Publications by authors named "Vicente Planelles"

101 Publications

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

J Virol 2021 Jan 20. Epub 2021 Jan 20.

Department of Microbiology, Boston University School of Medicine, Boston, MA 02118

Macrophages are infected by HIV-1 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 (GC) and the gut-associated microbe (), 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 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. The 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 .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.02141-20DOI Listing
January 2021

Neutralization assay with SARS-CoV-1 and SARS-CoV-2 spike pseudotyped murine leukemia virions.

Virol J 2021 01 4;18(1). Epub 2021 Jan 4.

Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, USA.

Background: Virus neutralization by antibodies 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 murine leukemia virus genome encoding firefly luciferase. This assay design is intended for use in laboratories with biocontainment level 2 and therefore circumvents the need for the biocontainment level 3 that would be required for replication-competent SARS-CoV-2 virus. To validate the pseudovirion assay, we set up comparisons with other available antibody tests including those from Abbott, Euroimmun and Siemens, using archived, known samples.

Results: 11 out of 12 SARS-CoV-2-infected patient serum samples showed neutralizing activity against SARS-CoV-2-spike pseudotyped MLV viruses, with neutralizing titers-50 (NT) that ranged from 1:25 to 1:1,417. Five historical samples from patients hospitalized for severe influenza infection in 2016 tested negative in the neutralization assay (NT < 25). Three serum samples with high neutralizing activity against SARS-CoV-2/MLV pseudoviruses showed no detectable neutralizing activity (NT < 25) against SARS-CoV-1/MLV pseudovirions. We also compared the semiquantitative Siemens SARS-CoV-2 IgG test, which measures binding of IgG to recombinantly expressed receptor binding domain of SARS-CoV-2 spike glycoprotein with the neutralization titers obtained in the pseudovirion assay and the results show high concordance between the two tests (R = 0.9344).

Conclusions: SARS-CoV-2 spike/MLV pseudovirions provide a practical means of assessing neutralizing activity of antibodies in serum or plasma from infected patients under laboratory conditions consistent with biocontainment level 2. This assay offers promise also in evaluating immunogenicity of spike glycoprotein-based candidate vaccines in the near future.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12985-020-01472-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7780907PMC
January 2021

Identification of Immunological Parameters as Predictive Biomarkers of Relapse in Patients with Chronic Myeloid Leukemia on Treatment-Free Remission.

J Clin Med 2020 Dec 25;10(1). Epub 2020 Dec 25.

Immunopathology Unit, National Center of Microbiology, Instituto de Salud Carlos III, 28220 Madrid, Spain.

BCR-ABL is an aberrant tyrosine kinase responsible for chronic myeloid leukemia (CML). Tyrosine kinase inhibitors (TKIs) induce a potent antileukemic response mostly based on the inhibition of BCR-ABL, but they also increase the activity of Natural Killer (NK) and CD8+ T cells. After several years, patients may interrupt treatment due to sustained, deep molecular response. By unknown reasons, half of the patients relapse during treatment interruption, whereas others maintain a potent control of the residual leukemic cells for several years. In this study, several immunological parameters related to sustained antileukemic control were analyzed. According to our results, the features more related to poor antileukemic control were as follows: low levels of cytotoxic cells such as NK, (Natural Killer T) NKT and CD8±TCRγβ+ T cells; low expression of activating receptors on the surface of NK and NKT cells; impaired synthesis of proinflammatory cytokines or proteases from NK cells; and HLA-E*0103 homozygosis and KIR haplotype BX. A Random Forest algorithm predicted 90% of the accuracy for the classification of CML patients in groups of relapse or non-relapse according to these parameters. Consequently, these features may be useful as biomarkers predictive of CML relapse in patients that are candidates to initiate treatment discontinuation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/jcm10010042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7795332PMC
December 2020

Broadly active zinc finger protein-guided transcriptional activation of HIV-1.

Mol Ther Methods Clin Dev 2021 Mar 27;20:18-29. Epub 2020 Oct 27.

Center for Gene Therapy, City of Hope, Beckman Research Institute and Hematological Malignancy and Stem Cell Transplantation Institute at the City of Hope, 1500 E. Duarte Rd., Duarte, CA 91010, USA.

Human immunodeficiency virus type 1 (HIV-1) causes a persistent viral infection resulting in the demise of immune regulatory cells. Clearance of HIV-1 infection results in integration of proviral DNA into the genome of host cells, which provides a means for evasion and long-term persistence. A therapeutic compound that specifically targets and sustainably activates a latent HIV-1 provirus could be transformative and is the goal for the "shock-and-kill" approach to a functional cure for HIV-1. Substantial progress has been made toward the development of recombinant proteins that target specific genomic loci for gene activation, repression, or inactivation by directed mutations. However, most of these modalities are too large or too complex for efficient therapeutic application. We describe here the development and testing of a novel recombinant zinc finger protein transactivator, ZFP-362-VPR, which specifically and potently enhances proviral HIV-1 transcription both in established latency models and activity across different viral clades. Additionally, ZFP-362-VPR-activated HIV-1 reporter gene expression in a well-established primary human CD4 T cell latency model and off-target pathways were determined by transcriptome analyses. This study provides clear proof of concept for the application of a novel, therapeutically relevant, protein transactivator to purge cellular reservoirs of HIV-1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.omtm.2020.10.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7726486PMC
March 2021

Pharmacological Activation of Non-canonical NF-κB Signaling Activates Latent HIV-1 Reservoirs .

