scholarly journals HIV-1 uncoating by release of viral cDNA from capsid-like structures in the nucleus of infected cells

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Thorsten G Müller ◽  
Vojtech Zila ◽  
Kyra Peters ◽  
Sandra Schifferdecker ◽  
Mia Stanic ◽  
...  
Keyword(s):  
Light And Electron Microscopy ◽  
Cell Types ◽  
Cellular Proteins ◽  
Viral Capsid ◽  
Target Cells ◽  
Physical Separation ◽  
Viral Cdna ◽  
Infected Cells ◽  
Hiv 1 ◽  
Over Time

HIV-1 replication commences inside the cone-shaped viral capsid, but timing, localization and mechanism of uncoating are under debate. We adapted a strategy to visualize individual reverse-transcribed HIV-1 cDNA molecules and their association with viral and cellular proteins using fluorescence and correlative-light-and-electron-microscopy (CLEM). We specifically detected HIV-1 cDNA inside nuclei, but not in the cytoplasm. Nuclear cDNA initially co-localized with a fluorescent integrase fusion (IN-FP) and the viral CA (capsid) protein, but cDNA-punctae separated from IN-FP/CA over time. This phenotype was conserved in primary HIV-1 target cells, with nuclear HIV-1 complexes exhibiting strong CA-signals in all cell types. CLEM revealed cone-shaped HIV-1 capsid-like structures and apparently broken capsid-remnants at the position of IN-FP signals and elongated chromatin-like structures in the position of viral cDNA punctae lacking IN-FP. Our data argue for nuclear uncoating by physical disruption rather than cooperative disassembly of the CA-lattice, followed by physical separation from the pre-integration complex.

scholarly journals HIV-1 uncoating by release of viral cDNA from capsid-like structures in the nucleus of infected cells

2020 ◽  
Author(s):  
Thorsten G. Müller ◽  
Vojtech Zila ◽  
Kyra Peters ◽  
Sandra Schifferdecker ◽  
Mia Stanic ◽  
...  
Keyword(s):  
Light And Electron Microscopy ◽  
Cell Types ◽  
Cellular Proteins ◽  
Viral Capsid ◽  
Target Cells ◽  
Physical Separation ◽  
Viral Cdna ◽  
Infected Cells ◽  
Hiv 1 ◽  
Over Time

AbstractHIV-1 replication commences inside the cone-shaped viral capsid, but timing, localization and mechanism of uncoating are under debate. We adapted a strategy to visualize individual reverse-transcribed HIV-1 cDNA molecules and their association with viral and cellular proteins using fluorescence and correlative-light-and-electron-microscopy (CLEM). We specifically detected HIV-1 cDNA inside nuclei, but not in the cytoplasm. Nuclear cDNA initially co-localized with a fluorescent integrase fusion (IN-FP) and the viral CA (capsid) protein, but cDNA-punctae separated from IN-FP/CA over time. This phenotype was conserved in primary HIV-1 target cells, with nuclear HIV-1 complexes exhibiting strong CA-signals in all cell types. CLEM revealed cone-shaped HIV-1 capsid-like structures and apparently broken capsid-remnants at the position of IN-FP signals and elongated chromatin-like structures in the position of viral cDNA punctae lacking IN-FP. Our data argue for nuclear uncoating by physical disruption rather than cooperative disassembly of the CA-lattice, followed by physical separation from the pre-integration complex.

scholarly journals Antibody-Dependent Cellular Cytotoxicity-Competent Antibodies against HIV-1-Infected Cells in Plasma from HIV-Infected Subjects

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Franck P. Dupuy ◽  
Sanket Kant ◽  
Alexandre Barbé ◽  
Jean-Pierre Routy ◽  
Julie Bruneau ◽  
...  
Keyword(s):  
Hiv Infection ◽  
Cell Types ◽  
Target Cells ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Adcc Assay ◽  
Cd4 Binding Site ◽  
Infected Cells ◽  
Bystander Cells ◽  
Hiv 1

