Cruceanu Margareta, Urbaneja Maria A, Hixson Catherine V, Johnson Donald G, Datta Siddhartha A, Fivash Matthew J, Stephen Andrew G, Fisher Robert J, Gorelick Robert J, Casas-Finet Jose R, Rein Alan, Rouzina Ioulia, Williams Mark C
Department of Physics, Northeastern University, 111 Dana Research Center, 110 Forsyth Street, Boston, MA 02115, USA.
Nucleic Acids Res. 2006 Jan 30;34(2):593-605. doi: 10.1093/nar/gkj458. Print 2006.
The Gag polyprotein of HIV-1 is essential for retroviral replication and packaging. The nucleocapsid (NC) protein is the primary region for the interaction 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 experiments. 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 approximately 10-fold higher than that of the NC proteins, and its destabilizing effect on dsDNA was negligible. These findings are consistent 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 chaperones. Also, our results suggest that NCp7's capability for fast sequence-nonspecific nucleic acid duplex destabilization, as well as its ability to facilitate nucleic 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 contrast, Gag's stronger DNA binding and aggregation capabilities likely make it an effective chaperone for processes that do not require significant duplex destabilization.
HIV-1的Gag多聚蛋白对于逆转录病毒的复制和包装至关重要。核衣壳（NC）蛋白是Gag与核酸相互作用的主要区域。在本研究中，我们使用溶液和单分子实验研究了Gag及其NC裂解产物（NCp15、NCp9和NCp7）与核酸的相互作用。NC裂解产物以相当的亲和力结合DNA，并强烈破坏DNA双链体的稳定性。相比之下，发现Gag与DNA的结合常数比NC蛋白高约10倍，并且其对双链DNA的破坏作用可忽略不计。这些发现与Gag作为核酸结合和包装蛋白的主要功能以及NC蛋白作为核酸伴侣的主要功能一致。此外，我们的结果表明，NCp7快速非序列特异性破坏核酸双链体的能力，以及其通过诱导链间静电吸引促进核酸链退火的能力，可能优化了完全加工的NC蛋白以促进复杂核酸二级结构重排。相比之下，Gag更强的DNA结合和聚集能力可能使其成为不需要显著破坏双链体稳定性的过程的有效伴侣。