Cell Rep Med 2020 Jun 23;1(3):100037. Epub 2020 Jun 23.

Infectious and Inflammatory Disease Center, Immunity and Pathogenesis Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA.

"Shock and kill" strategies focus on purging the latent HIV-1 reservoir by treating infected individuals with therapeutics that activate the latent virus and subsequently eliminating infected cells. We have previously reported that induction of non-canonical nuclear factor κB (NF-κB) signaling through a class of small-molecule antagonists known as Smac mimetics can reverse HIV-1 latency. Here, we describe the development of Ciapavir (SBI-0953294), a molecule specifically optimized for HIV-1 latency reversal that was found to be more efficacious as a latency-reversing agent than other Smac mimetics under clinical development for cancer. Critically, this molecule induced activation of HIV-1 reservoirs in a bone marrow, liver, thymus (BLT) humanized mouse model without mediating systemic T cell activation. This study provides proof of concept for the efficacy and safety of Ciapavir and indicates that Smac mimetics can constitute a critical component of a safe and efficacious treatment strategy to eliminate the latent HIV-1 reservoir.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.xcrm.2020.100037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7659604PMC
June 2020

Interferon-Mediated Long Non-Coding RNA Response in Macrophages in the Context of HIV.

Int J Mol Sci 2020 Oct 19;21(20). Epub 2020 Oct 19.

HIV Cure Research Center, Department of Internal Medicine and Pediatrics, Ghent University and Ghent University Hospital, 9000 Ghent, Belgium.

Interferons play a critical role in the innate immune response against a variety of pathogens, such as HIV-1. Recent studies have shown that long non-coding genes are part of a reciprocal feedforward/feedback relationship with interferon expression. They presumably contribute to the cell type specificity of the interferon response, such as the phenotypic and functional transition of macrophages throughout the immune response. However, no comprehensive understanding exists today about the IFN-lncRNA interplay in macrophages, also a sanctuary for latent HIV-1. Therefore, we completed a poly-A+ RNAseq analysis on monocyte-derived macrophages (MDMs) treated with members of all three types of IFNs (IFN-α, IFN-ε, IFN-γ or IFN-λ) and on macrophages infected with HIV-1, revealing an extensive non-coding IFN and/or HIV-1 response. Moreover, co-expression correlation with mRNAs was used to identify important (long) non-coding hub genes within IFN- or HIV-1-associated gene clusters. This study identified and prioritized IFN related hub lncRNAs for further functional validation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms21207741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589721PMC
October 2020

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

bioRxiv 2020 Sep 21. Epub 2020 Sep 21.

Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.07.17.207563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523104PMC
September 2020

Recommendations for measuring HIV reservoir size in cure-directed clinical trials.

Nat Med 2020 09 7;26(9):1339-1350. Epub 2020 Sep 7.

The Wistar Institute, Philadelphia, PA, USA.

Therapeutic strategies are being clinically tested either to eradicate latent HIV reservoirs or to achieve virologic control in the absence of antiretroviral therapy. Attaining this goal will require a consensus on how best to measure the numbers of persistently infected cells with the potential to cause viral rebound after antiretroviral-therapy cessation in assessing the results of cure-directed strategies in vivo. Current measurements assess various aspects of the HIV provirus and its functionality and produce divergent results. Here, we provide recommendations from the BEAT-HIV Martin Delaney Collaboratory on which viral measurements should be prioritized in HIV-cure-directed clinical trials.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41591-020-1022-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7703694PMC
September 2020

Cytotoxic cell populations developed during treatment with tyrosine kinase inhibitors protect autologous CD4+ T cells from HIV-1 infection.

Biochem Pharmacol 2020 12 20;182:114203. Epub 2020 Aug 20.

