Biology subjects

Planelles, V.

Publications and source records attributed to Planelles, V..

10 recordsLinked to original sources

bioRxiv2023

Reduced Monocyte Proportions and Responsiveness in Convalescent COVID-19 Patients

The clinical manifestations of acute severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection and COVID-19 suggest a dysregulation of the host immune response that leads to inflammation, thrombosis, and organ dysfunction. It is less clear whether these dysregulated processes persist during the convalescent phase of disease or during long COVID. We investigated the effects of SARS-CoV-2 infection on the proportions of classical, intermediate, and non-classical monocytes, their activation status, and their functional properties in convalescent COVID-19 patients and uninfected control subjects. We found that the percentage of total monocytes was decreased in convalescent COVID-19 patients compared to uninfected controls. This was due to decreased intermediate and non-classical monocytes. Classical monocytes from convalescent COVID-19 patients demonstrated a decrease in activation markers, such as CD56, in response to stimulation with bacterial lipopolysaccharide (LPS). In addition, classical monocytes from convalescent COVID-19 patients showed decreased expression of CD142 (tissue factor), which can initiate the extrinsic coagulation cascade, in response to LPS stimulation. Finally, we found that monocytes from convalescent COVID-19 patients produced less TNF- and IL-6 in response to LPS stimulation, than those from uninfected controls. In conclusion, SARS-CoV-2 infection exhibits a clear effect on the relative proportions of monocyte subsets, the activation status of classical monocytes, and proinflammatory cytokine production that persists during the convalescent phase of disease.

immunology↗

bioRxiv2021

ADAP1 promotes latent HIV-1 reactivation by selectively tuning a T cell signaling-transcriptional axis

Immune stimulation fuels cell signaling-transcriptional programs inducing biological responses to eliminate virus-infected cells. Yet, retroviruses that integrate into host cell chromatin, such as HIV-1, co-opt these programs to switch between latent and reactivated states; however, the regulatory mechanisms are still unfolding. Here, we implemented a functional screen leveraging HIV-1s dependence on CD4+ T cell signaling-transcriptional programs and discovered ADAP1 is an undescribed modulator of HIV-1 proviral fate. Specifically, we report ADAP1 (ArfGAP with dual PH domain-containing protein 1), a previously thought neuronal-restricted factor, is an amplifier of select T cell signaling programs. Using complementary biochemical and cellular assays, we demonstrate ADAP1 inducibly interacts with the immune signalosome to directly stimulate KRAS GTPase activity thereby augmenting T cell signaling through targeted activation of the ERK-AP-1 axis. Single cell transcriptomics analysis revealed loss of ADAP1 function blunts gene programs upon T cell stimulation consequently dampening latent HIV-1 reactivation. Our combined experimental approach defines ADAP1 as an unexpected tuner of T cell programs co-opted by HIV-1 for latency escape.

microbiology↗

bioRxiv2021

HIV-1 provirus transcription and translation in macrophages differs from macrophages containing pre-integrated cDNA complexes and requires E2F transcriptional programs

HIV-1 cDNA pre-integration complexes have been shown to persist for weeks in macrophages and to be transcriptionally active. Early and late gene transcripts are produced, along with some viral proteins, yet whole virus is not. While previous work has focused on the transcription and translation of HIV-1 genes; our understanding of cellular milieu that accompanies viral production is incomplete. We have used an in vitro system to model HIV-1 infection of macrophages, and single cell RNA sequencing (scRNA-seq) to compare the transcriptomes of uninfected cells, cells harboring pre-integration HIV-1 complexes (PIC) and those containing integrated provirus and actively making late HIV proteins. These are also compared to control cells, not exposed to virus. Several observations provide new perspective on the effects of HIV-1 transcription from pre-integrated cDNA versus from integrated provirus. First, HIV-1 transcript levels do not necessarily correlate with virus production, cells harboring PIC cDNA have transcript loads comparable to cells transcribing from provirus and making p24, mCherry, and vpu proteins. Second, all HIV-1 transcripts are easily detectable in abundance from PIC cDNA transcription, as is the case with cells transcribing from provirus, although the frequency of PIC cells with detectable gag-pol, tat, env, and nef transcripts is higher than the corresponding frequencies observed for "Provirus cells". Third, the background transcriptomes of cells harboring pre- integrated HIV-1 cDNA are not otherwise detectably altered from cells not containing any HIV- 1 transcript. Fourth, integration and production of p24, mCherry, and Vpu proteins is accompanied by a switch from transcriptomes characterized by NFkB and AP-1 promoted transcription to a transcriptome characterized by E2F family transcription products. While some of these observations may seem heretical, single cell analysis provides a more nuanced understanding of PIC cDNA transcription and the transcriptomic changes that support HIV-1 protein production from integrated provirus. Author SummarySingle cell analysis is able to distinguish between HIV-1 infected macrophage cells that are transcribing pre-integrated HIV-1 cDNA and those transcribing HIV-1 provirus. Only cells transcribing HIV-1 provirus are making p24, marker mCherry and Vpu proteins, which corresponds with a change in the host cells background transcriptome from one expressing viral restriction and immunological response genes to one that is expressing genes associated with cell replication and oxidative phosphorylation.