ABSTRACT Measuring Envelope (Env)-specific antibody (Ab)-dependent cellular cytotoxicity (ADCC)-competent Abs in HIV+ plasma is challenging because Env displays distinctive epitopes when present in a native closed trimeric conformation on infected cells or in a CD4-bound conformation on uninfected bystander cells. We developed an ADCC model which distinguishes Env-specific ADCC-competent Abs based on their capacity to eliminate infected, bystander, or Env rgp120-coated cells as a surrogate for shed gp120 on bystander cells. A panel of monoclonal Abs (MAbs), used to opsonize these target cells, showed that infected cells were preferentially recognized/eliminated by MAbs to CD4 binding site, V3 loop, and viral spike epitopes whereas bystander/coated cells were preferentially recognized/eliminated by Abs to CD4-induced (CD4i) epitopes. In HIV-positive (HIV+) plasma, Env-specific Abs recognized and supported ADCC of infected cells, though a majority were directed toward CD4i epitopes on bystander cells. For ADCC activity to be effective in HIV control, ADCC-competent Abs need to target genuinely infected cells. IMPORTANCE HIV Env-specific nonneutralizing Abs (NnAbs) able to mediate ADCC have been implicated in protection from HIV infection. However, Env-specific NnAbs have the capacity to support ADCC of both HIV-infected and HIV-uninfected bystander cells, potentially leading to misinterpretations when the assay used to measure ADCC does not distinguish between the two target cell types present in HIV cultures. Using a novel ADCC assay, which simultaneously quantifies the killing activity of Env-specific Abs on both infected and uninfected bystander cells, we observed that only a minority of Env-specific Abs in HIV+ plasma mediated ADCC of genuinely HIV-infected cells displaying Env in its native closed conformation. This assay can be used for the development of vaccine strategies aimed at eliciting Env-specific Ab responses capable of controlling HIV infection.

scholarly journals Vpr Enhances Tumor Necrosis Factor Production by HIV-1-Infected T Cells

2015 ◽  
Vol 89 (23) ◽  
pp. 12118-12130 ◽  
Author(s):  
Ferdinand Roesch ◽  
Léa Richard ◽  
Réjane Rua ◽  
Françoise Porrot ◽  
Nicoletta Casartelli ◽  
...  
Keyword(s):  
Immune Responses ◽  
Proinflammatory Cytokine ◽  
Tumor Necrosis ◽  
Cellular Proteins ◽  
Accessory Protein ◽  
Infected Cells ◽  
Hiv 1 ◽  
Necrosis Factor ◽  
Stimulation Of

ABSTRACTThe HIV-1 accessory protein Vpr displays different activities potentially impacting viral replication, including the arrest of the cell cycle in the G2phase and the stimulation of apoptosis and DNA damage response pathways. Vpr also modulates cytokine production by infected cells, but this property remains partly characterized. Here, we investigated the effect of Vpr on the production of the proinflammatory cytokine tumor necrosis factor (TNF). We report that Vpr significantly increases TNF secretion by infected lymphocytes.De novoproduction of Vpr is required for this effect. Vpr mutants known to be defective for G2cell cycle arrest induce lower levels of TNF secretion, suggesting a link between these two functions. Silencing experiments and the use of chemical inhibitors further implicated the cellular proteins DDB1 and TAK1 in this activity of Vpr. TNF secreted by HIV-1-infected cells triggers NF-κB activity in bystander cells and allows viral reactivation in a model of latently infected cells. Thus, the stimulation of the proinflammatory pathway by Vpr may impact HIV-1 replicationin vivo.IMPORTANCEThe role of the HIV-1 accessory protein Vpr remains only partially characterized. This protein is important for viral pathogenesis in infected individuals but is dispensable for viral replication in most cell culture systems. Some of the functions described for Vpr remain controversial. In particular, it remains unclear whether Vpr promotes or instead prevents proinflammatory and antiviral immune responses. In this report, we show that Vpr promotes the release of TNF, a proinflammatory cytokine associated with rapid disease progression. Using Vpr mutants or inhibiting selected cellular genes, we show that the cellular proteins DDB1 and TAK1 are involved in the release of TNF by HIV-infected cells. This report provides novel insights into how Vpr manipulates TNF production and helps clarify the role of Vpr in innate immune responses and inflammation.

scholarly journals The versatile role of exosomes in human retroviral infections: from immunopathogenesis to clinical application

