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. 2012;8(7):e1002818.
doi: 10.1371/journal.ppat.1002818. Epub 2012 Jul 26.

Directly infected resting CD4+T cells can produce HIV Gag without spreading infection in a model of HIV latency

Matthew J Pace  1 , Erin H Graf, Luis M Agosto, Angela M Mexas, Frances Male, Troy Brady, Frederic D Bushman, Una O'Doherty
Affiliations

Directly infected resting CD4+T cells can produce HIV Gag without spreading infection in a model of HIV latency

Matthew J Pace et al. PLoS Pathog. 2012.

Abstract

Despite the effectiveness of highly active antiretroviral therapy (HAART) in treating individuals infected with HIV, HAART is not a cure. A latent reservoir, composed mainly of resting CD4+T cells, drives viral rebound once therapy is stopped. Understanding the formation and maintenance of latently infected cells could provide clues to eradicating this reservoir. However, there have been discrepancies regarding the susceptibility of resting cells to HIV infection in vitro and in vivo. As we have previously shown that resting CD4+T cells are susceptible to HIV integration, we asked whether these cells were capable of producing viral proteins and if so, why resting cells were incapable of supporting productive infection. To answer this question, we spinoculated resting CD4+T cells with or without prior stimulation, and measured integration, transcription, and translation of viral proteins. We found that resting cells were capable of producing HIV Gag without supporting spreading infection. This block corresponded with low HIV envelope levels both at the level of protein and RNA and was not an artifact of spinoculation. The defect was reversed upon stimulation with IL-7 or CD3/28 beads. Thus, a population of latent cells can produce viral proteins without resulting in spreading infection. These results have implications for therapies targeting the latent reservoir and suggest that some latent cells could be cleared by a robust immune response.

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Conflict of interest statement

This work was funded in part by a grant from Merck. There are no other connections (patents, consultation, products in development etc). This does not alter our adherence to all PLoS Pathogens policies on sharing data and materials.

