scholarly journals Human Immunodeficiency Virus Type 1 Gag Polyprotein Multimerization Requires the Nucleocapsid Domain and RNA and Is Promoted by the Capsid-Dimer Interface and the Basic Region of Matrix Protein

1999 ◽  
Vol 73 (10) ◽  
pp. 8527-8540 ◽  
Author(s):  
Mark T. Burniston ◽  
Andrea Cimarelli ◽  
John Colgan ◽  
Sean P. Curtis ◽  
Jeremy Luban
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Protein Protein Interactions ◽  
Virion Assembly ◽  
Dimer Interface ◽  
Gag Polyprotein ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein directs the formation of virions from productively infected cells. Manygag mutations disrupt virion assembly, but little is known about the biochemical effects of many of these mutations. Protein-protein interactions among Gag monomers are believed to be necessary for virion assembly, and data suggest that RNA may modify protein-protein interactions or even serve as a bridge linking Gag polyprotein monomers. To evaluate the primary sequence requirements for HIV-1 Gag homomeric interactions, a panel of HIV-1 Gag deletion mutants was expressed in bacteria and evaluated for the ability to associate with full-length Gag in vitro. The nucleocapsid protein, the major RNA-binding domain of Gag, exhibited activity comparable to that of the complete polyprotein. In the absence of the nucleocapsid protein, relatively weak activity was observed that was dependent upon both the capsid-dimer interface and basic residues within the matrix domain. The relevance of the in vitro findings was confirmed with an assay in which nonmyristylated mutant Gags were assessed for the ability to be incorporated into virions produced by wild-type Gag expressed intrans. Evidence of the importance of RNA for Gag-Gag interaction was provided by the demonstration that RNase impairs the Gag-Gag interaction and that HIV-1 Gag interacts efficiently with Gags encoded by distantly related retroviruses and with structurally unrelated RNA-binding proteins. These results are consistent with models in which Gag multimerization involves indirect contacts via an RNA bridge as well as direct protein-protein interactions.

scholarly journals Basic Residues in Human Immunodeficiency Virus Type 1 Nucleocapsid Promote Virion Assembly via Interaction with RNA

2000 ◽  
Vol 74 (7) ◽  
pp. 3046-3057 ◽  
Author(s):  
Andrea Cimarelli ◽  
Sara Sandin ◽  
Stefan Höglund ◽  
Jeremy Luban
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Rna Binding ◽  
Zinc Fingers ◽  
Genomic Rna ◽  
Virion Assembly ◽  
Immunodeficiency Virus

ABSTRACT Retroviral Gag polyproteins drive virion assembly by polymerizing to form a spherical shell that lines the inner membrane of nascent virions. Deletion of the nucleocapsid (NC) domain of the Gag polyprotein disrupts assembly, presumably because NC is required for polymerization. Human immunodeficiency virus type 1 NC possesses two zinc finger motifs that are required for specific recognition and packaging of viral genomic RNA. Though essential, zinc fingers and genomic RNA are not required for virion assembly. NC promiscuously associates with cellular RNAs, many of which are incorporated into virions. It has been hypothesized that Gag polymerization and virion assembly are promoted by nonspecific interaction of NC with RNA. Consistent with this model, we found an inverse relationship between the number of NC basic residues replaced with alanine and NC's nonspecific RNA-binding activity, Gag's ability to polymerize in vitro and in vivo, and Gag's capacity to assemble virions. In contrast, mutation of NC's zinc fingers had only minor effects on these properties.

scholarly journals A Novel Fluorescence Resonance Energy Transfer Assay Demonstrates that the Human Immunodeficiency Virus Type 1 Pr55Gag I Domain Mediates Gag-Gag Interactions

