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

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.

Download Full-text

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

10.1101/2021.03.01.433343 ◽

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.

Download Full-text

Characterization of immune responses elicited in mice by intranasal co-immunization with HIV-1 Tat, gp140 ΔV2Env and/or SIV Gag proteins and the nontoxicogenic heat-labile Escherichia coli enterotoxin

Vaccine ◽

10.1016/j.vaccine.2007.12.030 ◽

2008 ◽

Vol 26 (9) ◽

pp. 1214-1227 ◽

Cited By ~ 16

Author(s):

Antonella Caputo ◽

Egidio Brocca-Cofano ◽

Arianna Castaldello ◽

Rebecca Voltan ◽

Riccardo Gavioli ◽

...

Keyword(s):

Escherichia Coli ◽

Immune Responses ◽

Gag Proteins ◽

Heat Labile ◽

Hiv 1

Download Full-text

Systematic characterization of the conformation and dynamics of budding yeast chromosome XII

The Journal of Cell Biology ◽

10.1083/jcb.201208186 ◽

2013 ◽

Vol 202 (2) ◽

pp. 201-210 ◽

Cited By ~ 45

Author(s):

Benjamin Albert ◽

Julien Mathon ◽

Ashutosh Shukla ◽

Hicham Saad ◽

Christophe Normand ◽

...

Keyword(s):

Large Scale ◽

Polymer Physics ◽

Rouse Model ◽

Transcriptional Reprogramming ◽

Tethered Polymers ◽

Yeast Chromosome ◽

Live Cell Microscopy ◽

Cell Microscopy ◽

Physical Principles

Chromosomes architecture is viewed as a key component of gene regulation, but principles of chromosomal folding remain elusive. Here we used high-throughput live cell microscopy to characterize the conformation and dynamics of the longest chromosome of Saccharomyces cerevisiae (XII). Chromosome XII carries the ribosomal DNA (rDNA) that defines the nucleolus, a major hallmark of nuclear organization. We determined intranuclear positions of 15 loci distributed every ∼100 kb along the chromosome, and investigated their motion over broad time scales (0.2–400 s). Loci positions and motions, except for the rDNA, were consistent with a computational model of chromosomes based on tethered polymers and with the Rouse model from polymer physics, respectively. Furthermore, rapamycin-dependent transcriptional reprogramming of the genome only marginally affected the chromosome XII internal large-scale organization. Our comprehensive investigation of chromosome XII is thus in agreement with recent studies and models in which long-range architecture is largely determined by the physical principles of tethered polymers and volume exclusion.

Download Full-text

Visualizing fusion of pseudotyped HIV-1 particles in real time by live cell microscopy

Retrovirology ◽

10.1186/1742-4690-6-84 ◽

2009 ◽

Vol 6 (1) ◽

pp. 84 ◽

Cited By ~ 25

Author(s):

Peter Koch ◽

Marko Lampe ◽

William J Godinez ◽

Barbara Müller ◽

Karl Rohr ◽

...

Keyword(s):

Real Time ◽

Live Cell ◽

Live Cell Microscopy ◽

Cell Microscopy ◽

Hiv 1

Download Full-text

Direct Visualization of HIV-1 with Correlative Live-Cell Microscopy and Cryo-Electron Tomography

Structure ◽

10.1016/j.str.2011.09.006 ◽

2011 ◽

Vol 19 (11) ◽

pp. 1573-1581 ◽

Cited By ~ 62

Author(s):

Sangmi Jun ◽

Danxia Ke ◽

Karl Debiec ◽

Gongpu Zhao ◽

Xin Meng ◽

...

Keyword(s):

Electron Tomography ◽

Live Cell ◽

Direct Visualization ◽

Cryo Electron Tomography ◽

Live Cell Microscopy ◽

Cell Microscopy ◽

Hiv 1

Download Full-text

Angiomotin functions in HIV-1 assembly and budding

eLife ◽

10.7554/elife.03778 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 24

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.

Download Full-text