Figures

Figure 1
Figure 1. Spinoculation, IL-7 and CCL19 do not alter the susceptibility of resting CD4+T cells to HIV integration.
An experimental schematic summarizing all experiments is shown in A. In B, a representative experiment shows the levels of integration measured at 0, 24, 48, and 72 hours post infection in resting and CD3/28 activated CD4+T cells. In C, purified resting cells were infected with HIV (MOI of 3) without spinoculation or were spinoculated at 300×g or 1200×g. Viral binding, total and integrated HIV DNA were measured. The average of 3 experiments in 3 different donors is shown. In D, purified resting cells or cells stimulated with CCL19, IL-7, or CD3/28 beads were spinoculated for 2 hours at 1200×g at an MOI of 3. The average total and integrated levels of 3 donors are shown. In E, cells were treated as in B. Cells were collected as described in A. The average ratio of integrated to total HIV DNA for 3 experiments with 3 different donors is shown. Error bars represent the standard error of the measurements. 1 This condition was modified in some later experiments and if so was noted. 2 The timepoint changed in some later experiments and if so was noted. *Statistically significant p<0.017. #p = 0.018, not statistically significant due to Holmes correction.
Figure 2
Figure 2. Infected resting CD4+T cells can produce HIV Gag.
Bulk unstimulated CD4+T cells were spinoculated and cultured in the presence of the protease inhibitor, saquinavir. Gag protein was measured in the endogenously activated (HLA-DR+, CD25+, or CD69+) and resting (HLA-DR−,CD25−,and CD69−) cells based on activation marker expression at 0, 24, 48, 72, and 96 hours post infection. Control cells were treated with the reverse transcriptase (RT) inhibitor, efavirenz, to establish background protein levels. An average of 3 experiments in 3 different donors is shown (A). In B, purified resting or CCL19 treated cells were infected with HIV with or without spinoculation at 1200×g. Controls and gates were made as in A. Cells were gated on the activation marker negative (HLA-DR−,CD25−,CD69−) population. Approximately 10,000 events were collected for spinoculated samples while approximately 100,000 events were collected for cells that were not spinoculated. Data is a representative of 2 experiments in 2 different donors.
Figure 3
Figure 3. Gag expressing resting CD4+T cells remain in a latent state.
In A, purified resting CD4+T cells were spinoculated with NL4-3 (MOI of 3) and cultured in the presence or absence of saquinavir for 4 days. Intracellular Gag was measured 96 hours post infection. Control cells were treated as in A and gates were set using an efavirenz treated control. Then resting cells cultured without saquinavir were stimulated with PHA+100 U/mL IL-2 for 48 hours in the presence or absence of saquinavir. Intracellular Gag was again measured. In B, cells were purified and treated as in Figure 1 but infected at a MOI of 0.2. After infection, half the cells were treated with saquinavir. Cells were collected at 72 hours for CD3/28 activated cells and at 7 days post infection for all other cells. Total DNA was measured in both fractions of cells. The average of 3 experiments with 3 different donors is shown. In C, resting and CD3/28 activated cells were spinoculated with HIV (MOI of 3). Supernatant was collected 96 hours post infection. CEMss-GFP cells were spinoculated with the collected supernatant from resting and activated cells. An efavirenz control was used to determine background GFP levels. A representative experiment is shown in C. An average of 2 experiments in 2 different donors is shown in D. Error bars represent the standard error of the measurements. *Statistically significant at p<0.05 level. ND = not detectable.
Figure 4
Figure 4. Resting CD4+T cells produce Gag but barely detectable levels of Env Cells were infected as in Figure 1 (MOI of 3) in the presence of 8 µg/mL polybrene.
Cells were cultured in the presence of 25 µg/mL anti-CD4 clone 19, and Gag and Env protein levels were measured at 72 hours (48 hours in CD3/28 treated cells). A representative experiment is shown in A. An efavirenz control was used to control for background levels. The average of 3 experiments with 3 different donors is shown in B. The average MFI (median fluorescence intensity) of the Gag+ cells in resting and CD3/28 activated cells is shown in C. Error bars represent the standard error of the measurements. *Statistically significant at p<0.05 level.
Figure 5
Figure 5. Protein expression differences are reflected at the transcript level.
A schematic showing the different HIV RNA species and the primers used to detect them is shown in A. The upper portion depicts the viral genome while the lower portion depicts the various RNA transcripts. For the different RNA species, black boxes represent regions present in all forms of the indicated viral transcript while white boxes represent regions that may or may not be present. GF and GR were used to detect gag levels. RF and ER were used to detect env. RF and VR were used to detect vif, and RF and TR were used to detect tat/rev. Primers were confirmed to solely detect their respective products by gel electrophoresis. Cells were treated and infected as in Figure 4. Cells were cultured in the presence or absence of the integrase inhibitor, raltegravir. RNA was collected at 72 hours (48 hours in CD3/28 treated cells). RNA levels were calculated per cell and were normalized to integration levels. gag levels were obtained by subtracting the levels of gag/cell in the raltegravir treated fraction from the levels in the - raltegravir fraction. An average of 3 experiments in 3 different donors is shown (B). In C, resting cells were infected with or without spinoculation and env and vif levels were measured as in B. An average of 3 experiments in 3 different donors is shown. Error bars represent the standard error of the measurements. *Statistically different at the p<0.05 level.
Figure 6
Figure 6. Differences in HIV integration site selection between Gag positive and negative cells.
Unstimulated cells were spinoculated with NL4-3. At 96 hours post infection, cells were stained with antibodies for the activation markers HLA-DR, CD25, and CD69 as well as for intracellular Gag. Cells were sorted into 4 populations: activated Gag−, activated Gag+, resting Gag−, and resting Gag+. Numbers reflect the percentage of each population in the total infected unstimulated cells (A). A heatmap showing relationships of integration site distributions to various genomic features is shown in (B). Each column represents a different population and each row a genomic feature. Darker shades of red indicate associations observed more than random and darker shades of blue indicate less than random. *p<0.05, **p<0.01, ***p<0.001.
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