Permeability of the HIV-1 capsid to metabolites modulates viral DNA synthesis

Reverse transcription, an essential event in the HIV-1 life cycle, requires deoxynucleotide triphosphates (dNTPs) to fuel DNA synthesis, thus requiring penetration of dNTPs into the viral capsid. The central cavity of the capsid protein (CA) hexamer reveals itself as a plausible channel that allows the passage of dNTPs into assembled capsids. Nevertheless, the molecular mechanism of nucleotide import into the capsid remains unknown. Employing all-atom molecular dynamics (MD) simulations, we established that cooperative binding between nucleotides inside a CA hexamer cavity results in energetically favorable conditions for passive translocation of dNTPs into the HIV-1 capsid. Furthermore, binding of the host cell metabolite inositol hexakisphosphate (IP6) enhances dNTP import, while binding of synthesized molecules like benzenehexacarboxylic acid (BHC) inhibits it. The enhancing effect on reverse transcription by IP6 and the consequences of interactions between CA and nucleotides were corroborated using atomic force microscopy, transmission electron microscopy, and virological assays. Collectively, our results provide an atomistic description of the permeability of the HIV-1 capsid to small molecules and reveal a novel mechanism for the involvement of metabolites in HIV-1 capsid stabilization, nucleotide import, and reverse transcription.

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Permeability of the HIV-1 capsid to metabolites modulates viral DNA synthesis

10.1101/2020.04.30.071217 ◽

2020 ◽

Cited By ~ 2

Author(s):

Chaoyi Xu ◽

Douglas K. Fischer ◽

Sanela Rankovic ◽

Wen Li ◽

Rob Dick ◽

...

Keyword(s):

Dna Synthesis ◽

Small Molecules ◽

Reverse Transcription ◽

Central Cavity ◽

Force Microscopy ◽

Atomic Force ◽

Transmission Electron ◽

Dynamics Simulations ◽

Favorable Conditions ◽

Hiv 1

AbstractReverse transcription, an essential event in the HIV-1 lifecycle, requires deoxynucleotide triphosphates (dNTPs) to fuel DNA synthesis, thus requiring penetration of dNTPs into the viral core. The central cavity of the capsid protein (CA) hexamer reveals itself as a plausible channel that allows the passage of dNTPs into assembled capsids. Nevertheless, the molecular mechanism of nucleotide import into the capsid remains unknown. Employing all-atom molecular dynamics simulations, we established that cooperative binding between nucleotides inside a CA hexamer cavity results in energetically-favorable conditions for passive translocation of dNTPs into the HIV-1 capsid. Furthermore, binding of the host cell metabolite inositol hexakisphosphate (IP6) enhances dNTP import, while binding of synthesized molecules like benzenehexacarboxylic acid (BHC) inhibits it. The enhancing effect on reverse transcription by IP6 and the consequences of interactions between CA and nucleotides were corroborated using atomic force microscopy, transmission electron microscopy, and virological assays. Collectively, our results provide an atomistic description of the permeability of the HIV-1 capsid to small molecules and reveal a novel mechanism for the involvement of metabolites in HIV-1 capsid stabilization, nucleotide import and reverse transcription.

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PF74 Reinforces the HIV-1 Capsid To Impair Reverse Transcription-Induced Uncoating

Journal of Virology ◽

10.1128/jvi.00845-18 ◽

2018 ◽

Vol 92 (20) ◽

Cited By ~ 21

Author(s):

Sanela Rankovic ◽

Ruben Ramalho ◽

Christopher Aiken ◽

Itay Rousso

Keyword(s):

