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Journal of Virology, January 2004, p. 52-60, Vol. 78, No. 1
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.1.52-60.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Nucleic Acid Binding-Induced Gag Dimerization in the Assembly of Rous Sarcoma Virus Particles In Vitro

Yu May Ma{dagger} and Volker M. Vogt*

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853

Received 3 July 2003/ Accepted 11 September 2003

As also found for other retroviruses, the Rous sarcoma virus structural protein Gag is necessary and sufficient for formation of virus-like particles (VLPs). Purified polypeptide fragments comprising most of Gag spontaneously assemble in vitro at pH 6.5 into VLPs lacking a membrane, a process that requires nucleic acid. We showed previously that the minimum length of a DNA oligonucleotide that can support efficient assembly is 16 nucleotides (nt), twice the protein's binding site size. This observation suggests that the essential role of nucleic acid in assembly is to promote the formation of Gag dimers. In order to gain further insight into the role of dimerization, we have studied the assembly properties of two proteins, a nearly full-length Gag ({Delta}MBD{Delta}PR) capable of proper in vitro assembly and a smaller Gag fragment (CTD-NC) capable of forming only irregular aggregates but with the same pH and oligonucleotide length requirements as for assembly with the larger protein. In analyses by sedimentation velocity and by cross-linking, both proteins remained monomeric in the absence of oligonucleotides or in the presence of an oligonucleotide of length 8 nt (GT8). At pH 8, which does not support assembly, binding to GT16 induced the formation of dimers of {Delta}MBD{Delta}PR but not of CTD-NC, implying that dimerization requires the N-terminal domain of the capsid moiety of Gag. Assembly of VLPs was induced by shifting the pH of dimeric complexes of {Delta}MBD{Delta}PR and GT16 from 8 to 6.5. An analogue of GT16 with a ribonucleotide linkage in the middle also supported dimer formation at pH 8. Even after quantitative cleavage of the oligonucleotide by treatment of the complex with RNase, these dimers could be triggered to undergo assembly by pH change. This result implies that protein-protein interactions stabilize the dimer. We propose that binding of two adjacent Gag molecules on a stretch of nucleic acid leads to protein-protein interactions that create a Gag dimer and that this species has an exposed surface not present in monomers which allows polymerization of the dimers into a spherical shell.

* Corresponding author. Mailing address: Department of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca, NY 14853. Phone: (607) 255-2443. Fax: (607) 255-2428. E-mail: vmv1{at}cornell.edu.

{dagger} Present address: Center for Blood Research, Harvard Medical School, Boston, MA 02115.

Journal of Virology, January 2004, p. 52-60, Vol. 78, No. 1
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.1.52-60.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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