2004 ◽  
Vol 78 (3) ◽  
pp. 1230-1242 ◽  
Author(s):  
Aaron Derdowski ◽  
Lingmei Ding ◽  
Paul Spearman
Keyword(s):  
Human Immunodeficiency Virus ◽  
Plasma Membrane ◽  
Energy Transfer ◽  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Protein Protein Interactions ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) assembly takes place at the plasma membrane of cells and is directed by the Pr55Gag polyprotein (Gag). One of the essential steps in the assembly process is the multimerization of Gag. We have developed a novel fluorescence resonance energy transfer (FRET) assay for the detection of protein-protein interactions between Gag molecules. We demonstrate that Gag multimerization takes place primarily on cellular membranes, with the majority of these interactions occurring on the plasma membrane. However, distinct sites of Gag-Gag interaction are also present at punctate intracellular locations. The I domain is a functional assembly domain within the nucleocapsid region of Gag that affects particle density, the subcellular localization of Gag, and the formation of detergent-resistant Gag protein complexes. Results from this study provide evidence that the I domain mediates Gag-Gag interactions. Using Gag-fluorescent protein fusion constructs that were previously shown to define the minimal I domain within HIV-1 Pr55Gag, we show by FRET techniques that protein-protein interactions are greatly diminished when Gag proteins lacking the I domain are expressed. Gag-Tsg101 interactions are also seen in living cells and result in a shift of Tsg101 to the plasma membrane. The results within this study provide direct evidence that the I domain mediates protein-protein interactions between Gag molecules. Furthermore, this study establishes FRET as a powerful tool for the detection of protein-protein interactions involved in retrovirus assembly.

scholarly journals Vif and the p55Gag Polyprotein of Human Immunodeficiency Virus Type 1 Are Present in Colocalizing Membrane-Free Cytoplasmic Complexes

1999 ◽  
Vol 73 (4) ◽  
pp. 2667-2674 ◽  
Author(s):  
James H. M. Simon ◽  
Elise A. Carpenter ◽  
Ron A. M. Fouchier ◽  
Michael H. Malim
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Protein Interactions ◽  
Subcellular Fractionation ◽  
Current Data ◽  
Protein Protein Interactions ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The Vif protein of human immunodeficiency virus type 1 (HIV-1) is a potent regulator of viral infectivity. Current data posit that Vif functions late in replication to modulate assembly, budding, and/or maturation. Consistent with this model, earlier indirect immunofluorescence analyses of HIV-1-infected cells demonstrated that Vif and Gag colocalize to a substantial degree (J. H. M. Simon, R. A. M. Fouchier, T. E. Southerling, C. B. Guerra, C. K. Grant, and M. H. Malim, J. Virol. 71:5259–5267, 1997). Here, we describe a series of subcellular fractionation studies which indicate that Vif and the p55Gag polyprotein are present in membrane-free cytoplasmic complexes that copurify in sucrose density gradients and are stable in nonionic detergents. Both Vif and Gag are targeted to these complexes independent of each other, and their association with them appears to be mediated by protein-protein interactions. We propose that these complexes may represent viral assembly intermediates and that Vif is appropriately localized to influence the final stages of the viral life cycle and, therefore, the infectivity of progeny virions.

scholarly journals PF74 and Its Novel Derivatives Stabilize Hexameric Lattice of HIV-1 Mature-Like Particles

Molecules ◽  
2020 ◽  
Vol 25 (8) ◽  
pp. 1895
Author(s):  
Alžběta Dostálková ◽  
Kryštof Škach ◽  
Filip Kaufman ◽  
Ivana Křížová ◽  
Romana Hadravová ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Early Stage ◽  
Protein A ◽  
Protein Protein Interactions ◽  
Particle Assembly ◽  
Gag Polyprotein ◽  
Polyprotein Precursor ◽  
Hiv 1 ◽  
Ca Protein

A major structural retroviral protein, capsid protein (CA), is able to oligomerize into two different hexameric lattices, which makes this protein a key component for both the early and late stages of HIV-1 replication. During the late stage, the CA protein, as part of the Gag polyprotein precursor, facilitates protein–protein interactions that lead to the assembly of immature particles. Following protease activation and Gag polyprotein processing, CA also drives the assembly of the mature viral core. In the early stage of infection, the role of the CA protein is distinct. It controls the disassembly of the mature CA hexameric lattice i.e., uncoating, which is critical for the reverse transcription of the single-stranded RNA genome into double stranded DNA. These properties make CA a very attractive target for small molecule functioning as inhibitors of HIV-1 particle assembly and/or disassembly. Of these, inhibitors containing the PF74 scaffold have been extensively studied. In this study, we reported a series of modifications of the PF74 molecule and its characterization through a combination of biochemical and structural approaches. Our data supported the hypothesis that PF74 stabilizes the mature HIV-1 CA hexameric lattice. We identified derivatives with a higher in vitro stabilization activity in comparison to the original PF74 molecule.