Atomic Force Microscopy ◽

Small Molecule ◽

Reverse Transcription ◽

Direct Evidence ◽

Main Body ◽

Force Microscopy ◽

The Core ◽

Atomic Force ◽

Capsid Stability ◽

Hiv 1

ABSTRACTThe RNA genome of human immunodeficiency virus type 1 (HIV-1) is enclosed in a cone-shaped capsid shell that disassembles following cell entry via a process known as uncoating. During HIV-1 infection, the capsid is important for reverse transcription and entry of the virus into the target cell nucleus. The small molecule PF74 inhibits HIV-1 infection at early stages by binding to the capsid and perturbing uncoating. However, the mechanism by which PF74 alters capsid stability and reduces viral infection is presently unknown. Here, we show, using atomic force microscopy (AFM), that binding of PF74 to recombinant capsid-like assemblies and to HIV-1 isolated cores stabilizes the capsid in a concentration-dependent manner. At a PF74 concentration of 10 μM, the mechanical stability of the core is increased to a level similar to that of the intrinsically hyperstable capsid mutant E45A. PF74 also prevented the complete disassembly of HIV-1 cores normally observed during 24 h of reverse transcription. Specifically, cores treated with PF74 only partially disassembled: the main body of the capsid remained intact and stiff, and a cap-like structure dissociated from the narrow end of the core. Moreover, the internal coiled structure that was observed to form during reverse transcriptionin vitropersisted throughout the duration of the measurement (∼24 h). Our results provide direct evidence that PF74 directly stabilizes the HIV-1 capsid lattice, thereby permitting reverse transcription while interfering with a late step in uncoating.IMPORTANCEThe capsid-binding small molecule PF74 inhibits HIV-1 infection at early stages and perturbs uncoating. However, the mechanism by which PF74 alters capsid stability and reduces viral infection is presently unknown. We recently introduced time-lapse atomic force microscopy to study the morphology and physical properties of HIV-1 cores during the course of reverse transcription. Here, we apply this AFM methodology to show that PF74 prevented the complete disassembly of HIV-1 cores normally observed during 24 h of reverse transcription. Specifically, cores with PF74 only partially disassembled: the main body of the capsid remained intact and stiff, but a cap-like structure dissociated from the narrow end of the core HIV-1. Our result provides direct evidence that PF74 directly stabilizes the HIV-1 capsid lattice.

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Reverse Transcription Mechanically Initiates HIV-1 Capsid Disassembly

Journal of Virology ◽

10.1128/jvi.00289-17 ◽

2017 ◽

Vol 91 (12) ◽

Cited By ~ 35

Author(s):

Sanela Rankovic ◽

Janani Varadarajan ◽

Ruben Ramalho ◽

Christopher Aiken ◽

Itay Rousso

Keyword(s):

Atomic Force Microscopy ◽

Reverse Transcription ◽

Target Cells ◽

Filamentous Structure ◽

Force Microscopy ◽

The Core ◽

Atomic Force ◽

Capsid Shell ◽

Hiv 1 ◽

Capsid Disassembly

ABSTRACT The HIV-1 core consists of the viral genomic RNA and several viral proteins encased within a conical capsid. After cell entry, the core disassembles in a process termed uncoating. Although HIV-1 uncoating has been linked to reverse transcription of the viral genome in target cells, the mechanism by which uncoating is initiated is unknown. Using time-lapse atomic force microscopy, we analyzed the morphology and physical properties of isolated HIV-1 cores during the course of reverse transcription in vitro. We found that, during an early stage of reverse transcription the pressure inside the capsid increases, reaching a maximum after 7 h. High-resolution mechanical mapping reveals the formation of a stiff coiled filamentous structure underneath the capsid surface. Subsequently, this coiled structure disappears, the stiffness of the capsid drops precipitously to a value below that of a pre-reverse transcription core, and the capsid undergoes partial or complete rupture near the narrow end of the conical structure. We propose that the transcription of the relatively flexible single-stranded RNA into a more rigid filamentous structure elevates the pressure within the core, which triggers the initiation of capsid disassembly. IMPORTANCE For successful infection, the HIV-1 genome, which is in the form of a single-stranded RNA enclosed inside a capsid shell, must be reverse transcribed into double-stranded DNA and released from the capsid (in a process known as uncoating) before it can be integrated into the target cell genome. The mechanism that triggers uncoating is a pivotal question of long standing. By using atomic force microscopy, we found that during reverse transcription the pressure inside the capsid increases until the internal stress exceeds the strength of the capsid structure and the capsid breaks open. The application of AFM technologies to study purified HIV-1 cores represents a new experimental platform for elucidating additional aspects of capsid disassembly and HIV-1 uncoating.

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HIV-1 uncoating occurs via a series of rapid biomechanical changes in the core related to individual stages of reverse transcription

Journal of Virology ◽

10.1128/jvi.00166-21 ◽

2021 ◽

Author(s):

Sanela Rankovic ◽

Akshay Deshpande ◽

Shimon Harel ◽

Christopher Aiken ◽

Itay Rousso

Keyword(s):

