Dataset Information

In vivo and in vitro proteome analysis of HIV-1 infected, human CD4+ T cells

ABSTRACT: Host directed therapies against HIV-1 are thought to be critical for long term containment of the HIV-1 pandemic but remain elusive. Since HIV-1 infects and manipulates important effectors of both the innate and adaptive immune system, identifying modulations of the host cell systems in humans during HIV-1 infection may be crucial for the development of immune based therapies. Here, we quantified the changes of the proteome in human CD4+ T cells upon HIV-1 infection, both in vitro and in vivo. A SWATH-MS approach was used to measure the proteome of human primary CD4+ T cells infected with HIV-1 in vitro as well as CD4+ T cells from HIV-1 infected patients with paired samples on and off antiretroviral treatment. In the in vitro experiment, the proteome of CD4+ T cells was quantified over a time course following HIV-1 infection. 1,725 host cell proteins and 4 HIV-1 proteins were quantified, with 145 proteins changing significantly during the time course. Changes in the proteome peaked 24 hours after infection, concomitantly with significant HIV-1 protein production. In the in vivo branch of the study, CD4+ T cells from viremic patients and those with no detectable viral load after treatment were sorted and the proteomes quantified. We consistently detected 895 proteins, 172 of which were considered to be significantly different between viraemic patients and patients undergoing successful treatment. The proteome of in vitro infected CD4+ T cells was modulated on multiple functional levels, including TLR-4 signalling and the type 1 interferon signalling pathway. Perturbations in the type 1 interferon signalling pathway were recapitulated in CD4+ T cells from patients. The study shows that proteome maps generated by SWATH-MS indicate a range of functionally significant changes in the proteome of HIV infected human CD4+ T cells. Exploring these perturbations in more detail may help identify new targets for immune based interventions.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood

SUBMITTER: Ben Collins

LAB HEAD: Ben Collins

PROVIDER: PXD005234 | Pride | 2017-02-28

REPOSITORIES: Pride

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Publications

In Vivo and in Vitro Proteome Analysis of Human Immunodeficiency Virus (HIV)-1-infected, Human CD4+ T Cells.

Nemeth Johannes J Vongrad Valentina V Metzner Karin J KJ Strouvelle Victoria P VP Weber Rainer R Pedrioli Patrick P Aebersold Ruedi R Günthard Huldrych F HF Collins Ben C BC

Molecular & cellular proteomics : MCP 20170221 4 suppl 1

Host-directed therapies against HIV-1 are thought to be critical for long term containment of the HIV-1 pandemic but remain elusive. Because HIV-1 infects and manipulates important effectors of both the innate and adaptive immune system, identifying modulations of the host cell systems in humans during HIV-1 infection may be crucial for the development of immune based therapies. Here, we quantified the changes of the proteome in human CD4+ T cells upon HIV-1 infection, both in vitr ...[more]

PMID: 28223351

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Project description:We assessed correlates of protection from disease progression in a rare subset of HIV-infected individuals, viremic non-progressors (VNPs). These individuals have high viral load for several years, but in contrast to the majority of infected individuals, they do not progress to AIDS. Here we found this lack of progression was associated with selective preservation of two essential subsets of memory CD4+ T cells, central memory (TCM) and stem-cell memory (TSCM) cells. Compared to HIV-infected putative progressors, VNPs had higher proliferation of these indispensable subsets of memory cells, which was associated with the number of TCM. In addition, the long-lived CD4+ TCM and TSCM cells in VNPs had decreased HIV infection compared to the less critical effector memory CD4+ T cells, which indicates a possible mechanism by which VNPs maintain their CD4+ T cell pool after several years of infection, and remain free from AIDS progression. 6 HIV-infected patients fitting the clinical criteria of Viremic Non-Progressors were identified. VNPs were defined as having confirmed HIV-1 infection for at least 9 years with sustained plasma HIV RNA levels >10,000 copies/ml and maintenance of peripheral blood CD4+ T cell counts >500 cells/mm3 and a CD4% (of all lymphocytes) >15%. As controls, 7 HIV-infected Putative Progressors were identified. PPs were defined as having plasma HIV RNA levels >10,000 copies/mL, CD4+ T cell counts >400 cells/mm3 and having been initially infected with HIV 2-24 months prior to the index visit. The estimated date of initial HIV infection was calculated according to published algorithms that incorporate âde-tunedâ anti-HIV-1 antibody ELISA results or by a documented sero-conversion window of <6 months. All participants were required to be antiretroviral therapy (ART)-naÃ¯ve. RNA from 6 VNP and 7 PPs was purified from PAXgene whole blood tubes and hybridized to Affymetrix U133 Plus 2.0 arrays. During data analysis, one VNP patient (PID 4015) was determined to be an outlier and removed from further analysis. Thus, 5 VNPs and 7 PPs are represented in this Series.

