Bosque, A and Planelles, V. Studies of HIV-1 latency in an ex vivo model that uses primary central memory T cells. Methods 53: 54-61

Department of Pathology, University of Utah, 15 North Medical Dr East #2100, Room 2520, Salt Lake City, UT 84112, USA.
Methods (Impact Factor: 3.65). 10/2010; 53(1):54-61. DOI: 10.1016/j.ymeth.2010.10.002
Source: PubMed


HIV-1 latency is considered the last hurdle toward viral eradication in the presence of antiretroviral therapy. Studies of viral latency in vivo are complicated by the low frequency of latently infected cells found in HIV-1 patients. To be able to study the signaling pathways and viral determinants of latency and reactivation, we have developed a novel method that generates high numbers of latently HIV-1 infected cells, which are derived from human primary CD4(+) T lymphocytes. This method allows for the study of different aspects of HIV-1 latency, such as the transcription factors needed for viral reactivation and the signaling pathways involved. In this review, we describe in detail an experimental protocol for the generation of HIV-1 latency using human primary CD4(+) T cells. We also present the salient points of other latency models in the field, along with key findings arising from each model.

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Available from: Alberto Bosque, Jan 06, 2014
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    • "Latently infected CD4 + T cells were generated according to the cytokine-polarized primary T cells model of latency (Bosque and Planelles, 2011) with few modifications. Briefly, naïve CD4 + T cells were activated with aCD3/aCD28 antibodies (1 lg/ml each; BD, Madrid, Spain) and supplemented with TGF-b1 (10 lg/mL, Peprotech ), aIL-12 (2 lg/mL) and aIL-4 (1 lg/mL, Peprotech). "
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    ABSTRACT: Antiretroviral therapy (ART) is unable to cure HIV infection. The ability of HIV to establish a subset of latent infected CD4+ T cells, which remain undetectable to the immune system, becomes a major roadblock to achieve viral eradication. Histone deacetylase inhibitors (HDACi) have been shown to potently induce the reactivation of latent HIV. Here, we show that a new thiol-based HDACi, the thioacetate-ω(γ-lactam carboxamide) derivative ST7612AA1, is a potent inducer of HIV reactivation. We evaluated HIV reactivation activity of ST7612AA1 compared to panobinostat (PNB), romidepsin (RMD) and vorinostat (VOR) in cell culture models of HIV-1 latency, in latently infected primary CD4+ T lymphocytes and in PBMCs from HIV+ patients. ST7612AA1 potently induced HIV-1 reactivation at submicromolar concentrations with comparable potency to panobinostat or superior to vorinostat. The presence of known antiretrovirals did not affect ST7612AA1-induced reactivation and their activity was not affected by ST7612AA1. Cell proliferation and cell activation were not affected by ST7612AA1, or any other HDACi used. In conclusion, our results indicate that ST7612AA1 is a potent activator of latent HIV and that reactivation activity of ST7612AA1 is exerted without activation or proliferation of CD4+ T cells. ST7612AA1 is a suitable candidate for further studies of HIV reactivation strategies and potential new therapies to eradicate the viral reservoirs.
    Antiviral research 09/2015; DOI:10.1016/j.antiviral.2015.09.004 · 3.94 Impact Factor
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    • "In contrast to earlier interpretations [8], cellular p24 positive staining after antigen stimulation is not an unequivocal sign of reactivated virus, but rather a combination of that and spreading viral replication. Therefore, the number of latently infected cells should be lower than initially inferred. "
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    ABSTRACT: The central memory T cell (TCM) model forms a unique HIV-1 latency model based on primary cells that closely resemble in vivo TCM. The virus employed in this model is based on an engineered vector incapable of replication after initial infection. We show that despite this strategy, replication competent viral particles are released into the culture medium due to recombination between overlapping sequences of the env deleted HIV genome that is co-transfected with intact env. This finding emphasizes the need for careful data analysis and interpretation if similar constructs are employed and urges for additional caution during laboratory work. Electronic supplementary material The online version of this article (doi:10.1186/s12977-014-0070-3) contains supplementary material, which is available to authorized users.
    Retrovirology 08/2014; 11(1):70. DOI:10.1186/PREACCEPT-1619001546133883 · 4.19 Impact Factor
    • "However, little is known about whether and how TLR ligands affect the latent reservoir of HIV infection in central memory CD4+ T cells. We have analyzed the potential ability of TLR agonists to transactivate the HIV-1 LTR using a previously described method for the generation of latently infected central memory T cells (TCM) [24,25]. We demonstrate that Pam3CSK4, a TLR-1/2 agonist, is able to reactivate latent HIV-1 in this in vitro model and in cells isolated from aviremic patients. "
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    ABSTRACT: Toll-like receptors (TLRs) are crucial for recognition of pathogen-associated molecular patterns by cells of the innate immune system. TLRs are present and functional in CD4+ T cells. Memory CD4+ T cells, predominantly central memory cells (TCM), constitute the main reservoir of latent HIV-1. However, how TLR ligands affect the quiescence of latent HIV within central memory CD4+ T cells has not been studied. We evaluated the ability of a broad panel of TLR agonists to reactivate latent HIV-1. The TLR-1/2 agonist Pam3CSK4 leads to viral reactivation of quiescent HIV in a model of latency based on cultured TCM and in resting CD4+ T cells isolated from aviremic patients. In addition, we investigated the signaling pathway associated with Pam3CSK4 involved in HIV-1 reactivation. We show that the transcription factors NFkappaB, NFAT and AP-1 cooperate to induce viral reactivation downstream of TLR-1/2 stimulation. Furthermore, increasing levels of cyclin T1 is not required for TLR-mediated viral reactivation, but induction of viral expression requires activated pTEFb. Finally, Pam3CSK4 reactivates latent HIV-1 in the absence of T cell activation or proliferation, in contrast to antigen stimulation. Our findings suggest that the signaling through TLR-1/2 pathway via Pam3CSK4 or other reagents should be explored as an anti-latency strategy either alone or in combination with others anti-latency drugs.
    Retrovirology 10/2013; 10(1):119. DOI:10.1186/1742-4690-10-119 · 4.19 Impact Factor
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