Reverse Transcription ◽

Viral Genome ◽

Viral Capsid ◽

Target Cells ◽

Force Microscopy ◽

The Core ◽

Atomic Force ◽

Successful Infection ◽

Capsid Shell ◽

Hiv 1

The HIV core consists of the viral genome and associated proteins encased by a cone-shaped protein shell termed the capsid. Successful infection requires reverse transcription of the viral genome and disassembly of the capsid shell within a cell in a process known as uncoating. The integrity of the viral capsid is critical for reverse transcription, yet the viral capsid must be breached to release the nascent viral DNA prior to integration. We employed atomic force microscopy to study the stiffness changes in HIV-1 cores during reverse transcription in vitro in reactions containing the capsid-stabilizing host metabolite IP6. Cores exhibited a series of stiffness spikes, with up to three spikes typically occurring between 10-30, 40-80, and 120-160 minutes after initiation of reverse transcription. Addition of the reverse transcriptase (RT) inhibitor efavirenz eliminated the appearance of these spikes and the subsequent disassembly of the capsid, thus establishing that both result from reverse transcription. Using timed addition of efavirenz, and analysis of an RNAseH-defective RT mutant, we established that the first stiffness spike requires minus-strand strong stop DNA synthesis, with subsequent spikes requiring later stages of reverse transcription. Additional rapid AFM imaging experiments revealed repeated morphological changes in cores that were temporally correlated with the observed stiffness spikes. Our study reveals discrete mechanical changes in the viral core that are likely related to specific stages of reverse transcription. These reverse-transcription-induced changes in the capsid progressively remodel the viral core to prime it for temporally accurate uncoating in target cells. Importance For successful infection, the HIV-1 genome, which is enclosed inside a capsid shell, must be reverse transcribed into double-stranded DNA and released from the capsid (in a process known as uncoating) before it can be integrated into the target cell genome. The mechanism of HIV-1 uncoating is a pivotal question of long standing. Using atomic force microscopy to analyze individual HIV-1 cores during reverse transcription, we observe a reproducible pattern of stiffness spikes. These spikes were shown to be associated with distinct stages of the reverse transcription reaction. Our findings suggest that these reverse-transcription-induced alterations gradually prepared the core for uncoating at the right time and location in target cells.

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Scanning tunneling microscopy and atomic force microscopy: New tools for biology

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100155864 ◽

1989 ◽

Vol 47 ◽

pp. 778-779

Author(s):

CE Bracker ◽

P. K. Hansma

Keyword(s):

Physical Property ◽

Scanning Probe ◽

Scanning Tunneling ◽

Small Scale ◽

Tunneling Microscopy ◽

Force Microscopy ◽

New Family ◽

Small Probe ◽

Atomic Force ◽

Transmission Electron

A new family of scanning probe microscopes has emerged that is opening new horizons for investigating the fine structure of matter. The earliest and best known of these instruments is the scanning tunneling microscope (STM). First published in 1982, the STM earned the 1986 Nobel Prize in Physics for two of its inventors, G. Binnig and H. Rohrer. They shared the prize with E. Ruska for his work that had led to the development of the transmission electron microscope half a century earlier. It seems appropriate that the award embodied this particular blend of the old and the new because it demonstrated to the world a long overdue respect for the enormous contributions electron microscopy has made to the understanding of matter, and at the same time it signalled the dawn of a new age in microscopy. What we are seeing is a revolution in microscopy and a redefinition of the concept of a microscope.Several kinds of scanning probe microscopes now exist, and the number is increasing. What they share in common is a small probe that is scanned over the surface of a specimen and measures a physical property on a very small scale, at or near the surface. Scanning probes can measure temperature, magnetic fields, tunneling currents, voltage, force, and ion currents, among others.

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The Electrical Characterization and Physical Failure Analysis for Transistor Gate Leakage

ISTFA 2006: Conference Proceedings from the 32nd International Symposium for Testing and Failure Analysis ◽

10.31399/asm.cp.istfa2006p0257 ◽

2006 ◽

Author(s):

Tsung-Te Li ◽

Chao-Chi Wu ◽

Jung-Hsiang Chuang ◽

Jon C. Lee

Keyword(s):

Failure Analysis ◽

Electrical Characterization ◽

Physical Analysis ◽

Gate Leakage ◽

Force Microscopy ◽

Atomic Force ◽

Transmission Electron ◽

Voltage Stress ◽

Leakage Site

Abstract This article describes the electrical and physical analysis of gate leakage in nanometer transistors using conducting atomic force microscopy (C-AFM), nano-probing, transmission electron microscopy (TEM), and chemical decoration on simulated overstressed devices. A failure analysis case study involving a soft single bit failure is detailed. Following the nano-probing analysis, TEM cross sectioning of this failing device was performed. A voltage bias was applied to exaggerate the gate leakage site. Following this deliberate voltage overstress, a solution of boiling 10%wt KOH was used to etch decorate the gate leakage site followed by SEM inspection. Different transistor leakage behaviors can be identified with nano-probing measurements and then compared with simulation data for increased confidence in the failure analysis result. Nano-probing can be used to apply voltage stress on a transistor or a leakage path to worsen the weak point and then observe the leakage site easier.