microbiology↗

bioRxiv2021

HIV-1 Vpr Induces Degradation of Nucleolar Protein CCDC137 as a Consequence of Cell Cycle Arrest

Expression of HIV-1 accessory proteins Vif and Vpr results in G2/M cell cycle arrest by hijacking the host ubiquitin-proteasome system. Vif directs cell cycle arrest by targeting protein phosphatase 2, regulatory subunit B alpha (PP2AB56) for degradation. However, the ubiquitination target(s) of Vpr that is directly responsible for G2/M arrest has remained elusive. Recently, Vpr directed degradation of nucleolar protein coiled-coil domain containing 137 (CCDC137), also known as retinoic acid resistance factor (RaRF), has been implicated as the proximal event leading to G2/M cell cycle arrest. In this study we aimed to further investigate this finding. We confirm that CCDC137 is targeted for degradation in the presence of Vpr with a requirement for the CUL4ADDB1.DCAF1 E3 ligase complex. However, degradation of CCDC137 is a general consequence, rather than a trigger, of G2/M arrest. Thus, whether induced by Vpr expression or pharmacologically via CDK1 inhibition, G2/M blockade results in degradation of CCDC137. Furthermore, siRNA-mediated depletion of CCDC137 failed to induce G2/M arrest.

microbiology↗

bioRxiv2020

Neutralization Assay with SARS-CoV-1 and SARS-CoV-2 Spike Pseudotyped Murine Leukemia Virions

Antibody neutralization is an important prognostic factor in many viral diseases. To easily and rapidly measure titers of neutralizing antibodies in serum or plasma, we developed pseudovirion particles composed of the spike glycoprotein of SARS-CoV-2 incorporated onto murine leukemia virus capsids and a modified minimal MLV genome encoding firefly luciferase. These pseudovirions provide a practical means of assessing immune responses under laboratory conditions consistent with biocontainment level 2.

microbiology↗

bioRxiv2020

Vpr R36W and R77Q Mutations Alter HIV-1 Replication and Cytotoxicity in T Lymphocytes

Chronic immune inflammation (CII) is a characteristic symptom of HIV-1 infection that contributes to acquired immunodeficiency syndrome (AIDS) progression in infected patients. Distinct AIDS development rates have shown that there are Rapid Progressor (RP) and Long-Term Non-Progressor (LTNP) patients, but the circumstances governing these differences in disease progression are poorly understood. Mutations in the Viral Protein R (Vpr) have been suggested to have a direct impact on disease progression. Studies have shown that Vpr interacts with both host and viral factors; these interactions affect cellular activities such as cell cycle progression and enhancement of apoptosis. The Vpr mutants R36W and R77Q have been associated with RP and LTNP phenotypes, respectively; however, these findings are still controversial. This study sheds light on the effects that Vpr mutations have in the context of HIV-1 infection of the HUT78 T cell line, using replication-competent CXCR4-tropic virus strains. Our results show a replication enhancement of the R36W mutant (increased viral load and percentage of p24+ cells) accompanied by increased cytotoxicity. Interestingly, the R77Q mutant showed a unique enhancement of apoptosis (measured by Annexin V and TUNEL staining) and G2 cell cycle arrest; these effects were not seen with WT or R36W viruses. Since necrosis is associated with the release of pro-inflammatory factors, the R36 mutation could lead to more robust CII and the RP phenotype. Conversely, the R77Q mutation leads to apoptosis, potentially avoiding CII and leading to a LTNP phenotype. Thus, Vpr mutations may impact HIV-1 related progression to AIDS. ImportanceThe vpr gene is thought to be an important virulence factor in Human Immunodeficiency Virus type 1 (HIV-1). vpr polymorphisms have been associated with different rates of acquired immunodeficiency syndrome (AIDS) progression. However, there is controversy about the cytopathic and virulence phenotypes of Vpr mutants, with contradictory conclusions about the same mutants. Here, we examine the replication capacity, apoptosis induction, and G2 cell cycle arrest phenotypes of three vpr mutants compared to wild-type HIV-1. One mutant associated with rapid AIDS progression replicated more efficiently and killed cells more rapidly than wild-type HIV-1. Another mutant associated with slow AIDS progression triggered apoptosis more efficiently than wild-type HIV-1. These results shed additional light on the role of vpr polymorphisms in T cell killing by HIV-1 and may help to explain the role of Vpr in different rates of AIDS progression.