AIDS Immunopathology Unit, National Center of Microbiology (CNM), Instituto de Salud Carlos III (ISCIII), Madrid, Spain. Electronic address:

Tyrosine kinase inhibitors (TKIs) are successfully used in clinic to treat chronic myeloid leukemia (CML). Our group previously described that CD4+ T cells from patients with CML on treatment with TKIs such as dasatinib were resistant to HIV-1 infection ex vivo. The main mechanism for this antiviral activity was primarily based on the inhibition of SAMHD1 phosphorylation, which preserves the activity against HIV-1 of this innate immune factor. Approximately 50% CML patients who achieved a deep molecular response (DMR) may safely withdraw TKI treatment without molecular recurrence. Therefore, it has been speculated that TKIs may induce a potent antileukemic response that is maintained in most patients even one year after treatment interruption (TI). Subsequent to in vitro T-cell activation, we observed that SAMHD1 was phosphorylated in CD4+ T cells from CML patients who withdrew TKI treatment more than one year earlier, which indicated that these cells were now susceptible to HIV-1 infection. Importantly, these patients were seronegative for HIV-1 and seropositive for cytomegalovirus (CMV), but without CMV viremia. Although activated CD4+ T cells from CML patients on TI were apparently permissive to HIV-1 infection ex vivo, the frequency of proviral integration was reduced more than 12-fold on average when these cells were infected ex vivo in comparison with cells isolated from untreated, healthy donors. This reduced susceptibility to infection could be related to an enhanced NK-dependent cytotoxic activity, which was increased 8-fold on average when CD4+ T cells were infected ex vivo with HIV-1 in the presence of autologous NK cells. Enhanced cytotoxic activity was also observed in CD8 + T cells from these patients, which showed 8-fold increased expression of TCRγδ and more than 18-fold increased production of IFNγ upon activation with CMV peptides. In conclusion, treatment with TKIs induced a potent antileukemic response that may also have antiviral effects against HIV-1 and CMV, suggesting that transient use of TKIs in HIV-infected patients could develop a sustained antiviral response that would potentially interfere with HIV-1 reservoir dynamics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bcp.2020.114203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686055PMC
December 2020

Structure-Activity Relationship Analysis of Benzotriazine Analogues as HIV-1 Latency-Reversing Agents.

Antimicrob Agents Chemother 2020 07 22;64(8). Epub 2020 Jul 22.

Department of Microbiology, Immunology, and Tropical Medicine, George Washington University, Washington, DC, USA

"Shock and kill" therapeutic strategies toward HIV eradication are based on the transcriptional activation of latent HIV with a latency-reversing agent (LRA) and the consequent killing of the reactivated cell by either the cytopathic effect of HIV or an arm of the immune system. We have recently found several benzotriazole and benzotriazine analogues that have the ability to reactivate latent HIV by inhibiting signal transducer and activator of transcription 5 (STAT5) SUMOylation and promoting STAT5 binding to the HIV long terminal repeat and increasing its transcriptional activity. To understand the essential structural groups required for biological activity of these molecules, we performed a systematic analysis of >40 analogues. First, we characterized the essential motifs within these molecules that are required for their biological activity. Second, we identified three benzotriazine analogues with similar activity. We demonstrated that these three compounds are able to increase STAT5 phosphorylation and transcriptional activity. All active analogues reactivate latent HIV in a primary cell model of latency and enhance the ability of interleukin-15 to reactivate latent HIV in cells isolated from aviremic participants. Third, this family of compounds also promote immune effector functions in the absence of toxicity or global immune activation. Finally, initial studies in mice suggest lack of acute toxicity A better understanding of the biological activity of these compounds will help in the design of improved LRAs that work via inhibition of STAT5 SUMOylation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.00888-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7526807PMC
July 2020

The landscape of alternative splicing in HIV-1 infected CD4 T-cells.

BMC Med Genomics 2020 04 3;13(Suppl 5):38. Epub 2020 Apr 3.

Department of Biomedical Informatics, University of Utah School of Medicine, Salt Lake City, UT, USA.

Background: Elucidating molecular mechanisms that are altered during HIV-1 infection may provide a better understanding of the HIV-1 life cycle and how it interacts with infected T-cells. One such mechanism is alternative splicing (AS), which has been studied for HIV-1 itself, but no systematic analysis has yet been performed on infected T-cells. We hypothesized that AS patterns in infected T-cells may illuminate the molecular mechanisms underlying HIV-1 infection and identify candidate molecular markers for specifically targeting infected T-cells.

Methods: We downloaded previously published raw RNA-seq data obtained from HIV-1 infected and non-infected T-cells. We estimated percent spliced in (PSI) levels for each AS exon, then identified differential AS events in the infected cells (FDR < 0.05, PSI difference > 0.1). We performed functional gene set enrichment analysis on the genes with differentially expressed AS exons to identify their functional roles. In addition, we used RT-PCR to validate differential alternative splicing events in cyclin T1 (CCNT1) as a case study.

Results: We identified 427 candidate genes with differentially expressed AS exons in infected T-cells, including 20 genes related to cell surface, 35 to kinases, and 121 to immune-related genes. In addition, protein-protein interaction analysis identified six essential subnetworks related to the viral life cycle, including Transcriptional regulation by TP53, Class I MHC mediated antigen, G2/M transition, and late phase of HIV life cycle. CCNT1 exon 7 was more frequently skipped in infected T-cells, leading to loss of the key Cyclin_N motif and affecting HIV-1 transcriptional elongation.