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jafar Rezaie ◽  
Cynthia Aslan ◽  
Mahdi Ahmadi ◽  
Naime Majidi Zolbanin ◽  
Fatah Kashanchi ◽  
...  
Keyword(s):  
Current Knowledge ◽  
Biological Fluids ◽  
Recipient Cell ◽  
Leukemia Virus ◽  
Target Cells ◽  
Modern Approach ◽  
Human T Cell ◽  
T Cell Leukemia Virus ◽  
Infected Cells ◽  
Hiv 1

AbstractEukaryotic cells produce extracellular vesicles (EVs) mediating intercellular communication. These vesicles encompass many bio-molecules such as proteins, nucleic acids, and lipids that are transported between cells and regulate pathophysiological actions in the recipient cell. Exosomes originate from multivesicular bodies inside cells and microvesicles shed from the plasma membrane and participate in various pathological conditions. Retroviruses such as Human Immunodeficiency Virus -type 1 (HIV-1) and Human T-cell leukemia virus (HTLV)-1 engage exosomes for spreading and infection. Exosomes from virus-infected cells transfer viral components such as miRNAs and proteins that promote infection and inflammation. Additionally, these exosomes deliver virus receptors to target cells that make them susceptible to virus entry. HIV-1 infected cells release exosomes that contribute to the pathogenesis including neurological disorders and malignancy. Exosomes can also potentially carry out as a modern approach for the development of HIV-1 and HTLV-1 vaccines. Furthermore, as exosomes are present in most biological fluids, they hold the supreme capacity for clinical usage in the early diagnosis and prognosis of viral infection and associated diseases. Our current knowledge of exosomes' role from virus-infected cells may provide an avenue for efficient retroviruses associated with disease prevention. However, the exact mechanism involved in retroviruses infection/ inflammation remains elusive and related exosomes research will shed light on the mechanisms of pathogenesis.

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

2021 ◽  
Author(s):  
Sanela Rankovic ◽  
Akshay Deshpande ◽  
Shimon Harel ◽  
Christopher Aiken ◽  
Itay Rousso
Keyword(s):  
Reverse Transcription ◽  
Viral Genome ◽  
Morphological Changes ◽  
Viral Capsid ◽  
Target Cells ◽  
Viral Core ◽  
Successful Infection ◽  
Capsid Shell ◽  
Hiv 1

AbstractThe 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. Our results suggest that reverse-transcription-induced changes in the capsid progressively remodel the viral core to prime it for temporally accurate uncoating in target cells.

scholarly journals Expression of Nef Downregulates CXCR4, the Major Coreceptor of Human Immunodeficiency Virus, from the Surfaces of Target Cells and Thereby Enhances Resistance to Superinfection

2006 ◽  
Vol 80 (22) ◽  
pp. 11141-11152 ◽  
Author(s):  
Stephanie Venzke ◽  
Nico Michel ◽  
Ina Allespach ◽  
Oliver T. Fackler ◽  
Oliver T. Keppler
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Lymphocytes ◽  
Maximum Level ◽  
Target Cells ◽  
Class I Mhc ◽  
Mhc I ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Stromal Cell Derived Factor ◽  
Hiv 1

ABSTRACT Lentiviral Nef proteins are key factors for pathogenesis and are known to downregulate functionally important molecules, including CD4 and major histocompatibility complex class I (MHC-I), from the surfaces of infected cells. Recently, we demonstrated that Nef reduces cell surface levels of the human immunodeficiency virus type 1 (HIV-1) entry coreceptor CCR5 (N. Michel, I. Allespach, S. Venzke, O. T. Fackler, and O. T. Keppler, Curr. Biol. 15:714-723, 2005). Here, we report that Nef downregulates the second major HIV-1 coreceptor, CXCR4, from the surfaces of HIV-infected primary CD4 T lymphocytes with efficiencies comparable to those of the natural CXCR4 ligand, stromal cell-derived factor-1 alpha. Analysis of a panel of mutants of HIV-1SF2 Nef revealed that the viral protein utilized the same signature motifs for downmodulation of CXCR4 and MHC-I, including the proline-rich motif P73P76P79P82 and the acidic cluster motif E66E67E68E69. Expression of wild-type Nef, but not of specific Nef mutants, resulted in a perinuclear accumulation of the coreceptor. Remarkably, the carboxy terminus of CXCR4, which harbors the classical motifs critical for basal and ligand-induced receptor endocytosis, was dispensable for the Nef-mediated reduction of surface exposure. Functionally, the ability of Nef to simultaneously downmodulate CXCR4 and CD4 correlated with maximum-level protection of Nef-expressing target cells from fusion with cells exposing X4 HIV-1 envelopes. Furthermore, the Nef-mediated downregulation of CXCR4 alone on target T lymphocytes was sufficient to diminish cells' susceptibility to X4 HIV-1 virions at the entry step. The downregulation of chemokine coreceptors is a conserved activity of Nef to modulate infected cells, an important functional consequence of which is an enhanced resistance to HIV superinfection.