Project description:Suppressive HAART does not eradicate HIV-1 and viral DNA persists as a stably integrated form in the absence of viral particle production. As a consequence, latent reservoirs are refractory to antiretroviral drugs and invisible to immune surveillance. The largest latent reservoir consists of resting memory CD4+ T cells. These cells can resume viral infection when activated through antigen recognition, causing bursts of viremia (blips). Current therapies targeting latent HIV-1 have focused primarily on the M-bM-^@M-^\shock and killM-bM-^@M-^] approach, which employs M-bM-^@M-^\anti-latencyM-bM-^@M-^] drugs M-bM-^@M-^S most notably histone deacetylase (HDAC) inhibitors M-bM-^@M-^S to reactivate and flush latent provirus from its cellular reservoirs in the absence of global T cell activation. This approach is predicated on the notions that viral reactivation will lead to the demise of the infected cell, and that HAART will prevent spreading of the infection. On the contrary, recent evidence indicates that latently infected CD4+ T cells of HIV-1 patients on HAART survive in vitro viral reactivation with the HDAC inhibitor, SAHA, even when co-cultured with autologous CD8+ cytotoxic T lymphocytes (CTL). Moreover, it remains to be addressed the impact of anti-latency drugs on viral reservoirs undergoing low-level ongoing replication, inherently more resistant to the cytopathic effects of HIV-1 and residing in anatomical sites hard to reach for some antiretroviral drugs (e.g. macrophages). As a consequence, there is a need to develop alternative therapeutic approaches aimed at eliminating or decreasing the latent reservoir. Progress in that direction has been hindered by the lack of biomarkers uniquely or differentially expressed on latently infected compared to their uninfected counterparts. To gain insight into the cellular mechanisms that take place in the context of latency, and with the goal of identifying distinctive markers that distinguish latently infected CD4+ T cells, we have used an in vitro model developed in our laboratory to study the expression profile of latently infected CD4+ T cells by microarray analysis. We have used a culture system, previously established in our laboratory, to generate and isolate quiescent latently infected CD4+ T cells in vitro. In this in vitro HIV-1 latency model, CD4+ T cells are activated, infected with full length, replication competent HIV-1, and then returned to quiescence in the presence of IL-7, yielding a culture of quiescent latently infected and uninfected cells. We showed that HIV-1 p24gag expressed during viral replication persists in the cytoplasm of latently infected cells for several days before being degraded. Therefore, we exploited the presence of cytoplasmic p24gag to sort latently infected from uninfected cells by FACS from the same initial cell culture. Total RNA was isolated from sorted latently infected and uninfected cells generated from CD4+ T cells of four different donors. Paired RNA samples from infected and uninfected cells were labeled with Cy3 and Cy5 to allow dual-color competitive hybridization. Moreover, to control for the dye bias in our experiments, we implemented a dye swap protocol (reciprocal labeling) for paired RNA samples from 2 donors. Samples were analyzed by dual-color competitive hybridization on the Agilent whole human genome microarrays (41,000 unique probes). This is the first comparative genomic profiling of primary latently infected resting memory CD4+ T cells versus their uninfected counterparts sorted from the same culture. Microarray analyses performed in this study revealed profound differences between latently infected and uninfected cells. Of relevance are genes involved, not only in previously described pathways related with transcriptional and post-transcriptional regulation, but affecting proliferation, survival, cell cycle progression and cell metabolism. This could explain why latently infected cells have been resistant to reactivation with current anti-latency approaches. Thus, targeting of more downstream steps, such as the ones identified in this study, may be able to enhance viral flushing from refractory latent reservoirs. In addition, we identified a panel of surface makers differentially expressed in latently infected cells, which seem worth investigating for their potential use as biomarkers. Indeed, they might allow the enrichment of this latent reservoir for molecular in depth studies, for monitoring the size of the latent reservoir in the clinical setting, as well as for the development of new therapeutic strategies aimed at eradicating this reservoir.

Project description:Our previous work demonstrated that HIV-1 infection progressively reduces TCR/CD3 expression due to a defect in CD3g gene transcripts. We further found that knocking down expression of the viral tat and/or nef genes was correlated with CD3g transcript and TCR/CD3 surface receptor levels on HIV-1 infected cells. This study was undertaken to investigate the direct effect of HIV-1 Tat expression on the TCR/CD3 machinery. Progressive downregulation from TCR/CD3hi to TCR/CD3lo to TCR/CD3â was observed on Tat expressing cells in a manner that emulated HIV-1 infection, with a lack of CD3g transcripts again responsible for the defect. When Tat cell cultures containing a mixture of TCR/CD3 surface densities were separated into TCR/CD3hi and TCR/CD3lo/â populations, they quickly reverted to a mixed CD3 phenotype. Thus, the progression TCR/CD3hi to TCR/CD3lo to TCR/CD3â is an active, reversible process with receptor levels fluctuating in response to intracellular dynamics. Examination of tat mutants found that the regions involved in Tat-mediated transactivation and TAR binding are required for TCR/CD3 downregulation while the lysine at position 28 and Tat exon 2 are dispensable. Global gene expression, assessed in association with TCR/CD3 downregulation in HIV-1 infected and Tat expressing cells, detected broad suppression of TCR/CD3 signaling, co-stimulation and negative regulatory genes along with target transcription factors, ligands and receptors. A significant subset of the genes altered in HIV-1 infected cells was specifically targeted by Tat in association with TCR/CD3 loss. Our finding that Tat negatively regulates many facets of the TCR/CD3 machinery has important implications for disease pathogenesis. We used microarrays to investigate changes in CD4+ T cell gene expression induced by HIV-1 infection for comparison with the Tat expressing cells. Examine changes in gene expression in TCR/CD3 negative HIV-1 infected cells compared to TCR/CD3 positive uninfected controls.

Dataset Information

In vivo and in vitro proteome analysis of HIV-1 infected, human CD4+ T cells

Publications

<i>In Vivo</i> and <i>in Vitro</i> Proteome Analysis of Human Immunodeficiency Virus (HIV)-1-infected, Human CD4<sup>+</sup> T Cells.

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