Conclusions: Our findings may provide new insight into systemic host AS regulation under HIV-1 infection and may provide useful initial candidates for the discovery of new markers for specifically targeting infected T-cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12920-020-0680-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7118826PMC
April 2020

Long non-coding RNAs and latent HIV - A search for novel targets for latency reversal.

PLoS One 2019 11;14(11):e0224879. Epub 2019 Nov 11.

San Diego VA Medical Center and Veterans Medical Research Foundation, San Diego, CA, United States of America.

The latent cellular reservoir of HIV is recognized as the major barrier to cure from HIV infection. Long non-coding RNAs (lncRNAs) are more tissue and cell type-specific than protein coding genes, and may represent targets of choice for HIV latency reversal. Using two in vitro primary T-cell models, we identified lncRNAs dysregulated in latency. PVT1 and RP11-347C18.3 were up-regulated in common between the two models, and RP11-539L10.2 was down-regulated. The major component of the latent HIV reservoir, memory CD4+ T-cells, had higher expression of these lncRNAs, compared to naïve T-cells. Guilt-by-association analysis demonstrated that lncRNAs dysregulated in latency were associated with several cellular pathways implicated in HIV latency establishment and maintenance: proteasome, spliceosome, p53 signaling, and mammalian target of rapamycin (MTOR). PVT1, RP11-347C18.3, and RP11-539L10.2 were down-regulated by latency reversing agents, suberoylanilide hydroxamic acid and Romidepsin, suggesting that modulation of lncRNAs is a possible secondary mechanism of action of these compounds. These results will facilitate prioritization of lncRNAs for evaluation as targets for HIV latency reversal. Importantly, our study provides insights into regulatory function of lncRNA during latent HIV infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0224879PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6844474PMC
March 2020

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

PLoS Pathog 2019 10 4;15(10):e1007903. Epub 2019 Oct 4.

Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.

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, with limited findings using CD4+ cells from donor blood consistent with observations in immortalized cells. 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 the sense orientation of genes 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1007903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6795456PMC
October 2019

Tyrosine Kinase Inhibition: a New Perspective in the Fight against HIV.

Curr HIV/AIDS Rep 2019 10;16(5):414-422

AIDS Immunopathology Unit, National Center of Microbiology, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo km2, 28220, Madrid, Spain.

Purpose Of Review: HIV-1 infection is incurable due to the existence of latent reservoirs that persist in the face of cART. In this review, we describe the existence of multiple HIV-1 reservoirs, the mechanisms that support their persistence, and the potential use of tyrosine kinase inhibitors (TKIs) to block several pathogenic processes secondary to HIV-1 infection.

Recent Findings: Dasatinib interferes in vitro with HIV-1 persistence by two independent mechanisms. First, dasatinib blocks infection and potential expansion of the latent reservoir by interfering with the inactivating phosphorylation of SAMHD1. Secondly, dasatinib inhibits the homeostatic proliferation induced by γc-cytokines. Since homeostatic proliferation is thought to be the main mechanism behind the maintenance of the latent reservoir, we propose that blocking this process will gradually reduce the size of the reservoir. TKIs together with cART will interfere with HIV-1 latent reservoir persistence, favoring the prospect for viral eradication.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11904-019-00462-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6814579PMC
October 2019

IFI16 Targets the Transcription Factor Sp1 to Suppress HIV-1 Transcription and Latency Reactivation.

Cell Host Microbe 2019 06 4;25(6):858-872.e13. Epub 2019 Jun 4.

Institute of Molecular Virology, Ulm University Medical Center, 89081 Ulm, Germany. Electronic address:

The interferon γ-inducible protein 16 (IFI16) is known as immune sensor of retroviral DNA intermediates. We show that IFI16 restricts HIV-1 independently of immune sensing by binding and inhibiting the host transcription factor Sp1 that drives viral gene expression. This antiretroviral activity and ability to bind Sp1 require the N-terminal pyrin domain and nuclear localization of IFI16, but not the HIN domains involved in DNA binding. Highly prevalent clade C HIV-1 strains are more resistant to IFI16 and less dependent on Sp1 than other HIV-1 subtypes. Furthermore, inhibition of Sp1 by IFI16 or pharmacologically by Mithramycin A suppresses reactivation of latent HIV-1 in CD4 T cells. Finally, IFI16 also inhibits retrotransposition of LINE-1, known to engage Sp1, and murine IFI16 homologs restrict Friend retrovirus replication in mice. Thus, IFI16 restricts retroviruses and retrotransposons by interfering with Sp1-dependent gene expression, and evasion from this restriction may facilitate spread of HIV-1 subtype C.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chom.2019.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6681451PMC
June 2019

Transcriptional Circuit Fragility Influences HIV Proviral Fate.

