Gene expression and viral prodution in latently infected, resting CD4+ T cells in viremic versus aviremic HIV-infected individuals

Abstract

The presence of HIV-1 in latently infected, resting CD4(+) T cells has been clearly demonstrated in infected individuals; however, the extent of viral expression and the underlying mechanisms of the persistence of HIV-1 in this viral reservoir have not been fully delineated. Here, we show that resting CD4(+) T cells from the majority of viremic patients are capable of producing cell-free HIV-1 spontaneously ex vivo. The levels of HIV-1 released by resting CD4(+) T cells were not significantly reduced in the presence of inhibitors of cellular proliferation and viral replication. However, resting CD4(+) T cells from the majority of aviremic patients failed to produce virions, despite levels of HIV-1 proviral DNA and cell-associated HIV-1 RNA comparable to viremic patients. The DNA microarray analysis demonstrated that a number of genes involving transcription regulation, RNA processing and modification, and protein trafficking and vesicle transport were significantly upregulated in resting CD4(+) T cells of viremic patients compared to those of aviremic patients. These results suggest that active viral replication has a significant impact on the physiologic state of resting CD4(+) T cells in infected viremic patients and, in turn, allows release of HIV-1 without exogenous activation stimuli. In addition, given that no quantifiable virions were produced by the latent viral reservoir in the majority of aviremic patients despite the presence of cell-associated HIV-1 RNA, evidence for transcription of HIV-1 RNA in resting CD4(+) T cells of aviremic patients should not necessarily be taken as direct evidence for ongoing viral replication during effective therapy.

Figure 1

Quantitation of cell-free HIV-1 virions released by latently infected, resting CD4⁺ T cells from representative viremic (A and B) and aviremic (C and D) patients. Cell-free supernatants collected from each culture at days 1 and 3 were subjected to Amplicor HIV-1 test (detection limit: 50 copies of HIV-1 RNA per ml) for quantitation of HIV-1 virions.

Figure 2

Levels of cell-free HIV-1 virions released by latently infected, resting CD4⁺ T cells from viremic and aviremic patients. Cell-free supernatants from each culture harvested at day 1 were subjected to Amplicor HIV-1 test (detection limit: 50 copies of HIV-1 RNA per ml). The geometric mean values are shown as black bars.

Figure 3

Frequency of latently infected, resting CD4⁺ T cells carrying HIV-1 proviral DNA (A) and cell-associated, unspliced HIV-1 RNA (B) from viremic and aviremic patients. Freshly isolated, resting CD4⁺ T cells were subjected to real-time PCR for detection of HIV-1 proviral DNA and cell-associated HIV-1 RNA. The geometric mean values are shown as black bars.

Figure 4

Clustering of differentially expressed genes in resting CD4⁺ T cells of viremic patients, aviremic patients, and healthy HIV-negative donors. Transcription profiles of resting cells from five viremic patients, five aviremic patients, and four HIV-negative donors were examined by using high-density microarrays. Levels of gene expression were assayed on Affymetrix U95A chips, and 535 differentially expressed genes were identified. Genes were grouped by using K-means clustering, and samples were grouped by hierarchical clustering. Clusters 4 and 5 indicate a set of 370 genes that were up-regulated in viremic patients relative to both aviremic and healthy individuals. Statistical analysis (Fisher exact test) of the genes in clusters 4 and 5 determined that the top three functional categories, transcription regulators, RNA processing/modification, and protein trafficking/vesicle, were overrepresented in this list of genes relative to all annotated genes on the chip. Differences in relative levels of gene expression (Z-score) are indicated in color, where red indicates up-regulation and green indicates down-regulation. Some genes were listed twice because certain probes recognize multiple regions of a single gene. Levels of expression of housekeeping genes are also shown.