HIV Cell-to-Cell Spread Results in Earlier Onset of Viral Gene Expression by Multiple Infections per Cell

Cell-to-cell spread of HIV, a directed mode of viral transmission, has been observed to be more rapid than cell-free infection. However, a mechanism for earlier onset of viral gene expression in cell-to-cell spread was previously uncharacterized. Here we used time-lapse microscopy combined with automated image analysis to quantify the timing of the onset of HIV gene expression in a fluorescent reporter cell line, as well as single cell staining for infection over time in primary cells. We compared cell-to-cell spread of HIV to cell-free infection, and limited both types of transmission to a two-hour window to minimize differences due to virus transit time to the cell. The mean time to detectable onset of viral gene expression in cell-to-cell spread was accelerated by 19% in the reporter cell line and by 35% in peripheral blood mononuclear cells relative to cell-free HIV infection. Neither factors secreted by infected cells, nor contact with infected cells in the absence of transmission, detectably changed onset. We recapitulated the earlier onset by infecting with multiple cell-free viruses per cell. Surprisingly, the acceleration in onset of viral gene expression was not explained by cooperativity between infecting virions. Instead, more rapid onset was consistent with a model where the fastest expressing virus out of the infecting virus pool sets the time for infection independently of the other co-infecting viruses

Intracellular growth of Mycobacterium tuberculosis after macrophage cell death leads to serial killing of host cells

A hallmark of pulmonary tuberculosis is the formation of macrophage-rich granulomas. These may restrict Mycobacterium tuberculosis (Mtb) growth, or progress to central necrosis and cavitation, facilitating pathogen growth. To determine factors leading to Mtb proliferation and host cell death, we used live cell imaging to track Mtb infection outcomes in individual primary human macrophages. Internalization of Mtb aggregates caused macrophage death, and phagocytosis of large aggregates was more cytotoxic than multiple small aggregates containing similar numbers of bacilli. Macrophage death did not result in clearance of Mtb. Rather, it led to accelerated intracellular Mtb growth regardless of prior activation or macrophage type. In contrast, bacillary replication was controlled in live phagocytes. Mtb grew as a clump in dead cells, and macrophages which internalized dead infected cells were very likely to die themselves, leading to a cell death cascade. This demonstrates how pathogen virulence can be achieved through numbers and aggregation states. DOI: http://dx.doi.org/10.7554/eLife.22028.00

Modelling faster onset of HIV gene expression by multiple infections per cell.

<p>(<b>A</b>) Proposed probabilistic mechanism for the more rapid onset of HIV gene expression. (<b>B</b>) Simulation of cell-free infection times at different MOI by random draws from a Gamma distribution parametrizing the best fit cell-free infection times of single virion infections. Circles are experimental data from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005964#ppat.1005964.g003" target="_blank">Fig 3A</a>, dashed lines represent the simulation results for MOI of 0.1 (blue), 0.5 (green), 2 (red), and 4 (purple). Fitted means±std of infection times were 26.4±5.2, 25.2±5.1, 22.7±4.8, and 21.9±4.9 hours. (<b>C</b>) Fit of coculture versus cell-free infection. Circles are experimental data from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005964#ppat.1005964.g001" target="_blank">Fig 1C</a>, dashed lines represent the simulation results. Means±std for the fits were 28.0±5.0 hours for coculture and 34.5±6.2 hours for cell-free infections. Best fit MOI for the coculture infection was 4.6.</p

Decreased sensitivity of coculture HIV spread to RAL predicts multiple infections per cell.

<p>Sensitivity of coculture (red) and cell-free (blue) infection to the integrase inhibitor raltegravir (RAL) in PBMCs. Points are means and standard errors of duplicates from one of three experiments with different blood donors. Transmission index (Tx) was calculated as the number of infected target cells with RAL divided by the number of infected target cells without RAL. Blue dashed line represents parametrization of the cell-free infection response to RAL in terms of IC₅₀ (1.9 nM) and hill coefficient (0.7). Red dashed line represents the fit of coculture infection using the decreased sensitivity to RAL (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005964#sec008" target="_blank">Materials and methods</a>). An effective MOI of 4.8 is predicted for coculture infection. Inset: Sensitivity of primary CD4⁺ T cell infection to RAL. Points are Tx means and standard errors of duplicates from one of three experiments with different blood donors. The drug sensitivity differences in CD4⁺ T cells between cell-free and coculture was statistically significant (p < 0.001, two-tailed unpaired t-test).</p

Earlier onset of HIV gene expression in PBMCs by coculture infection.

<p>(<b>A</b>) Gating strategy for detectably infected target cell frequency. Donors were labelled with CTFR and infection was assayed by flow cytometry following p24 staining for HIV Gag. Top row is coculture infection, bottom row is cell-free infection. Percent of infected targets in the population (bottom right quadrant) shown in red, and values for other subpopulations in black. (<b>B</b>) Timing of coculture (red) versus cell-free (blue) infection. Shown are means and standard errors of duplicates from one of three experiments with different blood donors. Frequencies of infected target cells were normalized by infected target cell frequency at 50 hours post infection. Means±std of best fit Gamma distributions were 22.1±9.3 hours for coculture and 34.2±9.1 hours for cell-free infection. Best fit MOI for the coculture infection was 5.0 (dashed red line). Inset: Infection of purified CD4⁺ T cells by cell-free HIV or autologous infected CD4⁺ T cells. Shown are mean and standard errors of duplicates of the frequencies of infected target cells normalized to the frequency of infected target cells at 48 hours post-infection. One of three experiments, each performed on purified CD4⁺ T cells from a different individual. Timing differences between cell-free and coculture in CD4⁺ T cells were statistically significant at 12, 24 and 36 hours post-infection (p<0.01, two-tailed unpaired t-test corrected for multiple comparisons using the Sidak-Bonferroni method).</p

HIV cell-to-cell spread leads to earlier onset of HIV gene expression.

<p>(<b>A</b>) Infection system. Left column shows uninfected (top plot) or HIV infected (bottom plot) E7 donor cells. Middle column shows uninfected (top plot) or HIV infected (bottom plot) G2 target cells. Right column shows the labelling of donor cells with CTFR for donor-target fusion exclusion. Numbers are percent of cells in the associated quadrant. (<b>B</b>) Time-lapse images from one field of view (FOV). At each time point post-infection, cells were imaged for GFP, mCherry, and CTFR fluorescence. Bar is 15μM. (<b>C</b>) Quantified timing of coculture (red) versus cell-free (blue) infection. Each point represents the frequency of infected target cells normalized to the mean frequency in the last three hours of the movie. Shown are means and standard errors over 25 FOVs. One of five independent experiments.</p

Acceleration of viral gene expression in coculture infection is not due to factors acting in trans.

<p>(<b>A</b>) Frequency of infected G2 target cells in coculture (red), cell-free (blue), coculture separated by transwell (green) or cell-free infection across the transwell (yellow). Shown are means and standard errors over 15 FOVs. One of three independent experiments. (<b>B</b>) Frequency of infected G2 target cells in coculture (red) versus cell-free infection in the absence (blue) or presence (green) of CD4⁺ primary T cells infected with NL-AD8 CCR5 tropic HIV which cannot infect G2 cells. Shown are means and standard errors over 25 FOVs. One of three independent experiments. For both experiments, frequencies of infected cells were normalized by mean infected target cell frequency in the last 3 hours of the movie. Fusion events leading to false positive target cells were excluded with CTFR.</p

How and When Retailers’ Sustainability Efforts Translate into Positive Consumer Responses: The Interplay Between Personal and Social Factors

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