scholarly journals Selective Decay of Intact HIV-1 Proviral DNA on Antiretroviral Therapy

2020 ◽  
Author(s):  
Rajesh T Gandhi ◽  
Joshua C Cyktor ◽  
Ronald J Bosch ◽  
Hanna Mar ◽  
Gregory M Laird ◽  
...  
Keyword(s):  
Antiretroviral Therapy ◽  
Half Life ◽  
Plasma Viremia ◽  
Art Initiation ◽  
Markers Of Inflammation ◽  
Infected Cells ◽  
Selective Decay ◽  
Hiv 1 ◽  
Over Time ◽  
Residual Plasma Viremia

Abstract Background HIV-1 proviruses persist in people on antiretroviral therapy (ART) but most are defective and do not constitute a replication-competent reservoir. The decay of infected cells carrying intact compared with defective HIV-1 proviruses has not been well defined in people on ART. Methods We separately quantified intact and defective proviruses, residual plasma viremia, and markers of inflammation and activation in people on long-term ART. Results Among 40 participants tested longitudinally from a median of 7.1 years to 12 years after ART initiation, intact provirus levels declined significantly over time (median half-life, 7.1 years; 95% confidence interval [CI], 3.9–18), whereas defective provirus levels did not decrease. The median half-life of total HIV-1 DNA was 41.6 years (95% CI, 13.6–75). The proportion of all proviruses that were intact diminished over time on ART, from about 10% at the first on-ART time point to about 5% at the last. Intact provirus levels on ART correlated with total HIV-1 DNA and residual plasma viremia, but there was no evidence for associations between intact provirus levels and inflammation or immune activation. Conclusions Cells containing intact, replication-competent proviruses are selectively lost during suppressive ART. Defining the mechanisms involved should inform strategies to accelerate HIV-1 reservoir depletion.

scholarly journals Flow Cytometry Analysis of HIV-1 Env Conformations at the Surface of Infected Cells and Virions: Role of Nef, CD4, and SERINC5

2019 ◽  
Vol 94 (6) ◽  
Author(s):  
Isabelle Staropoli ◽  
Jérémy Dufloo ◽  
Anaïs Ducher ◽  
Pierre-Henri Commere ◽  
Anna Sartori-Rupp ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Cellular Proteins ◽  
Viral Infectivity ◽  
Viral Particles ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Env Protein ◽  
The Impact ◽  
Hiv 1

ABSTRACT The HIV-1 Env protein is exposed at the surface of virions and infected cells. Env fluctuates between different closed and open structural states and these conformations influence both viral infectivity and sensitivity to antibody binding and neutralization. We established a flow virometry assay to visualize Env proteins at the surface of human immunodeficiency virus type 1 (HIV-1) virions. The assay is performed on ultracentrifuged fluorescent viral particles that are stained with a panel of broadly neutralizing antibodies (bNAbs) and nonneutralizing antibodies (nnAbs) that probe different epitopes of Env. We used this assay to compare Env at the surface of producer cells and viral particles and to analyze the effect of Nef, CD4, and SERINC5 on Env accessibility to antibodies. We studied the laboratory-adapted strain NL4-3 and two transmitted/founder viruses, THRO and CH058. We confirm that antibody accessibility varies between viral strains and show that Nef, CD4, and SERINC5 additively impact Env conformations. We further demonstrate that the Env accessibility profile on virions is globally similar to that observed on HIV-1-infected cells, with some noticeable differences. For instance, nnAbs bind to virions more efficiently than to producer cells, likely reflecting changes in Env conformational states on mature viral particles. This test complements other techniques and provides a convenient and simple tool for quantifying and probing the structure of Env at the virion surface and to analyze the impact of viral and cellular proteins on these parameters. IMPORTANCE HIV-1 Env conformation is one of the key parameters determining viral infectivity. The flow virometry-based assay developed in this study allows for the characterization of proteins incorporated in HIV-1 particles. We studied the conformation of HIV-1 Env and the impact that the viral protein Nef and the cellular proteins CD4 and SERINC5 have on Env accessibility to antibodies. Our assay permitted us to highlight some noticeable differences in the conformation of Env between producer cells and viral particles. It contributes to a better understanding of the actual composition of HIV-1 particles.

