstract
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.
Highlights
Retroviral replication involves a complex series of coordinated steps that are catalyzed by the viral reverse transcriptase (RT).1 Reverse transcription of the viral RNA genome into double-stranded DNA is a necessary step during the viral life cycle, generating a terminally redundant DNA product that subsequently becomes integrated into the host cell genome
Analysis of RNA Fragment Thermostabilities— sequence recognition has been demonstrated to play a role in facilitating the selection of the correct RNA fragment for plus strand initiation by RT (26 –30), removal of all potentially competing RNA fragments by the RNase H activity of RT, from the long terminal repeats (LTR) region, could provide a mechanism to help ensure accurate initiation
To begin to investigate whether genomic RNA could be effectively removed from the minus strand DNA through RT-catalyzed RNase H cleavage, we determined the predicted Tm for RNA fragments derived from viral sequences using thermodynamic parameters based on the nearest-neighbor model for thermostability prediction of RNA/DNA hybrid duplexes [25]
Summary
Retroviral systems, indicates that priming from regions upstream of the PPT in addition to PPT priming may not be detrimental to the virus [12,13,14,15,16,17,18,19]. Priming from within the LTR is predicted to result in the generation of products with one incorrect end, lacking the requisite integration signal normally present at the terminus of the LTR. These considerations raise the possibility that sequences with the potential to generate extendable primers following RNase H cleavage have been selectively excluded from the LTR region. Our results indicate that some non-PPT RNA fragments generated by RNase H from within the LTR region or from elsewhere in the genome have the capacity to prime synthesis by the polymerase, the efficiency of extension is significantly less than from the PPT primer
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