scholarly journals Derivation and Characterization of an inositol phosphate-independent HIV-1

2021 ◽  
Author(s):  
Daniel Poston ◽  
Trinity Zang ◽  
Paul Bieniasz
Keyword(s):  
Small Molecule ◽  
Protein Interactions ◽  
Inositol Phosphate ◽  
Critical Role ◽  
Primary Amines ◽  
Virion Assembly ◽  
Gag Proteins ◽  
Live Cell Microscopy ◽  
The Stability ◽  
Hiv 1

AbstractA critical step in the HIV-1 replication cycle is the assembly of Gag proteins to form virions at the plasma membrane. Virion assembly and maturation is facilitated by the cellular polyanion inositol hexaphosphate (IP6), which is proposed to stabilize both the immature Gag lattice and the mature capsid lattice by binding to rings of primary amines at the center of Gag or capsid protein (CA) hexamers. The amino acids comprising these rings are critical for proper virion formation and their substitution results in assembly deficits or impaired infectiousness. To better understand the nature of the deficits that accompany IP6-deficiency, we passaged HIV-1 mutants that had substitutions in IP6-coordinating residues to select for compensatory mutations. We found a mutation, a threonine to isoleucine substitution at position 371 (T371I) in Gag, that restored replication competence to an IP6-binding-deficient HIV-1 mutant. Notably, unlike wild-type HIV-1, the assembly and infectiousness of resulting virus was not impaired when IP6 biosynthetic enzymes were genetically ablated. Surprisingly, we also found that the maturation inhibitor Bevirimat (BVM) could restore the assembly and replication of an IP6-binding deficient mutant. Moreover, using BVM-dependent mutants we were able to image the BVM-inducible assembly of individual HIV-1 particles assembly in living cells. Overall these results suggest that IP6-Gag and Gag-Gag contacts are finely tuned to generate a Gag lattice of optimal stability, and that under certain conditions BVM can functionally replace IP6.Author SummaryA key step in HIV-1 replication is the assembly of virions that are released from the infected cell. Previous work has suggested that a small molecule called IP6 is critical role in this process, promoting both HIV-1 assembly and the stability of mature fully infectious virions. Since IP6 is required for multiple steps in HIV-1 assembly and maturation, it is a candidate for the development of anti-retroviral therapies. Here, we identify an HIV-1 mutant that replicates independently of IP6, and show that a different small molecule can functionally substitute for IP6 under certain conditions. These findings suggest that IP6 regulates the stability of protein interactions during virion assembly and that the precise degree stability of these interactions is finely tuned and important for generating infectious virions. Finally, our work identifies an inducible virion assembly system that can be utilized to visualize HIV-1 assembly events using live cell microscopy.

scholarly journals Derivation and characterization of an HIV-1 mutant that rescues IP6 binding deficiency

Retrovirology ◽  
2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Daniel Poston ◽  
Trinity Zang ◽  
Paul Bieniasz
Keyword(s):  
Primary Amines ◽  
Virion Assembly ◽  
Gag Proteins ◽  
Maturation Inhibitor ◽  
Live Cell Microscopy ◽  
Compensatory Mutations ◽  
Virion Formation ◽  
Cell Microscopy ◽  
Hiv 1

Abstract Background A critical step in the HIV-1 replication cycle is the assembly of Gag proteins to form virions at the plasma membrane. Virion assembly and maturation are facilitated by the cellular polyanion inositol hexaphosphate (IP6), which is proposed to stabilize both the immature Gag lattice and the mature capsid lattice by binding to rings of primary amines at the center of Gag or capsid protein (CA) hexamers. The amino acids comprising these rings are critical for proper virion formation and their substitution results in assembly deficits or impaired infectiousness. To better understand the nature of the deficits that accompany IP6 binding deficiency, we passaged HIV-1 mutants that had substitutions in IP6 coordinating residues to select for compensatory mutations. Results We found a mutation, a threonine to isoleucine substitution at position 371 (T371I) in Gag, that restored replication competence to an IP6-binding-deficient HIV-1 mutant. Notably, unlike wild-type HIV-1, the assembly and infectiousness of resulting virus was not impaired when IP6 biosynthetic enzymes were genetically ablated. Surprisingly, we also found that the maturation inhibitor Bevirimat (BVM) could restore the assembly and replication of an IP6-binding deficient mutant. Moreover, using BVM-dependent mutants we were able to image BVM-induced assembly of individual HIV-1 particles assembly in living cells. Conclusions Overall these results suggest that IP6-Gag and Gag-Gag contacts are finely tuned to generate a Gag lattice of optimal stability, and that under certain conditions BVM can rescue IP6 deficiency. Additionally, our work identifies an inducible virion assembly system that can be utilized to visualize HIV-1 assembly events using live cell microscopy.

scholarly journals Guide snoRNAs: Drivers or Passengers in Human Disease?

