Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 77 (2), 1392-402

Cellular Gene Expression Upon Human Immunodeficiency Virus Type 1 Infection of CD4(+)-T-cell Lines

Affiliations

Cellular Gene Expression Upon Human Immunodeficiency Virus Type 1 Infection of CD4(+)-T-cell Lines

Angélique B van 't Wout et al. J Virol.

Abstract

The expression levels of approximately 4,600 cellular RNA transcripts were assessed in CD4(+)-T-cell lines at different times after infection with human immunodeficiency virus type 1 strain BRU (HIV-1(BRU)) using DNA microarrays. We found that several classes of genes were inhibited by HIV-1(BRU) infection, consistent with the G(2) arrest of HIV-1-infected cells induced by Vpr. These included genes involved in cell division and transcription, a family of DEAD-box proteins (RNA helicases), and all genes involved in translation and splicing. However, the overall level of cell activation and signaling was increased in infected cells, consistent with strong virus production. These included a subgroup of transcription factors, including EGR1 and JUN, suggesting they play a specific role in the HIV-1 life cycle. Some regulatory changes were cell line specific; however, the majority, including enzymes involved in cholesterol biosynthesis, of changes were regulated in most infected cell lines. Compendium analysis comparing gene expression profiles of our HIV-1 infection experiments to those of cells exposed to heat shock, interferon, or influenza A virus indicated that HIV-1 infection largely induced specific changes rather than simply activating stress response or cytokine response pathways. Thus, microarray analysis confirmed several known HIV-1 host cell interactions and permitted identification of specific cellular pathways not previously implicated in HIV-1 infection. Continuing analyses are expected to suggest strategies for impacting HIV-1 replication in vivo by targeting these pathways.

Figures

FIG. 1.
FIG. 1.
Infection of CD4+-T-cell line CCRF-CEM with HIV-1BRU. (A and B) Cells were exposed to infectious virus at a multiplicity of infection of 2, heat-inactivated virus, or mock-infected cell supernatant. At 1 and 24 h postexposure, cells were fixed and permeabilized for intracellular p24 staining. Only cells exposed to the infectious virus stock showed detectable levels of p24 at 1 h postinfection (A) and increased p24 expression after 24 h (B). (C) Expression of HIV-1 RNA transcripts during infection. The graph depicts results for 17 HIV-1BRU infection experiments: a time course using CCRF-CEM cells and after 24 h of infection of CEMfh, CEMss, Jurkat, and SupT1 cells. Twelve PCR products representing different regions of the HIV-1BRU genome were spotted on each slide; nucleotide positions relative to the HIV-1BRU genome were 317 to 1281 (gag), 853 to 2471 (gag), 2179 to 3097 (pol), 3015 to 4363 (pol), 4337 to 5598 (pol), 5538 to 6974 (env), 6583 to 7404 (env), 7115 to 8631 (env), 8276 to 9225 (nef), 5402 to 5666 (tat), 7951 to 8272 (rev), 4619 to 5257 (vif), and 5124 to 5446 (vpr). Plotted are hybridization intensities of each of the 12 spots in the treated cells. HI (-) refers to mock infection versus heat-inactivated BRU, and HI (+) refers to heat-inactivated HIV-1BRU versus infectious HIV-1BRU.
FIG. 2.
FIG. 2.
Genes found to be differentially regulated under different selection criteria. (A) Effect of signature P on number of genes selected. (B) Number of down-regulated genes in each experiment using a signature P of ≤0.005. Depicted are genes down-regulated by at least 2-fold (white bars) or 1.5-fold (grey bars), and all differentially regulated genes (black bars). (C) Number of up-regulated genes in each experiment using a signature P of ≤0.005 and depicted as in panel B. (D) Effect of signature P and relative change on the correlation coefficient between duplicate experiments.
FIG. 3.
FIG. 3.
Gene groups up- or down-regulated by HIV-1BRU infection of CCRF-CEM cells. (A) Up-regulated genes; (B) down-regulated genes. Genes were divided into seven broad categories based on their function (middle pie charts), with each category depicted by a separate pattern and numbers reflecting percentages of genes in that category compared to total number of genes regulated. Genes involved in cell signaling and communication were mostly up-regulated; this category was further divided into seven classes (pie charts at left). Genes involved in gene and protein expression were mostly down-regulated; this category was further divided into eight classes (pie charts at right).
FIG. 4.
FIG. 4.
Gene clusters impacted by HIV-1BRU infection of five CD4+-T-cell lines. Log10 ratios are depicted in green (down-regulated) or red (up-regulated). The magnitude of the regulation is illustrated by the intensity of the color. HI, heat-inactivated BRU versus BRU. (A) Cell-type-specific responses to HIV-1 infection. Three hundred nineteen genes exhibited >2-fold change in expression in at least one of the eight HIV-1-infected cell line experiments (P ≤ 0.005). (B and C) Homogeneous responses to HIV-1 infection. Seventy-one genes were regulated in all cell lines with greater than 90% confidence (P ≤ 0.10); 46 genes were down-regulated (B), and 25 genes were up-regulated (C).
FIG. 5.
FIG. 5.
Verification of RNA expression and comparison to changes in protein levels. Array results were compared to semiquantitative RT-PCR for eight genes (A) and for three membrane antigens by FACS (B). (A) Total RNA samples from a 24-h infection of CEMfh cells with HIV-1BRU or mock infection were tested with primers for 18S internal control and gene-specific primers for eight different genes at two different cycles. The graph depicts results obtained using microarrays on the x axis and those obtained by RT-PCR on the y axis. (B) Five CD4+-T-cell lines were infected with HIV-1BRU or mock infected with culture supernatant from uninfected cells. After 24 h, cells were washed and stained with specific purified monoclonal antibodies or isotype controls. The level of regulation corresponded with the level of p24 expression in each cell line (data not shown). The graph depicts results obtained using microarrays on the x axis and those obtained by FACS on the y axis.
FIG. 6.
FIG. 6.
Impact of HIV infection compared to influenza A virus infection, heat shock, and interferon treatment. Two-dimensional cluster analysis of 271 genes in 34 array experiments comparing treatment of A549 cells with influenza A virus (16); HeLa, HFH, or HH2 cells with interferon (Geiss et al., unpublished observations); heat shock in CD4+-T-cell lines (van 't Wout et al., unpublished observations); and HIV-1 infection in CD4+-T-cell lines. Ratios were expressed as described in Fig. 4. Highlighted branches depict genes specifically regulated in HIV-1 infection experiments.

Similar articles

See all similar articles

Cited by 76 articles

See all "Cited by" articles

Publication types

Feedback