microbiology↗

bioRxiv2020

Interactions with Commensal and Pathogenic Bacteria Induce HIV-1 Latency in Macrophages through Altered Transcription Factor Recruitment to the LTR

Macrophages are infected by HIV-1 in vivo and contribute to both viral spread and pathogenesis. Recent human and animal studies suggest that HIV-1-infected macrophages serve as a reservoir that contributes to HIV-1 persistence during anti-retroviral therapy. The ability of macrophages to serve as persistent viral reservoirs is likely influenced by the local tissue microenvironment, including interactions with pathogenic and commensal microbes. Here we show that the sexually transmitted pathogen Neisseria gonorrhoeae (GC) and the gut-associated microbe Escherichia coli (E. coli), which encode ligands for both Toll-like receptor 2 (TLR2) and TLR4, repressed HIV-1 replication in macrophages and thereby induced a state reminiscent of viral latency. This repression was mediated by signaling through TLR4 and the adaptor protein TRIF and was associated with increased production of type I interferons. Inhibiting TLR4 signaling, blocking type 1 interferon, or knocking-down TRIF reversed LPS- and GC-mediated repression of HIV-1. Finally, the repression of HIV-1 in macrophages was associated with the recruitment of interferon regulatory factor 8 (IRF8) to the interferon stimulated response element (ISRE) downstream of the 5' HIV-1 long terminal repeat (LTR). Our data indicate that IRF8 is responsible for repression of HIV-1 replication in macrophages in response to TRIF-dependent signaling during GC and E. coli co-infection. These findings highlight the potential role of macrophages as HIV-1 reservoirs as well as the role of the tissue microenvironment and co-infections as modulators of HIV-1 persistence. IMPORTANCEThe major barrier toward the eradication of HIV-1 infection is the presence of a small reservoir of latently infected cells, which include CD4+ T cells and macrophages that escape immune-mediated clearance and the effects of anti-retroviral therapy. There remain crucial gaps in our understanding of the molecular mechanisms that lead to transcriptionally silent or latent HIV-1 infection of macrophages. The significance of our research is in identifying microenvironmental factors, such as commensal and pathogenic microbes, that can contribute to the establishment and maintenance of latent HIV-1 infection in macrophages. It is hoped that identifying key processes contributing to HIV-1 persistence in macrophages may ultimately lead to novel therapeutics to eliminate latent HIV-1 reservoirs in vivo.

microbiology↗

bioRxiv2019

Stable integrant-specific differences in bimodal HIV-1 expression patterns revealed by high-throughput analysis

HIV-1 gene expression is regulated by host and viral factors that interact with viral motifs and is influenced by proviral integration sites. Here, expression variation among integrants was followed for hundreds of individual proviral clones within polyclonal populations throughout successive rounds of virus and cultured cell replication. Initial findings in immortalized cells were validated using CD4+ cells from donor blood. Tracking clonal behavior by proviral \"zip codes\" indicated that mutational inactivation during reverse transcription was rare, while clonal expansion and proviral expression states varied widely. By sorting for provirus expression using a GFP reporter in the nef open reading frame, distinct clone-specific variation in on/off proportions were observed that spanned three orders of magnitude. Tracking GFP phenotypes over time revealed that as cells divided, their progeny alternated between HIV transcriptional activity and non-activity. Despite these phenotypic oscillations, the overall GFP+ population within each clone was remarkably stable, with clones maintaining clone-specific equilibrium mixtures of GFP+ and GFP-cells. Integration sites were analyzed for correlations between genomic features and the epigenetic phenomena described here. Integrants inserted in genes sense orientation were more frequently found to be GFP negative than those in the antisense orientation, and clones with high GFP+ proportions were more distal to repressive H3K9me3 peaks than low GFP+ clones. Clones with low frequencies of GFP positivity appeared to expand more rapidly than clones for which most cells were GFP+, even though the tested proviruses were Vpr-. Thus, much of the increase in the GFP-population in these polyclonal pools over time reflected differential clonal expansion. Together, these results underscore the temporal and quantitative variability in HIV-1 gene expression among proviral clones that are conferred in the absence of metabolic or cell-type dependent variability, and shed light on cell-intrinsic layers of regulation that affect HIV-1 population dynamics.\n\nSummaryVery few HIV-1 infected cells persist in patients for more than a couple days, but those that do pose life-long health risks. Strategies designed to eliminate these cells have been based on assumptions about what viral properties allow infected cell survival. However, such approaches for HIV-1 eradication have not yet shown therapeutic promise, possibly because much of the research underlying assumptions about virus persistence has been focused on a limited number of infected cell types, the averaged behavior of cells in diverse populations, or snapshot views. Here, we developed a high-throughput approach to study hundreds of distinct HIV-1 infected cells and their progeny over time in an unbiased way. This revealed that each virus established its own pattern of gene expression that, upon infected cell division, was stably transmitted to all progeny cells. Expression patterns consisted of alternating waves of activity and inactivity, with the extent of activity differing among infected cell families over a 1000-fold range. The dynamics and variability among infected cells and within complex populations that the work here revealed has not previously been evident, and may help establish more accurate correlates of persistent HIV-1 infection.

