scholarly journals Mapping of pseudouridine residues on cellular and viral transcripts using a novel antibody-based technique

2021 ◽  
Author(s):  
Cecilia Martínez-Campos ◽  
Kevin Tsai ◽  
David G Courtney ◽  
Hal P Bogerd ◽  
Christopher L Holley ◽  
...  
Keyword(s):  
Innate Immune ◽  
Mapping Technique ◽  
Pseudouridine Synthase ◽  
Antiviral Responses ◽  
Rna Transcripts ◽  
Exogenous Rna ◽  
Cellular Mrnas ◽  
Infected Cells ◽  
Non Coding Rnas ◽  
Hiv 1

Pseudouridine (ψ) is the most common non-canonical ribonucleoside present on mammalian non-coding RNAs (ncRNAs), including rRNAs, tRNAs and snRNAs, where it contributes ~7% of the total uridine level. However, ψ constitutes only ~0.1% of the uridines present on mRNAs and its effect on mRNA function remains unclear. Ψ residues have been shown to inhibit the detection of exogenous RNA transcripts by host innate immune factors, thus raising the possibility that viruses might have subverted the addition of ψ residues to mRNAs by host pseudouridine synthase (PUS) enzymes as a way to inhibit antiviral responses in infected cells. Here, we describe and validate a novel antibody-based ψ mapping technique called photo-crosslinking assisted ψ sequencing (PA-ψ-seq) and use it to map ψ residues on not only multiple cellular RNAs but also on the mRNAs and genomic RNA encoded by HIV-1. We describe several 293T-derived cell lines in which human PUS enzymes previously reported to add ψ residues to human mRNAs, specifically PUS1, PUS7 and TRUB1/PUS4, were inactivated by gene editing. Surprisingly, while this allowed us to assign several sites of ψ addition on cellular mRNAs to each of these three PUS enzymes, the ψ sites present on HIV-1 transcripts remained unaffected. Moreover, loss of PUS1, PUS7 or TRUB1 function did not significantly reduce the level of ψ residues detected on total human mRNA below the ~0.1% level seen in wild type cells, thus implying that the PUS enzyme(s) that adds the bulk of ψ residues to human mRNAs remains to be defined.

scholarly journals COMMD1/Murr1 Reinforces HIV-1 Latent Infection through IκB-α Stabilization

2014 ◽  
Vol 89 (5) ◽  
pp. 2643-2658 ◽  
Author(s):  
Manabu Taura ◽  
Eriko Kudo ◽  
Ryusho Kariya ◽  
Hiroki Goto ◽  
Kouki Matsuda ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Primary Infection ◽  
Latent Infection ◽  
Myeloid Cells ◽  
Innate Immune ◽  
Lymphoid Cells ◽  
Inhibition Mechanism ◽  
Major Barrier ◽  
Infected Cells ◽  
Hiv 1

ABSTRACTThe transcription factor NF-κB is important for HIV-1 transcription initiation in primary HIV-1 infection and reactivation in latently HIV-1-infected cells. However, comparative analysis of the regulation and function of NF-κB in latently HIV-1-infected cells has not been done. Here we show that the expression of IκB-α, an endogenous inhibitor of NF-κB, is enhanced by latent HIV-1 infection via induction of the host-derived factor COMMD1/Murr1 in myeloid cells but not in lymphoid cells by using four sets of latently HIV-1-infected cells and the respective parental cells. IκB-α protein was stabilized by COMMD1, which attenuated NF-κB signaling during Toll-like receptor ligand and tumor necrosis factor alpha treatment and enhanced HIV-1 latency in latently HIV-1-infected cells. Activation of the phosphoinositol 3-kinase (PI3K)–JAK pathway is involved in COMMD1 induction in latently HIV-1-infected cells. Our findings indicate that COMMD1 induction is the NF-κB inhibition mechanism in latently HIV-1-infected cells that contributes to innate immune deficiency and reinforces HIV-1 latency. Thus, COMMD1 might be a double-edged sword that is beneficial in primary infection but not beneficial in latent infection when HIV-1 eradication is considered.IMPORTANCEHIV-1 latency is a major barrier to viral eradication in the era of combination antiretroviral therapy. In this study, we found that COMMD1/Murr1, previously identified as an HIV-1 restriction factor, inhibits the proteasomal degradation of IκB-α by increasing the interaction with IκB-α in latently HIV-1-infected myeloid cells. IκB-α protein was stabilized by COMMD1, which attenuated NF-κB signaling during the innate immune response and enhanced HIV-1 latency in latently HIV-1-infected cells. Activation of the PI3K-JAK pathway is involved in COMMD1 induction in latently HIV-1-infected cells. Thus, the host-derived factor COMMD1 is beneficial in suppressing primary infection but enhances latent infection, indicating that it may be a double-edged sword in HIV-1 eradication.