Cell Rep 2019 04;27(1):154-171.e9

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. Electronic address:

Transcriptional circuit architectures in several organisms have been evolutionarily selected to dictate precise given responses. 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, although the host phase is regulated by a checkpoint whereby KAP1 mediates transcription activation, the virus evolved a minimalist system bypassing KAP1. Given the complex circuit's 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, although HIV drives molecular innovation to fuel robust gene activation, it experiences transcriptional fragility, thereby influencing viral fate and cure efforts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2019.03.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6461408PMC
April 2019

Sex Influences SAMHD1 Activity and Susceptibility to Human Immunodeficiency Virus-1 in Primary Human Macrophages.

J Infect Dis 2019 02;219(5):777-785

Department of Pathology, Division of Microbiology and Immunology, University of Utah School of Medicine.

Background: Macrophages are major targets for HIV-1, contribute to viral propagation in vivo, and are instrumental in the pathogenesis of HAND. While it is known that host sex affects HIV-1 viremia and influences the severity of HIV-1-associated neurocognitive disease, a cellular or molecular basis for these findings remains elusive.

Methods: We explored whether sex affects HIV-1 infectivity of primary human macrophages and CD4+ T cells in vitro.

Results: Macrophages derived from female donors were less susceptible to HIV-1 infection than those derived from males. This sex-dependent difference in macrophage infectivity was independent of the requirement for CD4/CCR5-mediated virus entry and was not observed in CD4+ T cells. Investigations into the mechanism governing these sex-dependent differences revealed that the host restriction factor SAMHD1 exists in a hyperphosphorylated, less active state in male-derived macrophages. In addition, the major kinase responsible for SAMHD1 phosphorylation, CDK1, exhibited lower levels of expression in female-derived macrophages in all tested donor pairs. The sex-dependent differences in viral restriction imposed by SAMHD1 were abrogated upon its depletion.

Conclusions: We conclude that SAMHD1 is an essential modulator of infectivity in a sex-dependent manner in macrophages, constituting a novel component of sex differences in innate immune control of HIV-1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/infdis/jiy583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6376916PMC
February 2019

Dual TLR2 and TLR7 agonists as HIV latency-reversing agents.

JCI Insight 2018 10 4;3(19). Epub 2018 Oct 4.

Department of Microbiology, Immunology, and Tropical Medicine, George Washington University, Washington, DC, USA.

The presence of a reservoir of latently infected cells in HIV-infected patients is a major barrier towards finding a cure. One active cure strategy is to find latency-reversing agents that induce viral reactivation, thus leading to immune cell recognition and elimination of latently infected cells, known as the shock-and-kill strategy. Therefore, the identification of molecules that reactivate latent HIV and increase immune activation has the potential to further these strategies into the clinic. Here, we characterized synthetic molecules composed of a TLR2 and a TLR7 agonist (dual TLR2/7 agonists) as latency-reversing agents and compared their activity with that of the TLR2 agonist Pam2CSK4 and the TLR7 agonist GS-9620. We found that these dual TLR2/7 agonists reactivate latency by 2 complementary mechanisms. The TLR2 component reactivates HIV by inducing NF-κB activation in memory CD4+ T cells, while the TLR7 component induces the secretion of TNF-α by monocytes and plasmacytoid dendritic cells, promoting viral reactivation in CD4+ T cells. Furthermore, the TLR2 component induces the secretion of IL-22, which promotes an antiviral state and blocks HIV infection in CD4+ T cells. Our study provides insight into the use of these agonists as a multipronged approach targeting eradication of latent HIV.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/jci.insight.122673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237480PMC
October 2018

Synthetic Ingenols Maximize Protein Kinase C-Induced HIV-1 Latency Reversal.

Antimicrob Agents Chemother 2018 11 24;62(11). Epub 2018 Oct 24.

Division of Microbiology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah, USA.

Antiretroviral therapy (ART) does not cure HIV-1 infection due to the persistence of proviruses in long-lived resting T cells. Strategies targeting these latently infected cells will be necessary to eradicate HIV-1 in infected individuals. Protein kinase C (PKC) activation is an effective mechanism to reactivate latent proviruses and allows for recognition and clearance of infected cells by the immune system. Several ingenol compounds, naturally occurring PKC agonists, have been described to have potent latency reversal activity. We sought to optimize this activity by synthesizing a library of novel ingenols via esterification of the C-3 hydroxyl group of the ingenol core, which itself is inactive for latency reversal. Newly synthesized ingenol derivatives were evaluated for latency reversal activity, cellular activation, and cytotoxicity alongside commercially available ingenols (ingenol-3,20-dibenzoate, ingenol 3-hexanoate, and ingenol-3-angelate) in HIV latency cell lines and resting CD4 T cells from aviremic participants. Among the synthetic ingenols that we produced, we identified several compounds that demonstrate high efficacy and represent promising leads as latency reversal agents for HIV-1 eradication.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.01361-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6201092PMC
November 2018

Influence of Biological Sex, Age, and HIV Status in an In Vitro Primary Cell Model of HIV Latency Using a CXCR4 Tropic Virus.

