Primary cell model for activation-inducible human immunodeficiency virus - PubMed (original) (raw)
Primary cell model for activation-inducible human immunodeficiency virus
Bryan Burke et al. J Virol. 2007 Jul.
Abstract
Quiescent T lymphocytes containing latent human immunodeficiency virus (HIV) provide a long-lived viral reservoir. This reservoir may be the source of active infection that is reinitiated following the cessation of antiretroviral therapy. Therefore, it is important to understand the mechanisms involved in latent infection to develop new strategies to eliminate the latent HIV reservoir. We have previously demonstrated that latently infected quiescent lymphocytes can be generated during thymopoiesis in vivo in the SCID-hu mouse system. However, there is still a pressing need for an in vitro model of HIV latency in primary human cells. Here, we present a novel in vitro model that recapitulates key aspects of dormant HIV infection. Using an enhanced green fluorescent protein-luciferase fusion protein-containing reporter virus, we have generated a stable infection in primary human CD4(+) CD8(+) thymocytes in the absence of viral gene expression. T-cell activation induces a >200-fold induction of reporter activity. The induced reporter activity originates from a fully reverse-transcribed and integrated genome. We further demonstrate that this model can be useful to study long terminal repeat regulation, as previously characterized NF-kappaB response element mutations decrease the activation of viral gene expression. This model can therefore be used to study intricate molecular aspects of activation-inducible HIV infection in primary cells.
Figures
FIG. 1.
In vitro model of activation-inducible HIV in primary cells. (A) Schematic of the HIV-based reporter virus NLEGFPLuc used in our model. The vector contains two functional LTRs and is highly attenuated, with the only functional genes being tat, rev, vpu, and EGFP-luciferase. EGFP and luciferase activities were assessed to measure gene expression driven from the HIV LTR. SD and SA, splice donor and acceptor sites, respectively; Ψ, packaging signal; Δgag, truncated gag sequence; RRE, _rev_-response element. (B) Schematic of the in vitro model used to study activation-inducible HIV infection. CD4+ CD8+ thymocytes were isolated and infected with NLEGFPLuc on day 0 and then cultured for 7 days to allow sufficient maturation. After 7 days in culture, the cells were divided into nonstimulated or costimulated (anti-CD3 and anti-CD28) conditions for an additional 3 days before the cells were harvested and reporter activity was assessed.
FIG. 2.
Assessment of viral gene expression. (A) Luciferase activity of CD4+ CD8+ thymocytes cultured for 10 days as described in the legend of Fig. 1B. Cells (105) were harvested from noninfected and infected cultures on the days indicated, and the luciferase activity at each time point was determined. Results are representative of two separate experiments. (B) EGFP expression in CD4+ CD8+ thymocytes infected and cultured for 10 days as described above. The left column shows the cells under phase-contrast microscopy, and the right column shows the cells expressing EGFP by using florescence microscopy. Results are representative of four separate experiments. (C) CD4+ CD8+ thymocytes infected with NLEGFPLuc and cultured in parallel with noninfected cells as described above were harvested and assayed for EGFP and CD25 expression by flow cytometry on day 10 p.i. Results are representative of two separate experiments.
FIG. 3.
Effects of thymocyte maturation on the ability to induce expression of dormant HIV. (A) Luciferase activity of CD4+ CD8+ thymocytes infected with NLEGFPLuc at an MOI of 0.1 and cultured for the indicated times before 5 × 105 cells were placed under nonstimulated or costimulated conditions for an additional 3 days in a 96-well format. Noninfected cells were cultured in parallel with infected cells. At the end of culture under each condition, the cells were harvested, and the luciferase activity was determined. Results are representative of two separate experiments performed in quadruplicate, and the error bars represent standard deviations. (B) CD4 and CD8 expression profiles of the thymocytes from over 7 days in culture. Results are representative of two separate experiments. (C) Maturation state of cells expressing virus. CD4 and CD8 expression profiles of total thymocytes were compared to those of cells expressing bright versus dim EGFP levels following costimulation. The panel on the left indicates the gating used in the EGFP-expressing panels to the right. Results are representative of two separate experiments. C illustrates the same experiment as that shown in Fig. 2C.
FIG. 4.
Reverse transcription and integration of NLEGFPLuc in infected CD4+ CD8+ thymocytes. (A) Luciferase assay of cells infected and cultured for 10 days as described in the legend of Fig. 1B. AZT was added at the indicated time periods following infection. Results are a representation of the average of two separate experiments, and the error bars represent the ranges. (B) _Alu_-PCR indicating levels of integration in thymocytes infected with NLEGFPLuc or an integrase-defective form of NLEGFPLuc on day 7 p.i. prior to costimulation. The results are shown as the number of integrated copies of HIV per 105 cells assessed in triplicate, and the error bars represent standard deviations. (C) Luciferase activity of cells from B that have been cultured for an additional 3 days under nonstimulated or costimulated conditions. Results are representation of two separate experiments assessed in quadruplicate, and the error bars represent standard deviations. Results shown in B and C are derived from the same experiment illustrated in Fig. 2C and 3C.
FIG. 5.
NF-κB response elements within the LTR of HIV greatly impact viral activation. (A) Sequence of the two NF-κB response elements within the LTR of HIV. Nucleotides mutated in the NLEGFPLucΔNFkB LTR are highlighted in boldface type. (B) EMSA using either the wild-type LTR probe or the NF-κB mutant probe on cellular extracts from nonstimulated (−) or costimulated (+) CD4+ CD8+ thymocytes from day 10. Three complexes induced by costimulation and disrupted by preincubation with antibodies to NF-κB subunits (“supershift”) or by NF-κB binding site mutations are indicated by arrows. One smaller complex unaffected by NF-κB antibodies or NF-κB binding site mutations is indicated by an asterisk. (C) Luciferase activity in cells infected with either wild-type or NF-κB mutant reporter virus and cultured as described in the legend of Fig. 1B. Results with the costimulated conditions are plotted as a percentage of expression from wild-type NLEGFPLuc. Results are a representation of three different experiments done in quadruplicate, and the error bars represent standard deviations.
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