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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.

ABSTRACT: 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-? 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.

SUBMITTER: White CH

PROVIDER: S-EPMC5127598 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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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.

White Cory H CH Moesker Bastiaan B Beliakova-Bethell Nadejda N Martins Laura J LJ Spina Celsa A CA Margolis David M DM Richman Douglas D DD Planelles Vicente V Bosque Alberto A Woelk Christopher H CH

PLoS pathogens 20161129 11

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 prim ...[more]

PMID: 27898737

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Project description:BackgroundThe regulatory cyclin, Cyclin T1 (CycT1), is a host factor essential for HIV-1 replication in CD4 T cells and macrophages. The importance of CycT1 and the Positive Transcription Elongation Factor b (P-TEFb) complex for HIV replication is well-established, but regulation of CycT1 expression and protein levels during HIV replication and latency establishment in CD4 T cells is less characterized.MethodsTo better define the regulation of CycT1 levels during HIV replication in CD4 T cells, multiparameter flow cytometry was utilized to study the interaction between HIV replication (intracellular p24) and CycT1 of human peripheral blood memory CD4 T cells infected with HIV in vitro. CycT1 was further examined in CD4 T cells of human lymph nodes.ResultsIn activated (CD3+CD28 costimulation) uninfected blood memory CD4 T cells, CycT1 was most significantly upregulated in maximally activated (CD69+CD25+ and HLA.DR+CD38+) cells. In memory CD4 T cells infected with HIV in vitro, two distinct infected populations of p24+CycT1+ and p24+CycT1- cells were observed during 7 days infection, suggestive of different phases of productive HIV replication and subsequent latency establishment. Intriguingly, p24+CycT1- cells were the predominant infected population in activated CD4 T cells, raising the possibility that productively infected cells may transition into latency subsequent to CycT1 downregulation. Additionally, when comparing infected p24+ cells to bystander uninfected p24- cells (after bulk HIV infections), HIV replication significantly increased T cell activation (CD69, CD25, HLA.DR, CD38, and Ki67) without concomitantly increasing CycT1 protein levels, possibly due to hijacking of P-TEFb by the viral Tat protein. Lastly, CycT1 was constitutively expressed at higher levels in lymph node CD4 T cells compared to blood T cells, potentially enhancing latency generation in lymphoid tissues.ConclusionsCycT1 is most highly upregulated in maximally activated memory CD4 T cells as expected, but may become less associated with T cell activation during HIV replication. The progression into latency may further be predicated by substantial generation of p24+CycT1- cells during HIV replication.

Project description:During antiretroviral therapy (ART), human immunodeficiency virus type 1 (HIV-1) persists as a latent reservoir in CD4+ T cell subsets in central memory (TCM), transitional memory (TTM), and effector memory (TEM) CD4+ T cells. We have identified differences in mechanisms underlying latency and responses to latency-reversing agents (LRAs) in ex vivo CD4+ memory T cells from virally suppressed HIV-infected individuals and in an in vitro primary cell model of HIV-1 latency. Our ex vivo and in vitro results demonstrate the association of transcriptional pathways of T cell differentiation, acquisition of effector function, and cell cycle entry in response to LRAs. Analyses of memory cell subsets showed that effector memory pathways and cell surface markers of activation and proliferation in the TEM subset are predictive of higher frequencies of cells carrying an inducible reservoir. Transcriptional profiling also demonstrated that the epigenetic machinery (known to control latency and reactivation) in the TEM subset is associated with frequencies of cells with HIV-integrated DNA and inducible HIV multispliced RNA. TCM cells were triggered to differentiate into TEM cells when they were exposed to LRAs, and this increase of TEM subset frequencies upon LRA stimulation was positively associated with higher numbers of p24+ cells. Together, these data highlight differences in underlying biological latency control in different memory CD4+ T cell subsets which harbor latent HIV in vivo and support a role for differentiation into a TEM phenotype in facilitating latency reversal.IMPORTANCE By performing phenotypic analysis of latency reversal in CD4+ T cells from virally suppressed individuals, we identify the TEM subset as the largest contributor to the inducible HIV reservoir. Differential responses of memory CD4+ T cell subsets to latency-reversing agents (LRAs) demonstrate that HIV gene expression is associated with heightened expression of transcriptional pathways associated with differentiation, acquisition of effector function, and cell cycle entry. In vitro modeling of the latent HIV reservoir in memory CD4+ T cell subsets identify LRAs that reverse latency with ranges of efficiency and specificity. We found that therapeutic induction of latency reversal is associated with upregulation of identical sets of TEM-associated genes and cell surface markers shown to be associated with latency reversal in our ex vivo and in vitro models. Together, these data support the idea that the effector memory phenotype supports HIV latency reversal in CD4+ T cells.

