Virus assembly and plasma membrane domains: Which came first?

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: Discussionsupporting

confidence: 91%

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

Barros

Heinrich

Datta

et al. 2016

“…Moreover, a recent study also showed the formation of an acidic interface, composed of plasma membrane acidic phospholipids, at the virological synapses between murine leukemia virusinfected and virus receptor-expressing cells (79). Altogether, our current findings and previous studies are consistent with a model in which basic residues within the HBR of the MA domain result in local clustering of acidic lipids (35,80), potentially driven by the multimerization of Gag, which in turn selectively recruits some host cell plasma membrane proteins on the basis of the presence of polybasic motifs in their cytoplasmic tails. It is also possible that a preexisting microdomain enriched in acidic phospholipids attracts both Gag and cellular transmembrane proteins with juxtamembrane polybasic sequences.…”

Section: Discussionsupporting

confidence: 91%

Basic Motifs Target PSGL-1, CD43, and CD44 to Plasma Membrane Sites Where HIV-1 Assembles

Grover

Veatch

Ono

2015

HIV-1 incorporates various host membrane proteins during particle assembly at the plasma membrane; however, the mechanisms mediating this incorporation process remain poorly understood. We previously showed that the HIV-1 structural protein Gag localizes to the uropod, a rear-end structure of polarized T cells, and that assembling Gag copatches with a subset, but not all, of the uropod-directed proteins, i.e., PSGL-1, CD43, and CD44, in nonpolarized T cells. The latter observation suggests the presence of a mechanism promoting virion incorporation of these cellular proteins. To address this possibility and identify molecular determinants, in the present study we examined coclustering between Gag and the transmembrane proteins in T and HeLa cells using quantitative two-color superresolution localization microscopy. Consistent with the findings of the T-cell copatching study, we found that basic residues within the matrix domain of Gag are required for Gag-PSGL-1 coclustering. Notably, the presence of a polybasic sequence in the PSGL-1 cytoplasmic domain significantly enhanced this coclustering. We also found that polybasic motifs present in the cytoplasmic tails of CD43 and CD44 also promote their coclustering with Gag. ICAM-1 and ICAM-3, uropod-directed proteins that do not copatch with Gag in T cells, and CD46, a non-uropod-directed protein, showed no or little coclustering with Gag. However, replacing their cytoplasmic tails with the cytoplasmic tail of PSGL-1 significantly enhanced their coclustering with Gag. Altogether, these results identify a novel mechanism for host membrane protein association with assembling HIV-1 Gag in which polybasic sequences present in the cytoplasmic tails of the membrane proteins and in Gag are the major determinants. IMPORTANCENascent HIV-1 particles incorporate many host plasma membrane proteins during assembly. However, it is largely unknown what mechanisms promote the association of these proteins with virus assembly sites within the plasma membrane. Notably, our previous study showed that HIV-1 structural protein Gag colocalizes with a group of uropod-directed transmembrane proteins, PSGL-1, CD43, and CD44, at the plasma membrane of T cells. The results obtained in the current study using superresolution localization microscopy suggest the presence of a novel molecular mechanism promoting the association of PSGL-1, CD43, and CD44 with assembling HIV-1 which relies on polybasic sequences in HIV-1 Gag and in cytoplasmic domains of the transmembrane proteins. This information advances our understanding of virion incorporation of host plasma membrane proteins, some of which modulate virus spread positively or negatively, and suggests a possible new strategy to enrich HIV-1-based lentiviral vectors with a desired transmembrane protein. HIV-1 assembles at the plasma membrane in most cell types. The viral structural protein Gag, synthesized as a precursor polyprotein (Pr55), initiates and coordinates viral assembly at the plasma membrane. Membrane binding and targeting of ...

“…Such a recruitment of negatively charged lipids by NC is in line with the recent hypothesis that Gag multimerization at the PM could induce the formation of acidic lipid-enriched microdomains (ALEM) (106,107). Interestingly, the binding constant of NC for negatively charged lipids is similar to that of the interaction of unmyristoylated MA and Gag proteins with DOPS-containing LUVs (91,92), which is mainly governed by electrostatic interactions through the HBR sequence of MA (59,60,85,106). However, in contrast to the myristoylated GagMA domain (67,91,108), which binds avidly and specifically to PI(4,5)P 2 through a hydrophobic interaction with the exposed myristyl chain, no specific binding was found for the NC-PI(4,5)P 2 interaction.…”

Section: Discussionsupporting

confidence: 85%

“…Importantly, while the number of binding sites was found to decrease with the percentage of negatively charged lipids in the LUV composition, the binding constant value was nearly independent of this percentage, suggesting that NC and NC-oligonucleotide complexes can recruit negatively charged lipids to ensure optimal binding. Such a recruitment of negatively charged lipids by NC is in line with the recent hypothesis that Gag multimerization at the PM could induce the formation of acidic lipid-enriched microdomains (ALEM) (106,107). Interestingly, the binding constant of NC for negatively charged lipids is similar to that of the interaction of unmyristoylated MA and Gag proteins with DOPS-containing LUVs (91,92), which is mainly governed by electrostatic interactions through the HBR sequence of MA (59,60,85,106).…”

Section: Discussionsupporting

confidence: 81%

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...