scholarly journals Serum extracellular vesicle depletion processes affect release and infectivity of HIV-1 in culture

2016 ◽  
Author(s):  
Zhaohao Liao ◽  
Dillon C. Muth ◽  
Erez Eitan ◽  
Meghan Travers ◽  
Elin Lehrmann ◽  
...  
Keyword(s):  
Culture Media ◽  
Virus Production ◽  
Cell Types ◽  
Extracellular Vesicle ◽  
Substantial Effect ◽  
Primary Cells ◽  
Antiviral Responses ◽  
Health And Disease ◽  
Infected Cells ◽  
Hiv 1

ABSTRACTExtracellular vesicles (EVs, including exosomes and microvesicles) are involved in intercellular communication in health and disease and affect processes including immune and antiviral responses. Ultracentrifuged serum is depleted of EVs and, when used in culture media, reduces growth and viability of some cell types. In this study, we examined the effects of serum EV depletion processes on HIV-1 replication in primary cells and cell lines, including two HIV-1 latency models. Increased HIV-1 production was observed in certain EV-depleted conditions, along with cell morphology changes and decreased cell viability. Add-back of ultracentrifuge pellets rescued baseline HIV-1 production. Primary cells appeared to be less sensitive to EV depletion. ACH-2 and U1 latency models produced more HIV-1 under EV-depleted conditions, while virus produced under processed serum conditions was more infectious. Finally, changes in cellular metabolism and gene expression were associated with EV-depleted culture. In conclusion, the EV environment of HIV-1 infected cells has a substantial effect on virus production and infectivity. EV-dependence of cell cultures should be examined carefully along with other experimental variables. However, EVs may not be the only particles depleted by ultracentrifugation or other processes. Effects of EVs may be accompanied by or confused with those of closely associated or physically similar particles.

scholarly journals Activation of Latent HIV-1 T Cell Reservoirs with a Combination of Innate Immune and Epigenetic Regulators

2019 ◽  
Vol 93 (21) ◽  
Author(s):  
Enrico Palermo ◽  
Chiara Acchioni ◽  
Daniele Di Carlo ◽  
Alessandra Zevini ◽  
Michela Muscolini ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Histone Deacetylase ◽  
Innate Immune ◽  
Viral Reservoir ◽  
Hiv Eradication ◽  
Shock And Kill ◽  
Infected Cells ◽  
Hiv 1

ABSTRACT The presence of T cell reservoirs in which human immunodeficiency virus (HIV) establishes latency by integrating into the host genome represents a major obstacle to an HIV cure and has prompted the development of strategies aimed at the eradication of HIV from latently infected cells. The “shock-and-kill” strategy is one of the most pursued approaches to the elimination of viral reservoirs. Although several latency-reversing agents (LRAs) have shown promising reactivation activity, they have failed to eliminate the cellular reservoir. In this study, we evaluated a novel immune system-mediated approach to clearing the HIV reservoir, based on a combination of innate immune stimulation and epigenetic reprogramming. The combination of the STING agonist cGAMP (cyclic GMP-AMP) and the FDA-approved histone deacetylase inhibitor resminostat resulted in a significant increase in HIV proviral reactivation and specific apoptosis in HIV-infected cells in vitro. Reductions in the proportion of HIV-harboring cells and the total amount of HIV DNA were also observed in CD4+ central memory T (TCM) cells, a primary cell model of latency, where resminostat alone or together with cGAMP induced high levels of selective cell death. Finally, high levels of cell-associated HIV RNA were detected ex vivo in peripheral blood mononuclear cells (PBMCs) and CD4+ T cells from individuals on suppressive antiretroviral therapy (ART). Although synergism was not detected in PBMCs with the combination, viral RNA expression was significantly increased in CD4+ T cells. Collectively, these results represent a promising step toward HIV eradication by demonstrating the potential of innate immune activation and epigenetic modulation for reducing the viral reservoir and inducing specific death of HIV-infected cells. IMPORTANCE One of the challenges associated with HIV-1 infection is that despite antiretroviral therapies that reduce HIV-1 loads to undetectable levels, proviral DNA remains dormant in a subpopulation of T lymphocytes. Numerous strategies to clear residual virus by reactivating latent virus and eliminating the reservoir of HIV-1 (so-called “shock-and-kill” strategies) have been proposed. In the present study, we use a combination of small molecules that activate the cGAS-STING antiviral innate immune response (the di-cyclic nucleotide cGAMP) and epigenetic modulators (histone deacetylase inhibitors) that induce reactivation and HIV-infected T cell killing in cell lines, primary T lymphocytes, and patient samples. These studies represent a novel strategy for HIV eradication by reducing the viral reservoir and inducing specific death of HIV-infected cells.