Project description:HIV can persist in people living with HIV (PLWH) on antiretroviral therapy (ART) in multiple CD4+ T cell subsets, including naive cells, central memory (CM) cells, transitional (TM) cells, and effector memory (EM) cells. Since these cells express different levels of the viral coreceptors CXCR4 and CCR5 on their surface, we sought to determine whether the HIV envelope protein (Env) was genotypically and phenotypically different between CD4+ T cell subsets isolated from PLWH on suppressive ART (n?=?8). Single genome amplification for the HIV env gene was performed on genomic DNA extracts from different CD4+ T cell subsets. We detected CXCR4-using (X4) strains in five of the eight participants studied, and in these participants, the prevalence of X4 strains was higher in naive CD4+ T cells than in the memory subsets. Conversely, R5 strains were mostly found in the TM and EM populations. Identical sets of env sequences, consistent with clonal expansion of some infected cells, were more frequent in EM cells. These expanded identical sequences could also be detected in multiple CD4+ T cell subsets, suggesting that infected cells can undergo T cell differentiation. These identical sequences largely encoded intact and functional Env proteins. Our results are consistent with a model in which X4 HIV strains infect and potentially establish latency in naive and CM CD4+ T cells through direct infection, in addition to maintenance of the reservoir through differentiation and proliferation of infected cells.IMPORTANCE In people living with HIV (PLWH) on suppressive ART, latent HIV can be found in a diverse range of CD4+ T cells, including quiescent naive and central memory cells that are typically difficult to infect in vitro It is currently unclear how latency is established in these cells in vivo We show that in CD4+ T cells from PLWH on suppressive ART, the use of the coreceptor CXCR4 was prevalent among viruses amplified from naive and central memory CD4+ T cells. Furthermore, we found that expanded numbers of identical viral sequences were most common in the effector memory population, and these identical sequences were also found in multiple different CD4+ T cell subsets. Our results help to shed light on how a range of CD4+ T cell subsets come to harbor HIV DNA, which is one of the major barriers to eradicating the virus from PLWH.

Project description:Purpose: Next-generation sequencing (NGS) has become a powerful microscope to study cell models of HIV. The goals of this study is to analyze latent HIV infection in the TCM model of HIV latency described in Martins et al 2015 and to evaluate potential markers for HIV infection. Methods: Peripheral blood mononuclear cells were collected from 4 healthy donors. Naive CD4 T cells were isolated and utilized to generate a model of latent HIV infection. During model generation, cells were sorted and collected for RNA extraction 10 days post infection along with their uninfected counterparts. 2 days after this, cells were activated with CD3/CD28 stimulation and collected for study for a total of 16 samples from 4 donors. After cell collection, RNA was extracted from infected cells and their uninfected counterparts for deep sequencing by Expression Analysis. Sequence reads that passed quality filters were mapped using Tophat and counted using HTSeq. In addition to human transcripts, we utilized 92 ERCC spikes as negative controls. Any human gene which did not achieve at least 1 count per million reads in at least 4 samples or any ERCC that did not achieve at least 5 reads in at least 4 samples was discarded. Differential expression for activated samples was not performed, but rather used to demonstrate upregulation of T-cell activation markers along with changing to the type and abundance of HIV transcripts produced. Results: Using an custom built data analysis pipeline, 82 million reads per sample to the human genome (build hg38) and identified 13,534 human transcripts 67 ERCC spike in transcripts, and HIV NL43 transcripts were identified with the Tophat/HTSeq workflow. Differential expression analysis was performed between uninfected and latently infected cells. 826 genes were found to be differentially expressed, 275 downregulated and 551 upregulated (FDR < 0.05) with EdgeR. GO and Pathway analysis of differential expressed genes revealed significant dysregulation of genes involved in the p53 signaling pathway. Subsequent studies with pifithrin demonstrated a reduction in latently infected cell during model generation. Conclusions: This study represents the first detailed analysis of HIV latency with this cell model using next generations sequencing. These results demonstrate that the TCM model of HIV latency of Martins et al 2015 is truly reflective of HIV latency. This study also provides a framework for which to analyze future cell models of HIV latency using next generation sequencing. Finally, this work demonstrates that the p53 signaling pathway is a key pathway dysregulated in latency for this cell model with several genes dysregulated in HIV latency.

Dataset Information

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.

Publications

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.

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