Solution Structure of the Human Immunodeficiency Virus Type 1 p6 Protein

Fossen, Torgils; Wray, Victor; Bruns, Karsten; Rachmat, Judhi; Henklein, Peter; Tessmer, Uwe; Maczurek, Annette; Klinger, Patricia; Schubert, Ulrich S.

doi:10.1074/jbc.m507375200

Cited by 56 publications

(74 citation statements)

References 69 publications

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“…The only sequences with known functions in this part of RSV Gag are the two late domains that interact with the ESCRT machinery, PPPY (62)(63)(64) and LYPSL (62). Retroviral late domains are known to be located in unstructured regions (65,66). Other biological functions of this long unstructured stretch of RSV Gag remain to be uncovered.…”

Section: Discussionmentioning

confidence: 99%

Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein

Dick

Datta

Nanda

et al. 2015

J Virol

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Previously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag protein and in parallel several truncation mutants of Gag and have studied their biophysical properties and membrane interactions in vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particles in vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small-angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag, which is compact. However, by neutron reflectometry measurements of RSV Gag bound to a supported bilayer, the protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. In contrast with full-length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain and a C-terminally truncated version that is missing the NC domain bind only weakly. These results imply that NC contributes to membrane interaction in vitro, either by directly contacting acidic lipids or by promoting Gag multimerization. IMPORTANCERetroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our results show that RSV Gag is highly flexible and can adopt a folded-over conformation on a lipid bilayer, implicating both the N and C termini in membrane binding. In addition, binding of Gag to membranes is diminished when either terminal domain is truncated. RSV Gag membrane association is significantly less sensitive than HIV Gag membrane association to lipid acyl chain saturation. These findings shed light on Gag assembly and membrane binding, critical steps in the viral life cycle and an untapped target for antiretroviral drugs. Many of the principles underlying retrovirus assembly are understood. For example, the retrovirus structural protein, Gag, is able to assemble into morphologically normal virus-like particles (VLPs) in cells (1) and as a purified protein in vitro (2). The structures and functions of the multiple Gag domains are known. Thus, the MA domain mediates binding to the plasma membrane (PM), where for most retroviruses the Gag lattice is formed and budding occurs, the CA domain and immediately adjoining sequences engage in critical contacts to form the hexagonal Gag lattice, t...

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Section: Discussionmentioning

confidence: 99%

Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein

Dick

Datta

Nanda

et al. 2015

J Virol

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“…Proteins separated by SDS-PAGE were blotted onto polyvinylidene difluoride membranes (GE Healthcare) and probed with the following Abs prior to ECL detection: polyclonal rabbit anti-p24 (Seramun Diagnostica), rabbit anti-p6 antiserum (37), monoclonal anti-b-actin (Sigma-Aldrich), human anti-HIV antiserum from pooled plasma of at least 20 HIV-positive donors, monoclonal anti-Tsg101 (GeneTex), monoclonal anti-Flag (SigmaAldrich), polyclonal rabbit anti-b5i Ab (BIOMOL), and polyclonal rabbit anti-b5i Ab (BIOMOL).…”

Section: Western Blottingmentioning

confidence: 99%

The PTAP Sequence within the p6 Domain of Human Immunodeficiency Virus Type 1 Gag Regulates Its Ubiquitination and MHC Class I Antigen Presentation

Hahn

Setz

Wild

et al. 2011

The Journal of Immunology

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Endogenous peptides presented by MHC class I (MHC-I) molecules are mostly derived from de novo synthesized, erroneous proteins, so-called defective ribosomal products (DRiPs), which are rapidly degraded via the ubiquitin–proteasome pathway. We have previously shown that the HIV-1 Gag protein represents a bona fide substrate for the DRiP pathway and that the amount of Gag-DRiPs can be enhanced by the introduction of an N-end rule degradation signal, leading to increased MHC-I presentation and immunogenicity of Gag. Based on these findings, we sought to identify a naturally occurring sequence motif within Gag that regulates its entry into the DRiP pathway. As the PTAP late assembly domain motif in the C-terminal p6 domain of Gag has been shown to negatively regulate the ubiquitination of Gag, we analyzed the correlation between ubiquitination and MHC-I presentation of PTAP-deficient Gag. Intriguingly, mutation of PTAP not only reduces the release of virus-like particles, but also increases ubiquitination of Gag and, consistently, enhances MHC-I presentation of a Gag-derived epitope. Although the half-life of the PTAP mutant was only mildly reduced, the entry into the DRiP pathway was significantly increased, as demonstrated by short-term pulse-chase analyses under proteasome inhibition. Collectively, these results indicate that, besides driving virus release, the PTAP motif regulates the entry of Gag into the DRiP pathway and, thus, into the MHC-I pathway. Although there are no naturally occurring PTAP mutants of HIV-1, mutations of PTAP might enhance the immunogenicity of Gag and, thus, be considered for the improvement of vaccine development.

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“…1B), which are proteolyzed into their freestanding mature forms during or after budding (14). The structures of most individual domains have been solved by X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy in both free and ligand-bound states (20,22,44,47), but the entire structure of Gag remains elusive (25). Each domain of Gag is multifunctional, serving interaction roles during assembly, structural roles in the mature virion, and facilitative roles during reverse transcription and integration (10,25,39).…”

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confidence: 99%

Matrix Domain Modulates HIV-1 Gag's Nucleic Acid Chaperone Activity via Inositol Phosphate Binding

Jones

Datta

Rein

et al. 2011

J Virol

124

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Retroviruses replicate by reverse transcribing their single-stranded RNA genomes into double-stranded DNA using specific cellular tRNAs to prime cDNA synthesis. In HIV-1, human tRNA 3 Lys serves as the primer and is packaged into virions during assembly. The viral Gag protein is believed to chaperone tRNA 3 Lys placement onto the genomic RNA primer binding site; however, the timing and possible regulation of this event are currently unknown. Composed of the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains, the multifunctional HIV-1 Gag polyprotein orchestrates the highly coordinated process of virion assembly, but the contribution of these domains to tRNA 3 Lys annealing is unclear. Here, we show that NC is absolutely essential for annealing and that the MA domain inhibits Gag's tRNA annealing capability. During assembly, MA specifically interacts with inositol phosphate (IP)-containing lipids in the plasma membrane (PM). Surprisingly, we find that IPs stimulate Gag-facilitated tRNA annealing but do not stimulate annealing in Gag variants lacking the MA domain or containing point mutations involved in PM binding. Moreover, we find that IPs prevent MA from binding to nucleic acids but have little effect on NC or Gag. We propose that Gag binds to RNA either with both NC and MA domains or with NC alone and that MA-IP interactions alter Gag's binding mode. We propose that MA's interactions with the PM trigger the switch between these two binding modes and stimulate Gag's chaperone function, which may be important for the regulation of events such as tRNA primer annealing.

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Solution Structure of the Human Immunodeficiency Virus Type 1 p6 Protein

Cited by 56 publications

References 69 publications

Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein

Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein

The PTAP Sequence within the p6 Domain of Human Immunodeficiency Virus Type 1 Gag Regulates Its Ubiquitination and MHC Class I Antigen Presentation

Matrix Domain Modulates HIV-1 Gag's Nucleic Acid Chaperone Activity via Inositol Phosphate Binding

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