microbiology↗

bioRxiv2018

Transcriptional Circuit Fragility Influences HIV Proviral Fate

Transcriptional circuit architectures can be evolutionarily selected to precisely dictate a given response. Unlike these cellular systems, HIV is regulated through a complex circuit composed of two successive phases (host and viral), which create a positive feedback loop facilitating viral replication. However, it has long remained unclear whether both phases operate identically and to what extent the host phase influences the entire circuit. Here we report that while the host phase is regulated by a checkpoint whereby KAP1 mediates transcription activation, the virus evolved a minimalist system bypassing KAP1. Given the complex circuits architecture, cell-to-cell KAP1 fluctuations impart heterogeneity in the host transcriptional responses thus affecting the feedback loop. Mathematical modeling of a complete circuit reveals how these oscillations ultimately influence homogeneous reactivation potential of a latent virus. Thus, while HIV drives molecular innovation to fuel robust gene activation, it experiences transcriptional fragility thereby influencing viral fate and cure efforts.\n\nIn BriefHIV evolved a minimalist but robust transcriptional circuit bypassing host regulatory checkpoints; however, the fragility of the circuit in the host phase (which primes HIV for activation) largely affects proviral transcription and fate.\n\nHighlightsO_LIThe host and viral phases of the HIV transcriptional circuit have different functional requirements\nC_LIO_LIHIV evolved a minimalist program to robustly bypass host cell regulatory checkpoints\nC_LIO_LIA mathematical model reveals that the host phase is subject to transcriptional circuit fragility\nC_LIO_LIHost transcriptional circuit fragility influences the viral feedback and latency reversal potential\nC_LI

molecular biology↗

bioRxiv2017

Histone deacetylase inhibition reduces deleterious cytokine release induced by ingenol stimulation

IntroductionLatency reversal agents (LRAs), such as protein kinase C (PKC) agonists, constitute a promising strategy for exposing and eliminating the HIV-1 latent reservoir. PKC agonists activate NF-{kappa}B and, in turn, induce deleterious pro-inflammatory cytokine production. Adjuvant pharmacological agents, such as ruxolitinib, a JAK inhibitor, and rapamycin, an mTOR inhibitor, have previously been combined with LRAs to reduce deleterious pro-inflammatory cytokine secretion without inhibiting HIV-1 viral reactivation in vitro. Histone deacetylase inhibitors (HDACi) are known to dampen pro-inflammatory cytokine secretion in the context of other diseases and can synergize with other LRAs to bring dormant proviruses out of latency. In this study we investigated whether a broad panel of epigenetic modifiers, including HDACi, could effectively dampen PKC-induced pro-inflammatory cytokine secretion during latency reversal. MethodsWe screened an epigenetic modifier library to identify compounds that reduced intracellular IL-6 production induced by the PKC agonist Ingenol-3,20-dibenzoate. We further tested the most promising epigenetic inhibitor class, HDACi, for their ability to reduce a broad panel of pro-inflammatory cytokines and reactivate latent HIV-1 ex vivo. ResultsWe identified nine epigenetic modulators that reduced PKC-induced intracellular IL-6. In cells from aviremic individuals living with HIV-1, the HDAC1-3 inhibitor, suberohydroxamic acid (SBHA), reduced secretion of pro-inflammatory cytokines TNF-, IL-5, IL-2r, and IL-17 but did not significantly reactivate latent HIV-1 when used in combination with Ingenol-3,20-dibenzoate. ConclusionThe addition of SBHA to Ingenol-3,20-dibenzoate reduces deleterious cytokine production during latency reversal but does not induce significant viral reactivation in aviremic donor PBMCs. The ability of SBHA to reduce PKC-induced pro-inflammatory cytokines when used in combination with Ingenol-3,20-dibenzoate suggests that SBHA can be used to reduced PKC induced pro-inflammatory cytokines but not to achieve latency reversal in the context of HIV-1.

microbiology↗