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Targeting Breast Cancer Cells with G4 PAMAM Dendrimers and Valproic Acid Derivative Complexes

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200423073812 ◽

2020 ◽

Vol 20 (15) ◽

pp. 1857-1872

Author(s):

Alberto M. Muñoz ◽

Manuel J. Fragoso-Vázquez ◽

Berenice P. Martel ◽

Alma Chávez-Blanco ◽

Alfonso Dueñas-González ◽

...

Keyword(s):

Valproic Acid ◽

Cell Lines ◽

Water Solubility ◽

Md Simulations ◽

Pamam Dendrimer ◽

Pamam Dendrimers ◽

Force Microscopy ◽

Micromolar Concentration ◽

Atomic Force

Background: Our research group has developed some Valproic Acid (VPA) derivatives employed as anti-proliferative compounds targeting the HDAC8 enzyme. However, some of these compounds are poorly soluble in water. Objective: Employed the four generations of Polyamidoamine (G4 PAMAM) dendrimers as drug carriers of these compounds to increase their water solubility for further in vitro evaluation. Methods: VPA derivatives were subjected to Docking and Molecular Dynamics (MD) simulations to evaluate their affinity on G4 PAMAM. Then, HPLC-UV/VIS, 1H NMR, MALDI-TOF and atomic force microscopy were employed to establish the formation of the drug-G4 PAMAM complexes. Results: The docking results showed that the amide groups of VPA derivatives make polar interactions with G4 PAMAM, whereas MD simulations corroborated the stability of the complexes. HPLC UV/VIS experiments showed an increase in the drug water solubility which was found to be directly proportional to the amount of G4 PAMAM. 1H NMR showed a disappearance of the proton amine group signals, correlating with docking results. MALDI-TOF and atomic force microscopy suggested the drug-G4 PAMAM dendrimer complexes formation. Discussion: In vitro studies showed that G4 PAMAM has toxicity in the micromolar concentration in MDAMB- 231, MCF7, and 3T3-L1 cell lines. VPA CF-G4 PAMAM dendrimer complex showed anti-proliferative properties in the micromolar concentration in MCF-7 and 3T3-L1, and in the milimolar concentration in MDAMB- 231, whereas VPA MF-G4 PAMAM dendrimer complex didn’t show effects on the three cell lines employed. Conclusion: These results demonstrate that G4 PAMAM dendrimers are capableof transporting poorly watersoluble aryl-VPA derivate compounds to increase its cytotoxic activity against neoplastic cell lines.

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Structural and Optical Properties of InAsSbBi Grown by Molecular Beam Epitaxy on Offcut GaSb Substrates

Photonics ◽

10.3390/photonics8060215 ◽

2021 ◽

Vol 8 (6) ◽

pp. 215

Author(s):

Rajeev R. Kosireddy ◽

Stephen T. Schaefer ◽

Marko S. Milosavljevic ◽

Shane R. Johnson

Keyword(s):

Molecular Beam Epitaxy ◽

Molecular Beam ◽

Optical Quality ◽

X Ray Diffraction ◽

Interface Quality ◽

Force Microscopy ◽

Atomic Force ◽

Transmission Electron ◽

Lateral Variations

Three InAsSbBi samples are grown by molecular beam epitaxy at 400 °C on GaSb substrates with three different offcuts: (100) on-axis, (100) offcut 1° toward [011], and (100) offcut 4° toward [011]. The samples are investigated using X-ray diffraction, Nomarski optical microscopy, atomic force microscopy, transmission electron microscopy, and photoluminescence spectroscopy. The InAsSbBi layers are 210 nm thick, coherently strained, and show no observable defects. The substrate offcut is not observed to influence the structural and interface quality of the samples. Each sample exhibits small lateral variations in the Bi mole fraction, with the largest variation observed in the on-axis growth. Bismuth rich surface droplet features are observed on all samples. The surface droplets are isotropic on the on-axis sample and elongated along the [011¯] step edges on the 1° and 4° offcut samples. No significant change in optical quality with offcut angle is observed.

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