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.

FIG. 1.

Expression of the HSA marker protein correlates with two phases of infection. (A) Jurkat 1.9 cells (10⁶) were infected with serial dilutions of HIV-1 NL4-3_HSA Env⁻ virus. Each histogram shows the HSA expression of live cells, and the percentage of gated HSA⁺ cells (compared to mock) is given at 8 and 23 h p.i. Virus was serially diluted, and the MOIs were as follows: (a) mock infected, MOI = 0; (b) 1:243 serial dilution of virus, MOI = 0.03; (c) 1:81 serial dilution of virus, MOI = 0.09; (d) 1:27 serial dilution of virus, MOI = 0.28; (e) 1:9 serial dilution of virus, MOI = 0.83; (f) 1:3 serial dilution of virus, MOI = 2.5; (g) undiluted virus stock, MOI = 7.5. In the bottom right corner (h) of each group of panels, the viability profile of sample g as detected by forward light scatter (x axis) and side light scatter (y axis) is shown. Viable cells are shown within the polygon, and the fraction of total events is given. (B) Representative experiment in which the sample identity and viral titer were the same as those described for panel A, analyzed 23 and 46 h after the start of infection. The units on the x axis are arbitrary fluorescence units, and the units on the y axis are event counts. A total of 5,000 events were collected for each sample.

FIG. 2.

Relationship of HSA expression levels to cell viability. (A) Thresholds of HSA staining distinguish levels of infection associated with different fates of the infected cell. Data are derived from sample number 4 on day 4 shown in panel B. Low-negative expression indicates HSA-negative cells or transient HSA passively acquired from virion-cell interaction. Cells expressing HSA at the medium level (Med) are productively infected but have not yet reached high levels of expression. High-level expressers (Hi) are at the peak of infection, which leads to cell death. (B) Quantification of samples gated on HSA expression as for panel A. The various times and MOIs are shown. (C) Viability profiles for the infected samples represented in panel B. Serial dilutions of NL4-3_HSA Env⁻ virus were used for Jurkat 1.9 cell infection, which was monitored by HSA expression.

FIG. 3.

HSA detected on the cell surface during infection represents two mechanisms of acquisition. (A) Jurkat cells were cultured in plain medium or in the presence of either the nucleoside reverse transcriptase inhibitor 3TC or the protease inhibitor SQV. 3TC (10 μM) was added for 24 h prior to the infection and replenished every 24 h. Alternatively, the protease inhibitor SQV (1 μM) was added to Jurkat cells at the time of infection and replenished every 48 h. Surface expression of HSA and the levels of intracellular p24 (inset) were determined by flow cytometry. (B) Mixing virus and cells at 3°C significantly reduces detectable HSA on the cell surface. A total of 5 × 10⁵ Jurkat 1.9 cells were spin infected with various dilutions of a concentrated stock of NL4-3_HSA for 2.5 h, at 800 × g, at either 3 or 37°C. Immediately following centrifugation, samples were analyzed for surface expression of HSA (virion HSA) by flow cytometry. The samples and all reagents were maintained at 3°C following the spin infection to prevent subsequent viral fusion. The average percentage of HSA-positive cells from four independent samples is graphed, with error bars indicating one standard deviation. (C) Cell surface HSA staining in early stages of infection is partially sensitive to proteinase K cleavage. A total of 5 × 10⁵ mock- or NL4-3_HSA-infected Jurkat 1.9 CD4⁺ T cells were washed in PBS, incubated for 45 min in a rotary shaker at 4°C in either PBS (left panels) or 0.5 mg of proteinase K/ml (right panels), washed again in PBS, and stained for HSA. Control stains showed that CD4 was completely removed, but CXCR4 was unaffected by protease treatment (data not shown). HSA levels on HIV-1-infected cells were analyzed at day 1 (upper panels) and day 3 (lower panels) p.i. The fraction of HSA⁺ viable cells is shown. Ten thousand live events were acquired for each sample.

FIG. 4.

Range of likely fits for uninfected cells. A time course of triplicate measurements of viability in cultures was made with uninfected cells (dots). The solid line is the maximum likelihood fit to the data. The dashed lines show fits with a likelihood that is a factor e lower than the maximum likelihood fit.

FIG. 5.

Inferred effective death rates for infected cells as a function of time, viral strain, and viral concentration. The fold increase q(t) (z axis) of the death rate of infected cells compared to uninfected cells as a function of time (x axis) and the amount of cell cycle blockage p(t) of infected cells (y axis). Each plot shows the results for a different initial viral concentration: 0.5- (A), 1.0- (B), or 1.5-ml titer (C). Each surface corresponds to a particular viral strain. The top surface in each plot corresponds to wild-type virus. The middle surface corresponds to the vpr mutant strain, and the bottom surface corresponds to mock infections. Time runs from day 1 to day 6 after infection on the x axis. The proliferation rate p(t) runs from 0 (complete cell cycle blockage) to 1 (no cell cycle blockage) on the y axis. The fold change runs from 0 to 120 on the z axis, e.g., a value of 50 on the z axis indicates that we inferred from the measurements of infectivity and viability that, at that point of time in the infection and assuming that particular amount of cell cycle blockage, the infected cells were dying at a 50 times higher rate than uninfected cells.

FIG. 6.

Inferred cytopathicity of different viral strains at different viral concentrations. The x-axis shows time in days and the y-axis shows the fold change in death rate of infected cells for the data in Fig. 5. Each bar indicates the inferred lower and upper bounds of the death rate that are obtained by setting p(t) = 0 and p(t) = 1, respectively. Green bars with triangles correspond to mock infection, blue bars with stars to the vpr mutant strain, and red bars with diamonds to wild-type virus. Each triplet of bars corresponds to cultures with viral-titers of 0.5 ml, 1 ml, and 1.5 ml, from left to right, respectively. Overlapping data sets were slightly shifted horizontally with respect with one another to make the figure easier to view. However, all samples for each set of viral titers were collected at the same time points.