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Analysis of human immunodeficiency virus cytopathicity by using a new method for quantitating viral dynamics in cell culture.

ABSTRACT: Human immunodeficiency virus (HIV) causes complex metabolic changes in infected CD4(+) T cells that lead to cell cycle arrest and cell death by necrosis. To study the viral functions responsible for deleterious effects on the host cell, we quantitated the course of HIV type 1 infection in tissue cultures by using flow cytometry for a virally encoded marker protein, heat-stable antigen (HSA). We found that HSA appeared on the surface of the target cells in two phases: passive acquisition due to association and fusion of virions with target cells, followed by active protein expression from transcription of the integrated provirus. The latter event was necessary for decreased target cell viability. We developed a general mathematical model of viral dynamics in vitro in terms of three effective time-dependent rates: those of cell proliferation, infection, and death. Using this model we show that the predominant contribution to the depletion of viable target cells results from direct cell death rather than cell cycle blockade. This allows us to derive accurate bounds on the time-dependent death rates of infected cells. We infer that the death rate of HIV-infected cells is 80 times greater than that of uninfected cells and that the elimination of the vpr protein reduces the death rate by half. Our approach provides a general method for estimating time-dependent death rates that can be applied to study the dynamics of other viruses.

SUBMITTER: Speirs C

PROVIDER: S-EPMC1061548 | biostudies-literature | 2005 Apr

REPOSITORIES: biostudies-literature

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Analysis of human immunodeficiency virus cytopathicity by using a new method for quantitating viral dynamics in cell culture.

Speirs Christina C van Nimwegen Erik E Bolton Diane D Zavolan Mihaela M Duvall Melody M Angleman Sara S Siegel Richard R Perelson Alan S AS Lenardo Michael J MJ

Journal of virology 20050401 7

Human immunodeficiency virus (HIV) causes complex metabolic changes in infected CD4(+) T cells that lead to cell cycle arrest and cell death by necrosis. To study the viral functions responsible for deleterious effects on the host cell, we quantitated the course of HIV type 1 infection in tissue cultures by using flow cytometry for a virally encoded marker protein, heat-stable antigen (HSA). We found that HSA appeared on the surface of the target cells in two phases: passive acquisition due to a ...[more]

PMID: 15767404

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Project description:Nuclear co-localization labels are critical to ocular research. Among these, the TUNEL assay has been established as a gold standard of cell death and apoptosis. While several validated computer-based methods exist to quantitate these markers, including ImageJ Retina Analysis (RA) Toolkit and ImagePro, none verify the count with the nuclear counter stain to confirm nuclear co-localization. We established a new ImageJ-based automated multichannel thresholding (MCT) method to quantitate nuclear co-localized labeling. The MCT method was validated by comparing it with the two published TUNEL analysis in TUNEL-positive photoreceptors in an experimental retinal detachment (RD) model. RDs were induced in murine eyes and cross-sectional images of TUNEL and DAPI counter stain were obtained. Images were classified as "typical" or high density "hotspot" TUNEL regions (n?=?10/group). Images were analyzed and compared between the MCT method and the published techniques including both "standard" and "high" settings of the RA Toolkit for detecting lower or higher TUNEL densities, respectively. Additional testing of the MCT method with built-in ImageJ thresholding algorithms was performed to produce fully automated measurements. All images were compared with Bland-Altman mean difference plots to assess the difference in counts and linear regression plots to assess correlation. Comparison between the MCT method and the ImagePro method were found to be well correlated (typical: R2?=?0.8972, hotspot: R2?=?0.9000) with minor to non-significant differences. The RA Toolkit settings were found to be mostly well correlated as well (standard/typical: R2?=?0.8036, standard/hotspot: R2?=?0.4309, high/typical: R2?=?0.7895, high/hotspot: R2?=?0.8738) but were often found to have significantly higher counts than the MCT. In conclusion, the MCT method compared favorably with validated computer-based methods of nuclear marker immunofluorescence quantitation and avoids staining artifacts through the incorporation of the nuclear counter stain to confirm positive cells.

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Analysis of human immunodeficiency virus cytopathicity by using a new method for quantitating viral dynamics in cell culture.

Publications

Analysis of human immunodeficiency virus cytopathicity by using a new method for quantitating viral dynamics in cell culture.

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