scholarly journals Differential Control of BST2 Restriction and Plasmacytoid Dendritic Cell Antiviral Response by Antagonists Encoded by HIV-1 Group M and O Strains

2016 ◽  
Vol 90 (22) ◽  
pp. 10236-10246 ◽  
Author(s):  
Mariana G. Bego ◽  
Lijun Cong ◽  
Katharina Mack ◽  
Frank Kirchhoff ◽  
Éric A. Cohen
Keyword(s):  
Dendritic Cell ◽  
Mononuclear Cells ◽  
Plasmacytoid Dendritic Cell ◽  
Viral Assembly ◽  
Effective Control ◽  
Type I ◽  
Antiviral Responses ◽  
Viral Spread ◽  
Infected Cells ◽  
Hiv 1

ABSTRACT BST2/tetherin is a type I interferon (IFN-I)-stimulated host factor that restricts the release of HIV-1 by entrapping budding virions at the cell surface. This membrane-associated protein can also engage and activate the plasmacytoid dendritic cell (pDC)-specific immunoglobulin-like transcript 7 (ILT7) inhibitory receptor to downregulate the IFN-I response by pDCs. Pandemic HIV-1 group M uses Vpu (M-Vpu) to counteract the two BST2 isoforms (long and short) that are expressed in human cells. M-Vpu efficiently downregulates surface long BST2, while it displaces short BST2 molecules away from viral assembly sites. We recently found that this attribute is used by M-Vpu to activate the BST2/ILT7-dependent negative-feedback pathway and to suppress pDC IFN-I responses during sensing of infected cells. However, whether this property is conserved in endemic HIV-1 group O, which has evolved Nef (O-Nef) to counteract specifically the long BST2 isoform, remains unknown. In the present study, we validated that O-Nefs have the capacity to downregulate surface BST2 and enhance HIV-1 particle release although less efficiently than M-Vpu. In contrast to M-Vpu, O-Nef did not efficiently enhance viral spread in T cell culture or displace short BST2 from viral assembly sites to prevent its occlusion by tethered HIV-1 particles. Consequently, O-Nef impairs the ability of BST2 to activate negative ILT7 signaling to suppress the IFN-I response by pDC-containing peripheral blood mononuclear cells (PBMCs) during sensing of infected cells. These distinctive features of BST2 counteraction by O-Nefs may in part explain the limited spread of HIV-1 group O in the human population. IMPORTANCE The geographical distributions and prevalences of different HIV-1 groups show large variations. Understanding drivers of distinctive viral spread may aid in the development of therapeutic strategies for controlling the spread of HIV-1 pandemic strains. The differential spread of HIV-1 groups appears to be linked to their capacities to antagonize the long and short isoforms of the BST2 restriction factor. We found that the endemic HIV-1 group O-encoded BST2 antagonist Nef is unable to counteract the restriction mediated by short BST2, a condition that impairs its ability to activate ILT7 and suppress pDC antiviral responses. This is in contrast to the pandemic HIV-1 group M-specified BST2 countermeasure Vpu, which displays a diverse array of mechanisms to counteract short and long BST2 isoforms, an attribute that allows the effective control of pDC antiviral responses. These findings may help explain the limited spread of HIV-1 group O as well as the continued predominance of HIV-1 group M throughout the world.

scholarly journals Pyrosequencing of small non-coding RNAs in HIV-1 infected cells: evidence for the processing of a viral-cellular double-stranded RNA hybrid