scholarly journals Unexpected Mutations in HIV-1 That Confer Resistance to the Tat Inhibitor Didehydro-Cortistatin A

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Andrew P. Rice
Keyword(s):  
Rna Polymerase Ii ◽  
Rna Binding ◽  
Latent Infection ◽  
Pol Ii ◽  
Immunodeficiency Virus ◽  
Tar Rna ◽  
Responsive Element ◽  
Hiv 1

ABSTRACT Didehydro-cortistatin A (dCA) is a human immunodeficiency virus type 1 (HIV-1) Tat inhibitor that functions by selectively binding to the RNA binding domain of Tat. In addition to inhibiting viral replication, dCA can drive HIV-1 into a state of “deep latency” in which latent viruses are refractory to reactivation. Mousseau et al. (G. Mousseau, R. Aneja, M. A. Clementz, S. Mediouni, et al., mBio 10:e01750-18, 2019, https://doi.org/10.1128/mBio.01750-18) have now selected dCA-resistant (dCAr) viruses in vitro. Remarkably, dCAr viruses do not contain mutations in Tat or the viral transactivation-responsive element (TAR) RNA element that is targeted by Tat. Rather, the viruses contain a combination of mutations in the viral long terminal repeat (LTR) and Nef and Vpr proteins that result in an increase in basal RNA polymerase II (Pol II) transcription of integrated HIV-1. Interestingly, dCAr viruses may be deficient in the establishment of latent infection because of their elevated basal Pol II transcription. dCA holds promise for strategies to achieve a functional cure of HIV-1 infection and justifies efforts to develop additional Tat inhibitors.

scholarly journals Structure, Function, and Interactions of the HIV-1 Capsid Protein

Life ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 100
Author(s):  
Eric Rossi ◽  
Megan E. Meuser ◽  
Camille J. Cunanan ◽  
Simon Cocklin
Keyword(s):  
Life Cycle ◽  
Capsid Protein ◽  
Adaptor Proteins ◽  
Restriction Factors ◽  
Gag Polyprotein ◽  
Immunodeficiency Virus ◽  
Host Cell Factors ◽  
Hiv 1

The capsid (CA) protein of the human immunodeficiency virus type 1 (HIV-1) is an essential structural component of a virion and facilitates many crucial life cycle steps through interactions with host cell factors. Capsid shields the reverse transcription complex from restriction factors while it enables trafficking to the nucleus by hijacking various adaptor proteins, such as FEZ1 and BICD2. In addition, the capsid facilitates the import and localization of the viral complex in the nucleus through interaction with NUP153, NUP358, TNPO3, and CPSF-6. In the later stages of the HIV-1 life cycle, CA plays an essential role in the maturation step as a constituent of the Gag polyprotein. In the final phase of maturation, Gag is cleaved, and CA is released, allowing for the assembly of CA into a fullerene cone, known as the capsid core. The fullerene cone consists of ~250 CA hexamers and 12 CA pentamers and encloses the viral genome and other essential viral proteins for the next round of infection. As research continues to elucidate the role of CA in the HIV-1 life cycle and the importance of the capsid protein becomes more apparent, CA displays potential as a therapeutic target for the development of HIV-1 inhibitors.

scholarly journals The Late-Domain-Containing Protein p6 Is the Predominant Phosphoprotein of Human Immunodeficiency Virus Type 1 Particles

2002 ◽  
Vol 76 (3) ◽  
pp. 1015-1024 ◽  
Author(s):  
Barbara Müller ◽  
Tilo Patschinsky ◽  
Hans-Georg Kräusslich
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Metabolic Labeling ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
A Minor ◽  
Hiv 1