AIDS Res Hum Retroviruses 2018 09 10;34(9):769-777. Epub 2018 Aug 10.

1 Department of Microbiology, Immunology, and Tropical Medicine, School of Medicine and Health Sciences, George Washington University , Washington, District of Columbia.

Primary cell models of human immunodeficiency virus (HIV) latency have become tools to both understand the mechanisms involved in establishment of latency and test preclinical strategies toward HIV-1 cure. These models rely on infection of CD4 T cells from healthy donors. As such, these models provide an opportunity to explore the role of biological sex, age, and HIV status on establishment and reactivation of latent HIV in vitro. We have used an established primary cell model of latency based on the generation of latently infected central memory CD4 T cells with the CXCR4 strain HIV-1 to address whether these variables influence (i) HIV-1 replication, (ii) establishment of latency, and (iii) latency reversal in CD4 T cells. Our results indicate that replication of HIV-1, but not establishment of latency, is influenced by the age of female, but not male, donors. Moreover, the frequency of latently infected cells in this model is directly correlated with levels of productive infection in both male and female donors independent of age. We did not find differences in the ability of five different latency-reversing agents to reactivate latent HIV-1. Finally, we have found that this model can be generated using cells from aviremic participants. In conclusion, we have further characterized the central memory T cell model of latency regarding biological sex and age and demonstrated that this model is suitable for use with cells isolated from aviremic participants, opening the opportunity to use this primary cell model to address cure approaches, including shock and kill, in HIV-infected individuals.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/AID.2018.0098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6152854PMC
September 2018

SAMHD1 Phosphorylation Coordinates the Anti-HIV-1 Response by Diverse Interferons and Tyrosine Kinase Inhibition.

mBio 2018 05 15;9(3). Epub 2018 May 15.

Division of Microbiology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah, USA

Macrophages are susceptible to human immunodeficiency virus type 1 (HIV-1) infection despite abundant expression of antiviral proteins. Perhaps the most important antiviral protein is the restriction factor sterile alpha motif domain and histidine/aspartic acid domain-containing protein 1 (SAMHD1). We investigated the role of SAMHD1 and its phospho-dependent regulation in the context of HIV-1 infection in primary human monocyte-derived macrophages and the ability of various interferons (IFNs) and pharmacologic agents to modulate SAMHD1. Here we show that stimulation by type I, type II, and to a lesser degree, type III interferons share activation of SAMHD1 via dephosphorylation at threonine-592 as a consequence of signaling. Cyclin-dependent kinase 1 (CDK1), a known effector kinase for SAMHD1, was downregulated at the protein level by all IFN types tested. Pharmacologic inhibition or small interfering RNA (siRNA)-mediated knockdown of CDK1 phenocopied the effects of IFN on SAMHD1. A panel of FDA-approved tyrosine kinase inhibitors potently induced activation of SAMHD1 and subsequent HIV-1 inhibition. The viral restriction imposed via IFNs or dasatinib could be overcome through depletion of SAMHD1, indicating that their effects are exerted primarily through this pathway. Our results demonstrate that SAMHD1 activation, but not transcriptional upregulation or protein induction, is the predominant mechanism of HIV-1 restriction induced by type I, type II, and type III IFN signaling in macrophages. Furthermore, SAMHD1 activation presents a pharmacologically actionable target through which HIV-1 infection can be subverted. Our experimental results demonstrate that SAMHD1 dephosphorylation at threonine-592 represents a central mechanism of HIV-1 restriction that is common to the three known families of IFNs. While IFN types I and II were potent inhibitors of HIV-1, type III IFN showed modest to undetectable activity. Regulation of SAMHD1 by IFNs involved changes in phosphorylation status but not in protein levels. Phosphorylation of SAMHD1 in macrophages occurred at least in part via CDK1. Tyrosine kinase inhibitors similarly induced SAMHD1 dephosphorylation, which protects macrophages from HIV-1 in a SAMHD1-dependent manner. SAMHD1 is a critical restriction factor regulating HIV-1 infection of macrophages.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/mBio.00819-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5954222PMC
May 2018

CD32 is expressed on cells with transcriptionally active HIV but does not enrich for HIV DNA in resting T cells.

Sci Transl Med 2018 04;10(437)

The Wistar Institute, Philadelphia, PA 19104, USA.