2009 ◽  
Vol 37 (19) ◽  
pp. 6575-6586 ◽  
Author(s):  
Man Lung Yeung ◽  
Yamina Bennasser ◽  
Koichi Watashi ◽  
Shu-Yun Le ◽  
Laurent Houzet ◽  
...  
Keyword(s):  
Double Stranded Rna ◽  
Infected Cells ◽  
Non Coding Rnas ◽  
Hiv 1

scholarly journals An Omics Approach to Extracellular Vesicles from HIV-1 Infected Cells

Cells ◽  
2019 ◽  
Vol 8 (8) ◽  
pp. 787 ◽  
Author(s):  
Robert A. Barclay ◽  
Pooja Khatkar ◽  
Gifty Mensah ◽  
Catherine DeMarino ◽  
Jeffery S. C. Chu ◽  
...  
Keyword(s):  
Extracellular Vesicle ◽  
Antiretroviral Drugs ◽  
Nucleic Acid Content ◽  
Cellular Mirnas ◽  
Rna Transcripts ◽  
Transcription Inhibitors ◽  
Infected Cells ◽  
Acquired Immunodeficiency ◽  
Immunodeficiency Syndrome ◽  
Hiv 1

Human Immunodeficiency Virus-1 (HIV-1) is the causative agent of Acquired Immunodeficiency Syndrome (AIDS), infecting nearly 37 million people worldwide. Currently, there is no definitive cure, mainly due to HIV-1′s ability to enact latency. Our previous work has shown that exosomes, a small extracellular vesicle, from uninfected cells can activate HIV-1 in latent cells, leading to increased mostly short and some long HIV-1 RNA transcripts. This is consistent with the notion that none of the FDA-approved antiretroviral drugs used today in the clinic are transcription inhibitors. Furthermore, these HIV-1 transcripts can be packaged into exosomes and released from the infected cell. Here, we examined the differences in protein and nucleic acid content between exosomes from uninfected and HIV-1-infected cells. We found increased cyclin-dependent kinases, among other kinases, in exosomes from infected T-cells while other kinases were present in exosomes from infected monocytes. Additionally, we found a series of short antisense HIV-1 RNA from the 3′ LTR that appears heavily mutated in exosomes from HIV-1-infected cells along with the presence of cellular noncoding RNAs and cellular miRNAs. Both physical and functional validations were performed on some of the key findings. Collectively, our data indicate distinct differences in protein and RNA content between exosomes from uninfected and HIV-1-infected cells, which can lead to different functional outcomes in recipient cells.

scholarly journals Innate Immune Activity Correlates with CD4 T Cell-Associated HIV-1 DNA Decline during Latency-Reversing Treatment with Panobinostat

2015 ◽  
Vol 89 (20) ◽  
pp. 10176-10189 ◽  
Author(s):  
Rikke Olesen ◽  
Selena Vigano ◽  
Thomas A. Rasmussen ◽  
Ole S. Søgaard ◽  
Zhengyu Ouyang ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Nk Cells ◽  
Expression Patterns ◽  
Innate Immune ◽  
Viral Reservoir ◽  
Immune Activity ◽  
Interferon Stimulated Genes ◽  
Infected Cells ◽  
Hiv 1 ◽  
Reversing Agents

ABSTRACTThe pharmaceutical reactivation of dormant HIV-1 proviruses by histone deacetylase inhibitors (HDACi) represents a possible strategy to reduce the reservoir of HIV-1-infected cells in individuals treated with suppressive combination antiretroviral therapy (cART). However, the effects of such latency-reversing agents on the viral reservoir size are likely to be influenced by host immune responses. Here, we analyzed the immune factors associated with changes in proviral HIV-1 DNA levels during treatment with the potent HDACi panobinostat in a human clinical trial involving 15 cART-treated HIV-1-infected patients. We observed that the magnitude, breadth, and cytokine secretion profile of HIV-1-specific CD8 T cell responses were unrelated to changes in HIV-1 DNA levels in CD4 T cells during panobinostat treatment. In contrast, the proportions of CD3−CD56+total NK cells and CD16+CD56dimNK cells were inversely correlated with HIV-1 DNA levels throughout the study, and changes in HIV-1 DNA levels during panobinostat treatment were negatively associated with the corresponding changes in CD69+NK cells. Decreasing levels of HIV-1 DNA during latency-reversing treatment were also related to the proportions of plasmacytoid dendritic cells, to distinct expression patterns of interferon-stimulated genes, and to the expression of theIL28BCC genotype. Together, these data suggest that innate immune activity can critically modulate the effects of latency-reversing agents on the viral reservoir and may represent a target for future immunotherapeutic interventions in HIV-1 eradication studies.IMPORTANCECurrently available antiretroviral drugs are highly effective in suppressing HIV-1 replication, but the virus persists, despite treatment, in a latent form that does not actively express HIV-1 gene products. One approach to eliminate these cells, colloquially termed the “shock-and-kill” strategy, focuses on the use of latency-reversing agents that induce active viral gene expression in latently infected cells, followed by immune-mediated killing. Panobinostat, a histone deacetylase inhibitor, demonstrated potent activities in reversing HIV-1 latency in a recent pilot clinical trial and reduced HIV-1 DNA levels in a subset of patients. Interestingly, we found that innate immune factors, such as natural killer cells, plasmacytoid dendritic cells, and the expression patterns of interferon-stimulated genes, were most closely linked to a decline in the HIV-1 DNA level during treatment with panobinostat. These data suggest that innate immune activity may play an important role in reducing the residual reservoir of HIV-1-infected cells.

