Mechanisms of human immunodeficiency virus-1 latency.
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CD4-Positive T-Lymphocytes↗
Biomedical subjects
Una O'Doherty
Publications and source records attributed to Una O'Doherty.
Human immunodeficiency virus type 1 can establish latent infection in resting CD4+ T cells in the absence of activating stimuli.
Resting CD4(+) T cells are the best-defined reservoir of latent human immunodeficiency virus type 1 (HIV-1) infection, but how the reservoir is formed is unclear. Understanding how the reservoir of latently infected cells forms is critical because it is a major barrier to curing HIV infection. The system described here may provide an in vitro model of latent HIV-1 infection in resting CD4(+) T cells. We demonstrated that HIV-1 integrates into the genomes of in vitro-inoculated resting CD4(+) T cells that have not received activating stimuli and have not entered cell cycle stage G(1b). A percentage of the resting CD4(+) T cells that contain integrated DNA produce virus upon stimulation, i.e., are latently infected. Our results show that latent HIV-1 infection occurs in unstimulated resting CD4(+) T cells and suggest a new route for HIV-1 reservoir formation.
Acquired Immunodeficiency Syndrome↗
Long HIV type 1 reverse transcripts can accumulate stably within resting CD4+ T cells while short ones are degraded.
We utilized quantitative methods to compare the efficiency of reverse transcription and stability of viral DNA within resting and activated T cells. Highly purified resting CD4(+) T cells and activated T cells from healthy donors were spinoculated with HIV-1(YU-2), then cultured in conditions that maintain both the viability and the quiescence of the resting cells. Spreading infection was suppressed, then kinetic PCR was used to relate the rates of synthesis of short (strong-stop, RU5) and long (gag or U3-gag second strand transfer) viral DNA to the mean number of virions initially bound to each type of cell. As shown previously, activated cells support an initial burst of high-level reverse transcription, which is then followed by a approximately 10-fold decay in cDNA levels over 4.5 days. In resting T cells, although the synthesis of late reverse transcripts was initially approximately 1000-fold less efficient than in activated T cells, the number of these cDNAs per bound input virion rose 10-fold as culture was extended to 4.5 days. The number of late reverse transcripts remained constant for 3 days after the addition of efavirinez, reflecting enhanced stability. In contrast, the short strong-step reverse transcripts were mostly degraded. Thus, late HIV-1 reverse transcripts can accumulate stably in resting T cells in the absence of detectable T cell activation. Defining the underlying basis for the stabilization of late reverse transcripts, and their associated nucleoprotein complexes, may be pertinent to the accumulation of reservoirs of latent HIV-1 in patients, and could provide a target for future therapies.
CD4-Positive T-Lymphocytes↗
Can digitally reconstructed radiographs (DRRS) replace simulation films in prostate cancer conformal radiotherapy?
PURPOSE: To evaluate the precision of using digitally reconstructed radiographs (DRRs) of either 3 mm or 6 mm slice separation vs. using simulator images for the setup verification of patients receiving CT planned conformal radiotherapy to the prostate. To calculate the transfer error between CT and simulator. METHODS AND MATERIALS: Twenty patients were CT scanned (3 mm slice spacing/width). DRRs were generated for both 3 mm (DRR 3) and 6 mm (DRR 6) separations. DRRs and a simulator image of an anterior and a lateral field were used as reference images. Five observers matched each of the reference images to treatment images using the Theraview "Target check" facility. It was assumed that poorer images would lead to a loss of precision of field placement estimations (FPE) between observers. The study was designed to detect a difference greater than 1.5 mm(2) in the precision of image placement. The transfer error was the mean difference in the setup error derived from the DRRs and the simulation films. RESULTS: The precision of evaluations for simulator films and 3 mm DRRs were similar. There was a trend for the DRR 6 mm to achieve less precise results which was greatest for craniocaudal examinations (variance: simulator 1.5 mm(2), DRR6 2.8 mm(2), p = 0.17), but this did not reach statistical significance. A range of transfer errors was identified, with standard deviations ranging from 1.7 to 4.2 mm. There was evidence of a significant systematic bias in anterior craniocaudal (1.3-1.9 mm, p < 0.004) and anterior posterior (-1.9 mm, p = 0.027). CONCLUSION: The precision of setup evaluations using DRRs is similar to that achieved by using simulator fields when planning conformal prostate radiotherapy. The use of DRRs could reduce systematic errors introduced in the planning process.
Computer Simulation↗
A sensitive, quantitative assay for human immunodeficiency virus type 1 integration.
Quantitative methods to measure human immunodeficiency virus type 1 (HIV-1) integration promise to be important tools in dissecting the mechanisms whereby latent reservoirs of provirus are established, most notably in the resting T cells of patients receiving antiretroviral therapy. Here we describe a fluorescence-monitored, nested PCR assay that is able to quantify the relatively rare integration events that occur within these cells. Following DNA extraction, a nonkinetic preamplification step is performed with primers that bind genomic Alu elements and HIV-1 gag sequences, under conditions where primers, deoxynucleoside triphosphates, and enzyme are not limiting. This is followed by a kinetic PCR that quantitates HIV-1 long terminal repeat sequences. A T-cell-based integration standard which reflects the randomness of HIV-1 integration is also described. The assay is 10 to 100 times more sensitive than previously reported quantitative Alu PCR-based integration assays. It is specific for integration events, since no proviruses are detected in cells infected either in the presence of an integrase inhibitor or with an integrase-deficient virus. This method promises to provide important new insights into the processes underlying the accumulation and persistence of latent HIV-1 reservoirs and may eventually be useful clinically in monitoring the eradication of latent virus by novel therapies.
CD4-Positive T-Lymphocytes↗