Biology ◽  
2018 ◽  
Vol 8 (1) ◽  
pp. 1 ◽  
Author(s):  
Manisha Deogharia ◽  
Mrinmoyee Majumder
Keyword(s):  
Protein Interactions ◽  
Human Disease ◽  
Critical Role ◽  
Mrna Translation ◽  
Causal Link ◽  
Rna Modification ◽  
Disease Manifestation ◽  
The Stability ◽  
Nucleoside Modification ◽  
Host Genes

In every domain of life, RNA-protein interactions play a significant role in co- and post-transcriptional modifications and mRNA translation. RNA performs diverse roles inside the cell, and therefore any aberrancy in their function can cause various diseases. During maturation from its primary transcript, RNA undergoes several functionally important post-transcriptional modifications including pseudouridylation and ribose 2′-O-methylation. These modifications play a critical role in the stability of the RNA. In the last few decades, small nucleolar RNAs (snoRNAs) were revealed to be one of the main components to guide these modifications. Due to their active links to the nucleoside modification, deregulation in the snoRNA expressions can cause multiple disorders in humans. Additionally, host genes carrying snoRNA-encoding sequences in their introns also show differential expression in disease. Although few reports support a causal link between snoRNA expression and disease manifestation, this emerging field will have an impact on the way we think about biomarkers or identify novel targets for therapy. This review focuses on the intriguing aspect of snoRNAs that function as a guide in post-transcriptional RNA modification, and regulation of their host genes in human disease.

Pharmacophore-Based Discovery of Small-Molecule Inhibitors of Protein-Protein Interactions between HIV-1 Integrase and Cellular Cofactor LEDGF/p75

ChemMedChem ◽  
2009 ◽  
Vol 4 (8) ◽  
pp. 1311-1316 ◽  
Author(s):  
Laura De Luca ◽  
Maria Letizia Barreca ◽  
Stefania Ferro ◽  
Frauke Christ ◽  
Nunzio Iraci ◽  
...  
Keyword(s):  
Small Molecule ◽  
Protein Interactions ◽  
Small Molecule Inhibitors ◽  
Protein Protein Interactions ◽  
Cellular Cofactor ◽  
Hiv 1

scholarly journals A Time-Resolved Fluorescence Assay to Identify Small-Molecule Inhibitors of HIV-1 Fusion

2007 ◽  
Vol 12 (6) ◽  
pp. 865-874 ◽  
Author(s):  
Géry Dams ◽  
Koen Van Acker ◽  
Emmanuel Gustin ◽  
Inge Vereycken ◽  
Lieve Bunkens ◽  
...  
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
Protein Interactions ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Coiled Coil ◽  
Heptad Repeat ◽  
Time Resolved ◽  
Helix Bundle ◽  
Hiv 1

Fusion of host cell and human immunodeficiency virus type 1 (HIV-1) membranes is mediated by the 2 “heptad-repeat” regions of the viral gp41 protein. The collapse of the C-terminal heptad-repeat regions into the hydrophobic grooves of a coiled-coil formed by the corresponding homotrimeric N-terminal heptad-repeat regions generates a stable 6-helix bundle. This brings viral and cell membranes together for membrane fusion, facilitating viral entry. The authors developed an assay based on soluble peptides derived from the gp41 N-terminal heptad-repeat region (IQN36) as well as from the C-terminal region (C34). Both peptides were labeled with fluorophores, IQN36 with allophycocyanin (APC) and C34 with the lanthanide europium (Eu3+). Formation of the 6-helix bundle brings both fluorophores in close proximity needed for Förster resonance energy transfer (FRET). Compounds that interfere with binding of C34-Eu with IQN36-APC suppress the FRET signal. The assay was validated with various peptides and small molecules, and quenching issues were addressed. Evaluation of a diversified compound collection in a high-throughput screening campaign enabled identification of small molecules with different chemical scaffolds that inhibit this crucial intermediate in the HIV-1 entry process. This study's observations substantiate the expediency of time-resolved FRET-based assays to identify small-molecule inhibitors of protein-protein interactions. ( Journal of Biomolecular Screening 2007:865-874)

scholarly journals The secrets of the stability of the HIV-1 capsid

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Martin Obr ◽  
Hans-Georg Kräusslich
Keyword(s):  
Small Molecule ◽  
Biophysical Studies ◽  
The Stability ◽  
Capsid Stability ◽  
Hiv 1

