HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 280, Issue 15, 14443-14452, April 15, 2005

This Article Full Text Full Text (PDF) All Versions of this Article: 280/15/14443    most recent M412190200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chen, Y. Articles by Bambara, R. A. Search for Related Content PubMed PubMed Citation Articles by Chen, Y. Articles by Bambara, R. A. Social Bookmarking                      What's this?

Acceptor RNA Cleavage Profile Supports an Invasion Mechanism for HIV-1 Minus Strand Transfer^*

Yan Chen¶, Mini Balakrishnan||, Bernard P. Roques**, and Robert A. Bambara

From the Department of Biochemistry and Biophysics and the Cancer Center, University of Rochester Medical Center, Rochester, New York 14642 and the ^**Departement de Pharmacochimie Moleculaire et Structurale, INSERM U266, CNRS UMR 8600, Faculte de Pharmacie 4, Avenue de l`Observatoire 75270 Paris Cedex 06, France

We previously proposed that HIV-1 minus strand transfer occurs by an acceptor invasion-initiated multi-step mechanism. During synthesis of minus strong stop DNA, reverse transcriptase (RT) transiently pauses at the base of TAR before continuing synthesis. Pausing promotes RT-RNase H cleavage of the donor RNA, exposing regions of the cDNA. The acceptor RNA then invades at these locations to interact with the minus strong stop DNA. Whereas primer extension continues on the donor RNA, the cDNA-acceptor hybrid expands by branch migration until transfer of the primer terminus is completed. We present results here showing that the interaction of the acceptor RNA and the cDNA can be determined by examining the time-dependent cleavage of the acceptor RNA by RNase H. Our approach utilizes a combination of RT-RNase H and Escherichia coli RNase H to allow assessment of acceptor-cDNA interactions at high sensitivity. Results show an initial interaction of the acceptor RNA with cDNA at the base of TAR. We observe a time-dependent shift in RNase H susceptibility along the length of the acceptor toward the 5' end, suggesting hybrid propagation from the initial invasion point. Control experiments validate that the RNase H cleavage profile represents the formation and expansion of the acceptor-DNA interaction and that the process is promoted by the nucleocapsid. Observations with this new approach lend additional support to the proposed multistep transfer mechanism.

Received for publication, October 27, 2004 , and in revised form, January 7, 2005.

^* This work was supported by National Institutes of Health Grant GM 049573 (to R. A. B). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors have contributed equally to this work.

^¶ Present address: Dept. of Molecular Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030.

^|| To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, Box 712, University of Rochester Medical Center, 601 Elmwood Ave., Box 712, Rochester, NY 14642. Tel.: 585-275-2764; Fax: 585-271-2683; E-mail: mini_balakrishnan{at}urmc.rochester.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				M. Song, V. P. Basu, M. N. Hanson, B. P. Roques, and R. A. Bambara Proximity and Branch Migration Mechanisms in HIV-1 Minus Strand Strong Stop DNA Transfer J. Biol. Chem., February 8, 2008; 283(6): 3141 - 3150. [Abstract] [Full Text] [PDF]

				L. Gao, M. Balakrishnan, B. P. Roques, and R. A. Bambara Insights into the Multiple Roles of Pausing in HIV-1 Reverse Transcriptase-promoted Strand Transfers J. Biol. Chem., March 2, 2007; 282(9): 6222 - 6231. [Abstract] [Full Text] [PDF]

				D. J. Operario, M. Balakrishnan, R. A. Bambara, and B. Kim Reduced dNTP Interaction of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Promotes Strand Transfer J. Biol. Chem., October 27, 2006; 281(43): 32113 - 32121. [Abstract] [Full Text] [PDF]

				M. Song, M. Balakrishnan, Y. Chen, B. P. Roques, and R. A. Bambara Stimulation of HIV-1 Minus Strand Strong Stop DNA Transfer by Genomic Sequences 3' of the Primer Binding Site J. Biol. Chem., August 25, 2006; 281(34): 24227 - 24235. [Abstract] [Full Text] [PDF]

				R. Galetto, V. Giacomoni, M. Veron, and M. Negroni Dissection of a Circumscribed Recombination Hot Spot in HIV-1 after a Single Infectious Cycle J. Biol. Chem., February 3, 2006; 281(5): 2711 - 2720. [Abstract] [Full Text] [PDF]

				V. Purohit, M. Balakrishnan, B. Kim, and R. A. Bambara Evidence That HIV-1 Reverse Transcriptase Employs the DNA 3' End-directed Primary/Secondary RNase H Cleavage Mechanism during Synthesis and Strand Transfer J. Biol. Chem., December 9, 2005; 280(49): 40534 - 40543. [Abstract] [Full Text] [PDF]