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

Abstract

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 (⌬MBD⌬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 ⌬MBD⌬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 ⌬MBD⌬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.

Dimeric Rous Sarcoma Virus Capsid Protein Structure Relevant to Immature Gag Assembly

“…Nevertheless, it is noteworthy that the 25-residue extension of CA into p10, the determinant for the assembly of Gag into spherical particles, creates a totally different dimer interface than occurs in crystal structures of the NTD CA domain and that the interface in the 25NTD structure is largely determined by these 25 residues. It is also relevant that both in vitro 28,29 and in vivo 30 studies of RSV Gag implicate dimerization as being critical for assembly. These observations suggest that the dimer interface seen in the 25NTD crystal structure also occurs in RSV Gag molecules when they are forming spherical, immature particles.…”

The structural biology of HIV assembly

“…1b) forms the innermost layer of the immature virion and also plays essential roles in particle formation [21,23,51,[78][79][80][81]. NC/RNA complex(es) do not follow the hexagonal symmetry of the CA and SP1 regions, and most experiments indicate that NC primarily "tethers" Gag molecules together, probably via RNA bridges, although additional NC-NC interactions may also occur [79,80,82,83]. NC/RNA tethers presumably increase the effective concentration of assembling Gag molecules, which could also play a more active role in orienting or otherwise facilitating CA-CA interactions.…”

Directionality of nucleocytoplasmic transport of the retroviral gag protein depends on sequential binding of karyopherins and viral RNA

“…CRM1 likely associates with a dimer of Gag, based on studies showing that nucleic acid binding promotes Gag dimerization (36,42) and Gag dimers form in the nucleus (30). However, the crystal structure of the p10-CA Gag dimer shows that the NES is buried in hydrophobic interactions with CA, and is therefore unavailable to bind to CRM1 (17).…”

“…Cellular proteins adopt related strategies, including the RNA-editing enzyme ADAR-1, for which binding of double-stranded RNA inhibits nuclear import and facilitates export (35). Thus, Gag-vRNA binding serves multiple critical roles, such as the molecular switch to trigger nuclear export of Gag, a platform for Gag dimerization (36), and the mechanism for specific encapsidation of the viral genome.…”