The persistence of HIV reservoirs, including latently infected, resting CD4 T cells, is the major obstacle to cure HIV infection. CD32a expression was recently reported to mark CD4 T cells harboring a replication-competent HIV reservoir during antiretroviral therapy (ART) suppression. We aimed to determine whether CD32 expression marks HIV latently or transcriptionally active infected CD4 T cells. Using peripheral blood and lymphoid tissue of ART-treated HIV or SIV subjects, we found that most of the circulating memory CD32 CD4 T cells expressed markers of activation, including CD69, HLA-DR, CD25, CD38, and Ki67, and bore a T2 phenotype as defined by CXCR3, CCR4, and CCR6. CD32 expression did not selectively enrich for HIV- or SIV-infected CD4 T cells in peripheral blood or lymphoid tissue; isolated CD32 resting CD4 T cells accounted for less than 3% of the total HIV DNA in CD4 T cells. Cell-associated HIV DNA and RNA loads in CD4 T cells positively correlated with the frequency of CD32 CD69 CD4 T cells but not with CD32 expression on resting CD4 T cells. Using RNA fluorescence in situ hybridization, CD32 coexpression with HIV RNA or p24 was detected after in vitro HIV infection (peripheral blood mononuclear cell and tissue) and in vivo within lymph node tissue from HIV-infected individuals. Together, these results indicate that CD32 is not a marker of resting CD4 T cells or of enriched HIV DNA-positive cells after ART; rather, CD32 is predominately expressed on a subset of activated CD4 T cells enriched for transcriptionally active HIV after long-term ART.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scitranslmed.aar6759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6282755PMC
April 2018

Novel Latency Reversal Agents for HIV-1 Cure.

Annu Rev Med 2018 01 3;69:421-436. Epub 2017 Nov 3.

Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah 84112; email:

Antiretroviral therapy (ART) has rendered HIV-1 infection a treatable illness; however, ART is not curative owing to the persistence of replication-competent, latent proviruses in long-lived resting T cells. Strategies that target these latently infected cells and allow immune recognition and clearance of this reservoir will be necessary to eradicate HIV-1 in infected individuals. This review describes current pharmacologic approaches to reactivate the latent reservoir so that infected cells can be recognized and targeted, with the ultimate goal of achieving an HIV-1 cure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1146/annurev-med-052716-031710DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5892446PMC
January 2018

Mycobacterium tuberculosis reactivates latent HIV-1 in T cells in vitro.

PLoS One 2017 26;12(9):e0185162. Epub 2017 Sep 26.

Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah, United States of America.

Following proviral integration into the host cell genome and establishment of a latent state, the human immunodeficiency virus type 1 (HIV-1) can reenter a productive life cycle in response to various stimuli. HIV-1 reactivation occurs when transcription factors, such as nuclear factor-κB (NF-κB), nuclear factor of activated T cells (NFAT), and activator protein -1 (AP-1), bind cognate sites within the long terminal repeat (LTR) region of the HIV-1 provirus to promote transcription. Interestingly, pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) can reactivate latent HIV-1 through activation of the transcription factor NF-κB. Some PRRs are expressed on central memory CD4+ T cells (TCM), which in HIV-1 patients constitute the main reservoir of latent HIV-1. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), interacts with PRRs through membrane components. However, the ability of Mtb to reactivate latent HIV-1 has not been extensively studied. Here we show that phosphatidylinositol mannoside 6 (PIM6), a component of the Mtb membrane, in addition to whole bacteria in co-culture, can reactivate HIV-1 in a primary TCM cell model of latency. Using a JLAT model of HIV-1 latency, we found this interaction to be mediated through Toll-like receptor-2 (TLR-2). Thus, we describe a mechanism by which Mtb can exacerbate HIV-1 infection. We hypothesize that chronic Mtb infection can drive HIV-1 reactivation. The phenomenon described here could explain, in part, the poor prognosis that characterizes HIV-1/Mtb co-infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0185162PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5614573PMC
October 2017

When DNA damage is acceptable.

Cell Cycle 2017 06 19;16(11):1020-1021. Epub 2017 May 19.

a Division of Microbiology and Immunology, Department of Pathology , University of Utah , Salt Lake City , UT , USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15384101.2017.1324125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499848PMC
June 2017

Benzotriazoles Reactivate Latent HIV-1 through Inactivation of STAT5 SUMOylation.

Cell Rep 2017 01;18(5):1324-1334

Division of Microbiology and Immunology, Department of Pathology, University of Utah, Salt Lake City, UT 84112, USA. Electronic address:

The presence of latent HIV-1 in infected individuals represents a major barrier preventing viral eradication. For that reason, reactivation of latent viruses in the presence of antiretroviral regimens has been proposed as a therapeutic strategy to achieve remission. We screened for small molecules and identified several benzotriazole derivatives with the ability to reactivate latent HIV-1. In the presence of IL-2, benzotriazoles reactivated and reduced the latent reservoir in primary cells, and, remarkably, viral reactivation was achieved without inducing cell proliferation, T cell activation, or cytokine release. Mechanistic studies showed that benzotriazoles block SUMOylation of phosphorylated STAT5, increasing STAT5's activity and occupancy of the HIV-1 LTR. Our results identify benzotriazoles as latency reversing agents and STAT5 signaling and SUMOylation as targets for HIV-1 eradication strategies. These compounds represent a different direction in the search for "shock and kill" therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2017.01.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5461578PMC
January 2017

Janus kinase inhibition suppresses PKC-induced cytokine release without affecting HIV-1 latency reversal ex vivo.

