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Nucleic Acids Research Advance Access originally published online on February 18, 2007
Nucleic Acids Research 2007 35(5):1649-1659; doi:10.1093/nar/gkm046
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Nucleic Acids Research, 2007, Vol. 35, No. 5 1649-1659
© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

RNA

The availability of the primer activation signal (PAS) affects the efficiency of HIV-1 reverse transcription initiation

Marcel Ooms, Daniel Cupac, Truus E. M. Abbink, Hendrik Huthoff and Ben Berkhout*

Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, The Netherlands

*To whom correspondence should be addressed. Tel: +31 205 664 822; Fax: +31 206 916 531; Email: b.berkhout{at}amc.uva.nl

Received October 10, 2006. Revised January 2, 2007. Accepted January 11, 2007.

Initiation of reverse transcription of a retroviral RNA genome is strictly regulated. The tRNA primer binds to the primer binding site (PBS), and subsequent priming is triggered by the primer activation signal (PAS) that also pairs with the tRNA. We observed that in vitro reverse transcription initiation of the HIV-1 leader RNA varies in efficiency among 3'-end truncated transcripts, despite the presence of both PBS and PAS motifs. As the HIV-1 leader RNA can adopt two different foldings, we investigated if the conformational state of the transcripts did influence the efficiency of reverse transcription initiation. However, mutant transcripts that exclusively fold one or the other structure were similarly active, thereby excluding the possibility of regulation of reverse transcription initiation by the structure riboswitch. We next set out to determine the availability of the PAS element. This sequence motif enhances the efficiency of reverse transcription initiation, but its activity is regulated because the PAS motif is initially base paired within the wild-type template. We measured that the initiation efficiency on different templates correlates directly with accessibility of the PAS motif. Furthermore, changes in PAS are critical to facilitate a primer-switch to a new tRNA species, demonstrating the importance of this enhancer element.


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M. M. Vrolijk, M. Ooms, A. Harwig, A. T. Das, and B. Berkhout
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