ABSTRACT The Gag-derived protein p6 of human immunodeficiency virus type 1 (HIV-1) plays a crucial role in the release of virions from the membranes of infected cells. It is presumed that p6 and functionally related proteins from other viruses act as adapters, recruiting cellular factors to the budding site. This interaction is mediated by so-called late domains within the viral proteins. Previous studies had suggested that virus release from the plasma membrane shares elements with the cellular endocytosis machinery. Since protein phosphorylation is known to be a regulatory mechanism in these processes, we have investigated the phosphorylation of HIV-1 structural proteins. Here we show that p6 is the major phosphoprotein of HIV-1 particles. After metabolic labeling of infected cells with [ortho- 32P]phosphate, we found that phosphorylated p6 from infected cells and from virus particles consisted of several forms, suggesting differential phosphorylation at multiple sites. Apparently, phosphorylation occurred shortly before or after the release of p6 from Gag and involved only a minor fraction of the total virion-associated p6 molecules. Phosphoamino acid analysis indicated phosphorylation at Ser and Thr, as well as a trace of Tyr phosphorylation, supporting the conclusion that multiple phosphorylation events do occur. In vitro experiments using purified virus revealed that endogenous or exogenously added p6 was efficiently phosphorylated by virion-associated cellular kinase(s). Inhibition experiments suggested that a cyclin-dependent kinase or a related kinase, most likely ERK2, was involved in p6 phosphorylation by virion-associated enzymes.

scholarly journals Ontogeny and Specificities of Mucosal and Blood Human Immunodeficiency Virus Type 1-Specific CD8+ Cytotoxic T Lymphocytes

2003 ◽  
Vol 77 (1) ◽  
pp. 291-300 ◽  
Author(s):  
L. Musey ◽  
Y. Ding ◽  
J. Cao ◽  
J. Lee ◽  
C. Galloway ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cell ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Cytotoxic T Lymphocyte ◽  
Infected Individual ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Induction of adaptive immunity to human immunodeficiency virus type 1 (HIV-1) at the mucosal site of transmission is poorly understood but crucial in devising strategies to control and prevent infection. To gain further understanding of HIV-1-specific T-cell mucosal immunity, we established HIV-1-specific CD8+ cytotoxic T-lymphocyte (CTL) cell lines and clones from the blood, cervix, rectum, and semen of 12 HIV-1-infected individuals and compared their specificities, cytolytic function, and T-cell receptor (TCR) clonotypes. Blood and mucosal CD8+ CTL had common HIV-1 epitope specificities and major histocompatibility complex restriction patterns. Moreover, both systemic and mucosal CTL lysed targets with similar efficiency, primarily through the perforin-dependent pathway in in vitro studies. Sequence analysis of the TCRβ VDJ region revealed in some cases identical HIV-1-specific CTL clones in different compartments in the same HIV-1-infected individual. These results clearly establish that a subset of blood and mucosal HIV-1-specific CTL can have a common origin and can traffic between anatomically distinct compartments. Thus, these effectors can provide immune surveillance at the mucosa, where rapid responses are needed to contain HIV-1 infection.

scholarly journals Gag sequence variation in a human immunodeficiency virus type 1 transmission cluster influences viral replication fitness

2013 ◽  
Vol 94 (2) ◽  
pp. 354-359 ◽  
Author(s):  
Esther F. Gijsbers ◽  
Ad C. van Nuenen ◽  
Hanneke Schuitemaker ◽  
Neeltje A. Kootstra
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Sequence Variation ◽  
Clinical Course ◽  
Immunodeficiency Virus ◽  
Compensatory Mutations ◽  
Replication Fitness ◽  
Hiv 1

Three men from a proven homosexual human immunodeficiency virus type 1 (HIV-1) transmission cluster showed large variation in their clinical course of infection. To evaluate the effect of evolution of the same viral variant in these three patients, we analysed sequence variation in the capsid protein and determined the impact of the observed variation on viral replication fitness in vitro. Viral gag sequences from all three patients contained a mutation at position 242, T242N or T242S, which have been associated with lower virus replication in vitro. Interestingly, HIV-1 variants from patients with a progressive clinical course of infection developed compensatory mutations within the capsid that restored viral fitness, instead of reversion of the T242S mutation. In HIV-1 variants from patient 1, an HLA-B57+ elite controller, no compensatory mutations emerged during follow-up.

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