Retrovirology 2016 Dec 20;13(1):88. Epub 2016 Dec 20.

Department of Pathology, University of Utah School of Medicine, Emma Eccles Jones Medical Research Building Room 2520, 15 North Medical Drive East, Salt Lake City, UT, 84112, USA.

Background: Despite the durable viral suppression afforded by antiretroviral therapy, HIV-1 eradication will require strategies to target latently infected cells that persist in infected individuals. Protein kinase C (PKC) activation is a promising strategy to reactivate latent proviruses and allow for subsequent recognition and clearance of infected cells by the immune system. Ingenol derivatives are PKC agonists that induce latency reversal but also lead to T cell activation and the release of pro-inflammatory cytokines, which would be undesirable in vivo. In this work, we sought to identify compounds that would suppress pro-inflammatory cytokine production in the context of PKC activation.

Design And Methods: We performed an in vitro screen to identify compounds that could dampen pro-inflammatory cytokine release associated with T cell activation, using IL-6 as a model cytokine. We then tested the ability of the most promising screening hit, the FDA-approved Janus Kinase (JAK) inhibitor ruxolitinib, to diminish release of multiple cytokines and its effect on latency reversal using cells from HIV-1-positive, aviremic participants.

Results: We demonstrate that co-administration of ruxolitinib with ingenol-3,20-dibenzoate significantly reduces pro-inflammatory cytokine release without impairing latency reversal ex vivo.

Conclusion: The combination of ingenol compounds and JAK inhibition represents a novel strategy for HIV-1 eradication.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12977-016-0319-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5175306PMC
December 2016

Transcriptomic Analysis Implicates the p53 Signaling Pathway in the Establishment of HIV-1 Latency in Central Memory CD4 T Cells in an In Vitro Model.

PLoS Pathog 2016 Nov 29;12(11):e1006026. Epub 2016 Nov 29.

Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, Hampshire, United Kingdom.

The search for an HIV-1 cure has been greatly hindered by the presence of a viral reservoir that persists despite antiretroviral therapy (ART). Studies of HIV-1 latency in vivo are also complicated by the low proportion of latently infected cells in HIV-1 infected individuals. A number of models of HIV-1 latency have been developed to examine the signaling pathways and viral determinants of latency and reactivation. A primary cell model of HIV-1 latency, which incorporates the generation of primary central memory CD4 T cells (TCM), full-length virus infection (HIVNL4-3) and ART to suppress virus replication, was used to investigate the establishment of HIV latency using RNA-Seq. Initially, an investigation of host and viral gene expression in the resting and activated states of this model indicated that the resting condition was reflective of a latent state. Then, a comparison of the host transcriptome between the uninfected and latently infected conditions of this model identified 826 differentially expressed genes, many of which were related to p53 signaling. Inhibition of the transcriptional activity of p53 by pifithrin-α during HIV-1 infection reduced the ability of HIV-1 to be reactivated from its latent state by an unknown mechanism. In conclusion, this model may be used to screen latency reversing agents utilized in shock and kill approaches to cure HIV, to search for cellular markers of latency, and to understand the mechanisms by which HIV-1 establishes latency.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1006026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127598PMC
November 2016

HIV Latency-Reversing Agents Have Diverse Effects on Natural Killer Cell Function.

Front Immunol 2016 21;7:356. Epub 2016 Sep 21.

UNC HIV Cure Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

In an effort to clear persistent HIV infection and achieve a durable therapy-free remission of HIV disease, extensive pre-clinical studies and early pilot clinical trials are underway to develop and test agents that can reverse latent HIV infection and present viral antigen to the immune system for clearance. It is, therefore, critical to understand the impact of latency-reversing agents (LRAs) on the function of immune effectors needed to clear infected cells. We assessed the impact of LRAs on the function of natural killer (NK) cells, the main effector cells of the innate immune system. We studied the effects of three histone deacetylase inhibitors [SAHA or vorinostat (VOR), romidepsin, and panobinostat (PNB)] and two protein kinase C agonists [prostratin (PROST) and ingenol] on the antiviral activity, cytotoxicity, cytokine secretion, phenotype, and viability of primary NK cells. We found that exposure to VOR had minimal impact on all parameters assessed, while PNB caused a decrease in NK cell viability, antiviral activity, and cytotoxicity. PROST caused non-specific NK cell activation and, interestingly, improved antiviral activity. Overall, we found that LRAs can alter the function and fate of NK cells, and these effects must be carefully considered as strategies are developed to clear persistent HIV infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2016.00356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5030263PMC
September 2016