Membrane Binding of the Rous Sarcoma Virus Gag Protein Is Cooperative and Dependent on the Spacer Peptide Assembly Domain

Abstract

The principles underlying membrane binding and assembly of retroviral Gag proteins into a lattice are understood. However, little is known about how these processes are related. Using purified Rous sarcoma virus Gag and Gag truncations, we studied the interrelation of Gag-Gag interaction and Gag-membrane interaction. Both by liposome binding and by surface plasmon resonance on a supported bilayer, Gag bound to membranes much more tightly than did matrix (MA), the isolated membrane binding domain. In principle, this difference could be explained either by protein-protein interactions leading to cooperativity in membrane binding or by the simultaneous interaction of the N-terminal MA and the C-terminal nucleocapsid (NC) of Gag with the bilayer, since both are highly basic. However, we found that NC was not required for strong membrane binding. Instead, the spacer peptide assembly domain (SPA), a putative 24-residue helical sequence comprising the 12-residue SP segment of Gag and overlapping the capsid (CA) C terminus and the NC N terminus, was required. SPA is known to be critical for proper assembly of the immature Gag lattice. A single amino acid mutation in SPA that abrogates assembly in vitro dramatically reduced binding of Gag to liposomes. In vivo, plasma membrane localization was dependent on SPA. Disulfide cross-linking based on ectopic Cys residues showed that the contacts between Gag proteins on the membrane are similar to the known contacts in virus-like particles. Taken together, we interpret these results to mean that Gag membrane interaction is cooperative in that it depends on the ability of Gag to multimerize. IMPORTANCEThe retroviral structural protein Gag has three major domains. The N-terminal MA domain interacts directly with the plasma membrane (PM) of cells. The central CA domain, together with immediately adjoining sequences, facilitates the assembly of thousands of Gag molecules into a lattice. The C-terminal NC domain interacts with the genome, resulting in packaging of viral RNA. For assembly in vitro with purified Gag, in the absence of membranes, binding of NC to nucleic acid somehow facilitates further Gag-Gag interactions that lead to formation of the Gag lattice. The contributions of MA-mediated membrane binding to virus particle assembly are not well understood. Here, we report that in the absence of nucleic acid, membranes provide a platform that facilitates Gag-Gag interactions. This study demonstrates that the binding of Gag, but not of MA, to membranes is cooperative and identifies SPA as a major factor that controls this cooperativity. L ate in the retroviral life cycle, the structural protein Gag localizes to the inner leaflet of the plasma membrane (PM), where it assembles around the viral genomic RNA, leading to budding from the cell. Gag proteins include three major domains, the Nterminal matrix (MA) domain, which mediates membrane binding; the central capsid (CA) domain, which mediates Gag-Gag interactions during virion assembly; and the C-terminal nucleocapsid (NC...

Effects of Membrane Charge and Order on Membrane Binding of the Retroviral Structural Protein Gag

“…All SUMO-tagged proteins were purified using standard bacterial ex- (10,53). In brief, Escherichia coli BL21 cultures were grown at 37°C to an optical density at 600 nm of 0.6.…”

“…SUMO-mNG-KRn (n ϭ 4 [RKKR], n ϭ 8 [RKKRKKRK], or n ϭ 12 [RKKRKKRKKRKK]) and SUMO-mNG-MARCKS (KKKKKRFSCKKS-FKLSGFSFKKNKK) (63, 64) were made by inserting annealed primers into SUMO-mNG using EagI and XhoI. The linker sequence between the tandem Evectin2 domains is VDGT (65).All SUMO-tagged proteins were purified using standard bacterial ex- (10,53). In brief, Escherichia coli BL21 cultures were grown at 37°C to an optical density at 600 nm of 0.6.…”

“…The LUVs we use are 100 nm in size, and LUVbound protein can be quantitated by flotation in a density gradient (21) or centrifugation to a pellet (liposome pelleting) (53). GUVs we use are cell sized (Ն10 m) and thus easily visualized by fluorescence microscopy when the membrane lipid phases or a protein are marked with a fluorescent dye (54,55).…”

“…The work described here represents the first systematic study of the effect of charge, phase, and lipid order on a purified retroviral Gag protein in two-phase GUVs with known PS concentrations in each phase. pression and affinity column techniques (10,53). In brief, Escherichia coli BL21 cultures were grown at 37°C to an optical density at 600 nm of 0.6.…”

Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation

“…It is well documented that CA-CA interactions drive Gag oligomerization (6,7), and artificial multimerization of MA enhances its affinity to membranes by an order of magnitude (8). Moreover, a recent study identifies protein-protein contacts in the SPA segment of RSV Gag that stabilize oligomers and increase membrane affinity, leading in turn to pronounced cooperativity in membrane binding (9).…”