Rous Sarcoma Virus Gag Has No Specific Requirement for Phosphatidylinositol-(4,5)-Bisphosphate for Plasma Membrane Association
            <i>In Vivo</i>
            or for Liposome Interaction
            <i>In Vitro</i>

By assembling in a protein lattice on the host's plasma membrane, the retroviral Gag polyprotein triggers formation of the viral protein/membrane shell. The MA domain of Gag employs multiple signals-electrostatic, hydrophobic, and lipid-specific-to bring the protein to the plasma membrane, thereby complementing protein-protein interactions, located in full-length Gag, in lattice formation. We report the interaction of myristoylated and unmyristoylated HIV-1 Gag MA domains with bilayers composed of purified lipid components to dissect these complex membrane signals and quantify their contributions to the overall interaction. Surface plasmon resonance on well-defined planar membrane models is used to quantify binding affinities and amounts of protein and yields free binding energy contributions, ⌬G, of the various signals. Charge-charge interactions in the absence of the phosphatidylinositide PI(4,5)P 2 attract the protein to acidic membrane surfaces, and myristoylation increases the affinity by a factor of 10; thus, our data do not provide evidence for a PI(4,5)P 2 trigger of myristate exposure. Lipid-specific interactions with PI(4,5)P 2 , the major signal lipid in the inner plasma membrane, increase membrane attraction at a level similar to that of protein lipidation. While cholesterol does not directly engage in interactions, it augments protein affinity strongly by facilitating efficient myristate insertion and PI(4,5)P 2 binding. We thus observe that the isolated MA protein, in the absence of protein-protein interaction conferred by the full-length Gag, binds the membrane with submicromolar affinities. IMPORTANCELike other retroviral species, the Gag polyprotein of HIV-1 contains three major domains: the N-terminal, myristoylated MA domain that targets the protein to the plasma membrane of the host; a central capsid-forming domain; and the C-terminal, genome-binding nucleocapsid domain. These domains act in concert to condense Gag into a membrane-bounded protein lattice that recruits genomic RNA into the virus and forms the shell of a budding immature viral capsid. In binding studies of HIV-1 Gag MA to model membranes with well-controlled lipid composition, we dissect the multiple interactions of the MA domain with its target membrane. This results in a detailed understanding of the thermodynamic aspects that determine membrane association, preferential lipid recruitment to the viral shell, and those aspects of Gag assembly into the membrane-bound protein lattice that are determined by MA. T he polyprotein Gag is an essential component for the reproduction of retroviruses, such as HIV-1, in infected cells. In the production of new viruses, many copies of Gag assemble laterally, either in the cytoplasm or on the plasma membrane (PM) of the host, thus acquiring their lipid envelope as they bud from the cell. Distinct retroviral genera encode Gag proteins which all show a similar domain architecture, with an N-terminal matrix (MA) domain that targets the PM, followed by the capsid (CA) and nucleocapsid ...

Section: Discussionmentioning

confidence: 99%

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

Barros

Heinrich

Datta

et al. 2016

“…Myristoylated full-length Gag synthesized in reticulocyte lysates does not bind negatively charged liposomes containing PS at the physiological concentration of this acidic lipid (ϳ30%) (12,19,20). Even at higher concentrations (ϳ50%), palmitoyl-oleoyl-PS (POPS), the most abundant form of PS in viral and plasma membranes (21), does not support efficient liposome binding although di-oleoyl-PS does (20).…”

mentioning

confidence: 99%

Evidence in Support of RNA-Mediated Inhibition of Phosphatidylserine-Dependent HIV-1 Gag Membrane Binding in Cells

Chukkapalli

Inlora

Todd

et al. 2013

The matrix domain promotes plasma-membrane-specific binding of HIV-1 Gag through interaction with an acidic lipid phosphatidylinositol-(4,5)-bisphosphate. In in vitro systems, matrix-bound RNA suppresses Gag interactions with phosphatidylserine, an acidic lipid prevalent in various cytoplasmic membranes, thereby enhancing the lipid specificity of the matrix domain. Here we provide in vitro and cell-based evidence supporting the idea that this RNA-mediated suppression occurs in cells and hence is a physiologically relevant mechanism that prevents Gag from binding promiscuously to phosphatidylserine-containing membranes.

“…Thus, RNAs and notably gRNA may act as negative regulators of nonspecific membrane binding, likely by reducing the electrostatic interactions of the HBR with acidic lipids and preventing myristate exposure (62,63). Phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P 2 ] and probably other phosphoinositides (64,65), which are essentially found on the cytoplasmic leaflet of the PM, can outcompete gRNA (61,66) and promote the anchoring of the myristoylated MA domain to the PM (62,67). The PI(4,5)P 2 -induced release of GagMA from the gRNA is also thought to promote Gag trimerization and a structural switch of Gag to its rodshaped conformation that is observed in virus particles (68)(69)(70).…”

mentioning

confidence: 99%

The HIV-1 Nucleocapsid Protein Recruits Negatively Charged Lipids To Ensure Its Optimal Binding to Lipid Membranes

Kempf

Postupalenko

Bora

et al. 2015

The HIV-1 Gag polyprotein precursor composed of the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains orchestrates virus assembly via interactions between MA and the cell plasma membrane (PM) on one hand and NC and the genomic RNA on the other hand. As the Gag precursor can adopt a bent conformation, a potential interaction of the NC domain with the PM cannot be excluded during Gag assembly at the PM. To investigate the possible interaction of NC with lipid membranes in the absence of any interference from the other domains of Gag, we quantitatively characterized by fluorescence spectroscopy the binding of the mature NC protein to large unilamellar vesicles (LUVs) used as membrane models. We found that NC, either in its free form or bound to an oligonucleotide, was binding with high affinity (ϳ10 7 M ؊1 ) to negatively charged LUVs. The number of NC binding sites, but not the binding constant, was observed to decrease with the percentage of negatively charged lipids in the LUV composition, suggesting that NC and NC/oligonucleotide complexes were able to recruit negatively charged lipids to ensure optimal binding. However, in contrast to MA, NC did not exhibit a preference for phosphatidylinositol-(4,5)-bisphosphate. These results lead us to propose a modified Gag assembly model where the NC domain contributes to the initial binding of the bent form of Gag to the PM. IMPORTANCEThe NC protein is a highly conserved nucleic acid binding protein that plays numerous key roles in HIV-1 replication. While accumulating evidence shows that NC either as a mature protein or as a domain of the Gag precursor also interacts with host proteins, only a few data are available on the possible interaction of NC with lipid membranes. Interestingly, during HIV-1 assembly, the Gag precursor is thought to adopt a bent conformation where the NC domain may interact with the plasma membrane. In this context, we quantitatively characterized the binding of NC, as a free protein or as a complex with nucleic acids, to lipid membranes and showed that the latter constitute a binding platform for NC. Taken together, our data suggest that the NC domain may play a role in the initial binding events of Gag to the plasma membrane during HIV-1 assembly.T he nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) is a small basic nucleic acid binding protein with two strictly conserved CCHC motifs that strongly bind zinc (1). Mature NC results from the protease-mediated cleavage of the Gag polyprotein precursor and plays key roles in HIV-1 replication (2-5). During the early steps, NC acts as a cofactor of the reverse transcriptase (RT), to promote the initiation of reverse transcription (6-8), as well as the two obligatory strand transfers (9, 10). Moreover, NC also reduces RT pauses at the initiation step (11-13), increases the overall RT processivity (14, 15), promotes synthesis through pause sites (16-19), helps to remove the RNA fragments resulting from the RT RNase H activity (20), and may contribute to the pro...