scholarly journals HIV-1 Vpr up-regulates expression of ligands for the activating NKG2D receptor and promotes NK cell–mediated killing

Blood ◽  
2010 ◽  
Vol 115 (7) ◽  
pp. 1354-1363 ◽  
Author(s):  
Jonathan Richard ◽  
Sardar Sindhu ◽  
Tram N. Q. Pham ◽  
Jean-Philippe Belzile ◽  
Éric A. Cohen
Keyword(s):  
Dna Damage ◽  
Cell Surface ◽  
Nk Cell ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Target Cells ◽  
Nkg2d Ligands ◽  
Infected Cells ◽  
Nkg2d Receptor ◽  
Hiv 1

AbstractHIV up-regulates cell-surface expression of specific ligands for the activating NKG2D receptor, including ULBP-1, -2, and -3, but not MICA or MICB, in infected cells both in vitro and in vivo. However, the viral factor(s) involved in NKG2D ligand expression still remains undefined. HIV-1 Vpr activates the DNA damage/stress-sensing ATR kinase and promotes G2 cell-cycle arrest, conditions known to up-regulate NKG2D ligands. We report here that HIV-1 selectively induces cell-surface expression of ULBP-2 in primary CD4+ T lymphocytes by a process that is Vpr dependent. Importantly, Vpr enhanced the susceptibility of HIV-1–infected cells to NK cell–mediated killing. Strikingly, Vpr alone was sufficient to up-regulate expression of all NKG2D ligands and thus promoted efficient NKG2D-dependent NK cell–mediated killing. Delivery of virion-associated Vpr via defective HIV-1 particles induced analogous biologic effects in noninfected target cells, suggesting that Vpr may act similarly beyond infected cells. All these activities relied on Vpr ability to activate the ATR-mediated DNA damage/stress checkpoint. Overall, these results indicate that Vpr is a key determinant responsible for HIV-1–induced up-regulation of NKG2D ligands and further suggest an immunomodulatory role for Vpr that may not only contribute to HIV-1–induced CD4+ T-lymphocyte depletion but may also take part in HIV-1–induced NK-cell dysfunction.

scholarly journals How Many Human Immunodeficiency Virus Type 1-Infected Target Cells Can a Cytotoxic T-Lymphocyte Kill?

2005 ◽  
Vol 79 (21) ◽  
pp. 13579-13586 ◽  
Author(s):  
W. David Wick ◽  
Otto O. Yang ◽  
Lawrence Corey ◽  
Steven G. Self
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Target Cells ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Hiv 1

ABSTRACT The antiviral role of CD8+ cytotoxic T lymphocytes (CTLs) in human immunodeficiency virus type 1 (HIV-1) infection is poorly understood. Specifically, the degree to which CTLs reduce viral replication by killing HIV-1-infected cells in vivo is not known. Here we employ mathematical models of the infection process and CTL action to estimate the rate that CTLs can kill HIV-1-infected cells from in vitro and in vivo data. Our estimates, which are surprisingly consistent considering the disparities between the two experimental systems, demonstrate that on average CTLs can kill from 0.7 to 3 infected target cells per day, with the variability in this figure due to epitope specificity or other factors. These results are compatible with the observed decline in viremia after primary infection being primarily a consequence of CTL activity and have interesting implications for vaccine design.

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