Structural and biophysical studies help to follow the disassembly of the HIV-1 capsid in vitro, and reveal the role of a small molecule called IP6 in regulating capsid stability.

scholarly journals Screening for Novel Small-Molecule Inhibitors Targeting the Assembly of Influenza Virus Polymerase Complex by a Bimolecular Luminescence Complementation-Based Reporter System

2016 ◽  
Vol 91 (5) ◽  
Author(s):  
Chunfeng Li ◽  
Zining Wang ◽  
Yang Cao ◽  
Lulan Wang ◽  
Jingyun Ji ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Virus Replication ◽  
Small Molecule ◽  
Protein Interactions ◽  
Critical Role ◽  
Protein Protein Interactions ◽  
Reporter System ◽  
Polymerase Complex ◽  
Virus Rna ◽  
Influenza Virus Replication

ABSTRACT Influenza virus RNA-dependent RNA polymerase consists of three viral protein subunits: PA, PB1, and PB2. Protein-protein interactions (PPIs) of these subunits play pivotal roles in assembling the functional polymerase complex, which is essential for the replication and transcription of influenza virus RNA. Here we developed a highly specific and robust bimolecular luminescence complementation (BiLC) reporter system to facilitate the investigation of influenza virus polymerase complex formation. Furthermore, by combining computational modeling and the BiLC reporter assay, we identified several novel small-molecule compounds that selectively inhibited PB1-PB2 interaction. Function of one such lead compound was confirmed by its activity in suppressing influenza virus replication. In addition, our studies also revealed that PA plays a critical role in enhancing interactions between PB1 and PB2, which could be important in targeting sites for anti-influenza intervention. Collectively, these findings not only aid the development of novel inhibitors targeting the formation of influenza virus polymerase complex but also present a new tool to investigate the exquisite mechanism of PPIs. IMPORTANCE Formation of the functional influenza virus polymerase involves complex protein-protein interactions (PPIs) of PA, PB1, and PB2 subunits. In this work, we developed a novel BiLC assay system which is sensitive and specific to quantify both strong and weak PPIs between influenza virus polymerase subunits. More importantly, by combining in silico modeling and our BiLC assay, we identified a small molecule that can suppress influenza virus replication by disrupting the polymerase assembly. Thus, we developed an innovative method to investigate PPIs of multisubunit complexes effectively and to identify new molecules inhibiting influenza virus polymerase assembly.

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 Angiomotin functions in HIV-1 assembly and budding

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Gaelle Mercenne ◽  
Steven L Alam ◽  
Jun Arii ◽  
Matthew S Lalonde ◽  
Wesley I Sundquist
Keyword(s):  
Adaptor Protein ◽  
Cellular Protein ◽  
Virus Release ◽  
Electron Microscopic ◽  
Gag Protein ◽  
Virion Assembly ◽  
Gag Proteins ◽  
Immature Virion ◽  
Late Assembly Domain ◽  
Hiv 1

Many retroviral Gag proteins contain PPXY late assembly domain motifs that recruit proteins of the NEDD4 E3 ubiquitin ligase family to facilitate virus release. Overexpression of NEDD4L can also stimulate HIV-1 release but in this case the Gag protein lacks a PPXY motif, suggesting that NEDD4L may function through an adaptor protein. Here, we demonstrate that the cellular protein Angiomotin (AMOT) can bind both NEDD4L and HIV-1 Gag. HIV-1 release and infectivity are stimulated by AMOT overexpression and inhibited by AMOT depletion, whereas AMOT mutants that cannot bind NEDD4L cannot function in virus release. Electron microscopic analyses revealed that in the absence of AMOT assembling Gag molecules fail to form a fully spherical enveloped particle. Our experiments indicate that AMOT and other motin family members function together with NEDD4L to help complete immature virion assembly prior to ESCRT-mediated virus budding.

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