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.
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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
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    • "This reporter virus was then used to establish a latent HIV-1 infection in primary human CD4+ T cells, isolated from peripheral blood mononuclear cells (PBMCs) collected from healthy donors, via two distinct methods. The first involved the infection of non-polar CD4+ T cells, which are considered to be the in vitro counterparts of latently infected central memory T cells in vivo[45]. Naïve human CD4+ T cells were isolated, activated in a non-polarizing environment, infected with the gGnΔ virus, and then cultured for seven days to establish a latent infection. "
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    ABSTRACT: Existing highly active antiretroviral therapy (HAART) effectively controls viral replication in human immunodeficiency virus type 1 (HIV-1) infected individuals but cannot completely eradicate the infection, at least in part due to the persistence of latently infected cells. One strategy that is being actively pursued to eliminate the latent aspect of HIV-1 infection involves therapies combining latency antagonists with HAART. However, discordant pharmacokinetics between these types of drugs can potentially create sites of active viral replication within certain tissues that might be impervious to HAART. A preliminary reverse genetic screen indicated that the proteasome might be involved in the maintenance of the latent state. This prompted testing to determine the effects of proteasome inhibitors (PIs) on latently infected cells. Experiments demonstrated that PIs effectively activated latent HIV-1 in several model systems, including primary T cell models, thereby defining PIs as a new class of HIV-1 latency antagonists. Expanding upon experiments from previous reports, it was also confirmed that PIs inhibit viral replication. Moreover, it was possible to show that PIs act as bifunctional antagonists of HIV-1. The data indicate that PIs activate latent provirus and subsequently decrease viral titers and promote the production of defective virions from activated cells. These results represent a proof-of-concept that bifunctional antagonists of HIV-1 can be developed and have the capacity to ensure precise tissue overlap of anti-latency and anti-replication functions, which is of significant importance in the consideration of future drug therapies aimed at viral clearance.
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