Nucleic Acids Research, 2006, Vol. 34, No. 2 593–605 doi:10.1093/nar/gkj458 Nucleic acid binding and chaperone properties of HIV-1 Gag and nucleocapsid proteins Margareta Cruceanu 1 , Maria A. Urbaneja2, Catherine V. Hixson 2 , Donald G. Johnson 2 , Siddhartha A. Datta3, Matthew J. Fivash 4 , Andrew G. Stephen 5 , Robert J. Fisher5, Robert J. Gorelick2, Jose R. Casas-Finet6, Alan Rein3, Ioulia Rouzina7 and Mark C. Williams1,8,* 1Department of Physics, Northeastern University, 111 Dana Research Center, 110 Forsyth Street, Boston, MA 02115, USA, 2AIDS Vaccine Program, SAIC-Frederick, Inc., NCI at Frederick, Frederick, MD 21702, USA, 3HIV Drug Resistance Program, NCI-Frederick, Frederick, MD 21702-1201, USA, 4Data Management Services, Inc., NCI-Frederick, Frederick, MD 2170, USA, 5Protein Chemistry Laboratory, SAIC Frederick, Inc., NCI at Frederick, Frederick, MD 2170, USA, 6MedImmune, Inc., 35 W. Watkins Mill Road, Gaithersburg, MD 20878, USA, 7Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 6-155 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA and 8Center for Interdisciplinary Research on Complex Systems, Northeastern University, 111 Dana Research Center, 110 Forsyth Street, Boston, MA 02115, USA Received December 5, 2005; Revised and Accepted January 5, 2006 ABSTRACT The Gag polyprotein of HIV-1 is essential for retro- viral replication and packaging. The nucleocapsid (NC) protein is the primary region for the interac- tion of Gag with nucleic acids. In this study, we examine the interactions of Gag and its NC cleavage products (NCp15, NCp9 and NCp7) with nucleic acids using solution and single molecule experi- ments. The NC cleavage products bound DNA with comparable affinity and strongly destabilized the DNA duplex. In contrast, the binding constant of Gag to DNA was found to be 10-fold higher than that of the NC proteins, and its destabilizing effect on dsDNA was negligible. These findings are con- sistent with the primary function of Gag as a nucleic acid binding and packaging protein and the primary function of the NC proteins as nucleic acid chaper- ones. Also, our results suggest that NCp7’s capability for fast sequence-nonspecific nucleic acid duplex destabilization, as well as its ability to facilitate nuc- leic acid strand annealing by inducing electrostatic attraction between strands, likely optimize the fully processed NC protein to facilitate complex nucleic acid secondary structure rearrangements. In con- trast, Gag’s stronger DNA binding and aggregation capabilities likely make it an effective chaperone for processes that do not require significant duplex destabilization. INTRODUCTION The human immunodeﬁciency virus type 1 (HIV-1) contains two copies of single-stranded RNA (ssRNA), which are tran- scribed into double-stranded DNA (dsDNA) by a complex reverse transcription process. The linear dsDNA that results from this process is integrated into the DNA of the host cell, and from there it will be transcribed and translated using the gene expression machinery of the host cell. The main structural proteins of HIV-1 are expressed as a single poly- protein Gag (Figure 1A). Gag alone is sufﬁcient for the for- mation of virus-like particles (VLP) in a mammalian cell (1). In fact, recombinant Gag, in the presence of nucleic acid and an additional small molecule cofactor, is sufﬁcient for the assembly of VLP in vitro (2–4) which, except for the absence of a surrounding membrane, are morphologically very similar to the immature viruses formed in vivo (3,5). However, this self-assembly does not occur in the absence of nucleic acid (2,4,6,7). Surprisingly, the self-assembly of structurally normal VLPs has been shown to be supported by the presence of virtually any RNA or DNA molecules (2,4,6–8). However, the HIV-1 Gag protein is also capable of preferentially pack- aging viral RNA from a large pool of cellular nucleic acid *To whom correspondence should be addressed. Tel: 1 617 373 7323; Fax: 1 617 373 2943; Email: mark@neu.edu Correspondence may also be addressed to Ioulia Rouzina. Tel: 1 612 624 7468; Fax: 1 612 624 5121; Email: rouzina@cbs.umn.edu The Author 2006. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org