Relationships between MA-RNA binding in cells and suppression of HIV-1 Gag mislocalization to intracellular membranes

Thornhill, Dishari; Olety, Balaji; Ono, Akira

doi:10.1101/629998

Cited by 3 publications

(1 citation statement)

References 75 publications

(96 reference statements)

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“…In addition to acidic phospholipids, the MA-HBR binds RNA [14][15][16][17][18][19]. This binding prevents Gag membrane binding in general; however, full-length Gag can bind membranes containing PI(4,5)P 2 , which outcompetes RNA for MA binding [20].…”

Section: Ma-hbr Also Interacts With Trna Which Regulates Membrane Bin...mentioning

confidence: 99%

The “basics” of HIV-1 assembly

Sumner,

Ono

2024

PLoS Pathog

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Section: Ma-hbr Also Interacts With Trna Which Regulates Membrane Bin...mentioning

confidence: 99%

The “basics” of HIV-1 assembly

Sumner,

Ono

2024

PLoS Pathog

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Structural basis for human T-cell leukemia virus type 1 Gag targeting to the plasma membrane for assembly

Herrmann

Zhou²,

Hm³

et al. 2021

Preprint

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During the late phase of retroviral replication, the virally encoded Gag polyprotein is targeted to the plasma membrane (PM) for assembly. GagPM binding is mediated by the N-terminal matrix (MA) domain of Gag. For many retroviruses, Gag binding to the PM was found to be dependent on phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. However, it was shown that for human T-cell leukemia virus type 1 (HTLV-1), Gag binding to membranes is less dependent on PI(4,5)P2, suggesting that other factors may modulate Gag assembly. To elucidate the mechanism by which HTLV-1 Gag binds to the PM, we employed NMR techniques to solve the structure of unmyristoylated MA (myr(−)MA) and to characterize its interactions with lipids and liposomes. The MA structure consists of four α-helices and unstructured N- and C-termini. We show that myr(−)MA binds to PI(4,5)P2 via the polar head and that myr(−)MA binding to inositol phosphates (IPs) is significantly enhanced by increasing the number of phosphate groups on the inositol ring, indicating that the MAIP binding is governed by chargecharge interactions. The IP binding site was mapped to a well-defined basic patch formed by lysine and arginine residues. Using a sensitive NMR-based liposome binding assay, we show that myr(−)MA binding to membranes is significantly enhanced by phosphatidylserine (PS). Confocal microscopy data show that Gag is localized to the inner leaflet of the PM of infected cells, while the Gag G2A mutant, lacking myristoylation, is diffuse and cytoplasmic. These findings advance our understanding of a key mechanism in retroviral assembly.

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Relationships between MA-RNA Binding in Cells and Suppression of HIV-1 Gag Mislocalization to Intracellular Membranes

Thornhill

Olety

Ono

2019

J Virol

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The HIV-1 Gag matrix (MA) domain mediates the localization of Gag to the plasma membrane (PM), the site for infectious virion assembly. The MA highly basic region (MA-HBR) interacts with phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2], a PM-specific acidic lipid. The MA-HBR also binds RNAs. To test whether acidic lipids alone determine PM-specific localization of Gag or whether MA-RNA binding also plays a role, we compared a panel of MA-HBR mutants that contain two types of substitutions at MA residues 25 and 26 or residues 29 and 31: Lys→Arg (KR) (25/26KR and 29/31KR) and Lys→Thr (KT) (25/26KT and 29/31KT). Consistent with the importance of the HBR charge in RNA binding, both KT mutants failed to bind RNA via MA efficiently, unlike the corresponding KR mutants. Both 25/26KT Gag-yellow fluorescent protein (YFP) and 29/31KT Gag-YFP bound nonspecifically to the PM and intracellular membranes, presumably via the myristoyl moiety and remaining MA basic residues. In contrast, 25/26KR Gag-YFP bound specifically to the PM, suggesting a role for the total positive charge and/or MA-bound RNA in navigating Gag to the PM. Unlike 29/31KT Gag-YFP, 29/31KR Gag-YFP was predominantly cytosolic and showed little intracellular membrane binding despite having a higher HBR charge. Therefore, it is likely that MA-RNA binding blocks promiscuous Gag membrane binding in cells. Notably, the introduction of a heterologous multimerization domain restored PI(4,5)P2-dependent PM-specific localization for 29/31KR Gag-YFP, suggesting that the blocking of PM binding is more readily reversed than that of intracellular membrane binding. Altogether, these cell-based data support a model in which MA-RNA binding ensures PM-specific localization of Gag via suppression of nonspecific membrane binding. IMPORTANCE The PM-specific localization of HIV-1 Gag is a crucial early step in infectious progeny production. The interaction between the MA highly basic region (MA-HBR) of Gag and the PM-specific lipid PI(4,5)P2 is critical for Gag localization to the PM. Additionally, in vitro evidence has indicated that MA-RNA binding prevents nonspecific binding of Gag to non-PI(4,5)P2-containing membranes. However, cell-based evidence supporting a role for HIV-1 MA-RNA binding in PM-specific subcellular localization has been scarce; thus, it remained possible that in cells, just the high basic charge or the PI(4,5)P2 binding ability is sufficient for MA to direct Gag specifically to the PM. The present study reveals for the first time an excellent correlation between RNA binding of the MA-HBR and inhibition of promiscuous Gag localization, both within the cells, and thereby provides cell-based evidence supporting a mechanism in which HIV-1 MA binding to RNA ensures the specific localization of Gag to the PM.

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Relationships between MA-RNA binding in cells and suppression of HIV-1 Gag mislocalization to intracellular membranes

Cited by 3 publications

References 75 publications

The “basics” of HIV-1 assembly

The “basics” of HIV-1 assembly

Structural basis for human T-cell leukemia virus type 1 Gag targeting to the plasma membrane for assembly

Relationships between MA-RNA Binding in Cells and Suppression of HIV-1 Gag Mislocalization to Intracellular Membranes

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