scholarly journals Human Immunodeficiency Virus Type 1 Mediates Global Disruption of Innate Antiviral Signaling and Immune Defenses within Infected Cells

2009 ◽  
Vol 83 (20) ◽  
pp. 10395-10405 ◽  
Author(s):  
Brian P. Doehle ◽  
Florian Hladik ◽  
John P. McNevin ◽  
M. Juliana McElrath ◽  
Michael Gale
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Virus Infection ◽  
Innate Immune ◽  
Immune Signaling ◽  
Innate Immune Signaling ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Hiv 1

ABSTRACT Interferon regulatory factor 3 (IRF-3) is essential for innate intracellular immune defenses that limit virus replication, but these defenses fail to suppress human immunodeficiency virus (HIV) infection, which can ultimately associate with opportunistic coinfections and the progression to AIDS. Here, we examined antiviral defenses in CD4+ cells during virus infection and coinfection, revealing that HIV type 1 (HIV-1) directs a global disruption of innate immune signaling and supports a coinfection model through suppression of IRF-3. T cells responded to paramyxovirus infection to activate IRF-3 and interferon-stimulated gene expression, but they failed to mount a response against HIV-1. The lack of response associated with a marked depletion of IRF-3 but not IRF-7 in HIV-1-infected cells, which supported robust viral replication, whereas ectopic expression of active IRF-3 suppressed HIV-1 infection. IRF-3 depletion was dependent on a productive HIV-1 replication cycle and caused the specific disruption of Toll-like receptor and RIG-I-like receptor innate immune signaling that rendered cells permissive to secondary virus infection. IRF-3 levels were reduced in vivo within CD4+ T cells from patients with acute HIV-1 infection but not from long-term nonprogressors. Our results indicate that viral suppression of IRF-3 promotes HIV-1 infection by disrupting IRF-3-dependent signaling pathways and innate antiviral defenses of the host cell. IRF-3 may direct an innate antiviral response that regulates HIV-1 replication and viral set point while governing susceptibility to opportunistic virus coinfections.

scholarly journals SARS-CoV-2 utilizes a multipronged strategy to suppress host protein synthesis

2020 ◽  
Author(s):  
Yaara Finkel ◽  
Avi Gluck ◽  
Roni Winkler ◽  
Aharon Nachshon ◽  
Orel Mizrahi ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Cellular Protein ◽  
Ribosome Profiling ◽  
Innate Immune ◽  
Host Protein ◽  
Immune Genes ◽  
Mrna Pool ◽  
Cellular Mrnas ◽  
Infected Cells ◽  
Innate Immune Genes

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 19 (COVID-19) pandemic. Despite the urgent need, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis and its ability to antagonize innate immune responses. Here, we use RNA-sequencing and ribosome profiling along SARS-CoV-2 infection and comprehensively define the mechanisms that are utilized by SARS-CoV-2 to shutoff cellular protein synthesis. We show SARS-CoV-2 infection leads to a global reduction in translation but that viral transcripts are not preferentially translated. Instead, we reveal that infection leads to accelerated degradation of cytosolic cellular mRNAs which facilitates viral takeover of the mRNA pool in infected cells. Moreover, we show that the translation of transcripts whose expression is induced in response to infection, including innate immune genes, is impaired, implying infection prevents newly transcribed cellular mRNAs from accessing the ribosomes. Overall, our results uncover the multipronged strategy employed by SARS-CoV-2 to commandeer the translation machinery and to suppress host defenses.

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