The simian immunodeficiency virus targets central cell cycle functions through transcriptional repression in vivo

Abstract

A massive and selective loss of CD4+ memory T cells occurs during the acute phase of immunodeficiency virus infections. The mechanism of this depletion is poorly understood but constitutes a key event with implications for progression. We assessed gene expression of purified T cells in Rhesus Macaques during acute SIVmac239 infection in order to define mechanisms of pathogenesis. We observe a general transcriptional program of over 1,600 interferon-stimulated genes induced in all T cells by the infection. Furthermore, we identify 113 transcriptional changes that are specific to virally infected cells. A striking downregulation of several key cell cycle regulator genes was observed and shared promotor-region E2F binding sites in downregulated genes suggested a targeted transcriptional control of an E2F regulated cell cycle program. In addition, the upregulation of the gene for the fundamental regulator of RNA polymerase II, TAF7, demonstrates that viral interference with the cell cycle and transcriptional regulation programs may be critical components during the establishment of a pathogenic infection in vivo.

Figure 1. The peak of infection occurs at day 7 post infection.

The plasma viral load assessed by a quantitative PCR measurement of viral RNA copies in sampled peripheral blood (A). The major viral production is attributed to CD4+ central memory cells, which constitutes the main target for the CCR5 tropic SIV mac239. At day 10 post infection the cell associated viral load is 13 fold higher in the central memory cells compared to the naive CD4+ cells, as assessed by quantitative PCR for integrated gag DNA (B). A preferential selective loss of central memory cells occurs over the course of the infection. Over half of the central memory cells are lost at day 10 post infection, illustrated here as the percent change in the ratio of central memory cells over naive CD4+ T cells; assessed by flowcytometry (C).

Figure 2. Cell sorting scheme.

Flowcytometric cell sorting was used to isolate T cell subsets from each animal at day 0, and at day 7 and day 10 post infection. Subsets of naive and central memory CD4+ T cells and naive CD8+ T cells were sorted using markers to selectively isolate viable CD14− CD3+ cells subsetted as CD4+ CD28+ CD95− CD45RA+ CCR7+ naive CD4+ T cells and CD4+ CD28+ CD95+ CD45RA− CCR7+ central memory CD4+ T cells and further CD8+ CD45RA+ CCR7+ CD28+ CD95− naive CD8+ T cells (see orange highlighted box). The general phenotypic features defined by these markers were maintained over the course of the infection (day 0 – day 10). Arrows indicates the gating strategy for selecting each subset to be sorted. This is a representative figure for one of the four animals (DA9A). See Figure S2 for the profiles of all 4 animals.

Figure 3. Schematic figure illustrating factors affecting the transcriptional changes in the three different sorted T cell subsets over the course of the infection.

Figure 4. Targeted changes affecting the CD4+ central memory cells.

A. Heat map representation of a total set of 113 up and down regulated changes returned by the robust F-test (FDR<0.05). 35 genes are upregulated and 78 genes are downregulated. B. Isomap representation of PCA 1, 2 and 3 of upregulated targeted changes returned by the F-test (FDR<0.05) illustrating how sets of genes forms distinct biologic networks. C. Representation of the more stringent F-test (FDR<0.01) showing a set of 36 up and down regulated genes (8 and 28 respectively). The heat map representations display data normalized to a mean of 0. Hierarchical clustering was performed on the smallest maximum pair wise distance (maximum linkage).