Unknown,Transcriptomics,Genomics,Proteomics

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Investigation of temporal changes in cellular gene expression upon infection with HIV-1.

ABSTRACT: The purpose was to study temporal changes in cellular gene expression upon infection with HIV-1. The T lymphoblastic leukemia cell line SUP-T1 was infected with HIV-1 The purpose was to study temporal changes in cellular gene expression upon infection with HIV-1. 1x10^6 homo sapien were infected with LAI/lab strain at MOI giving infection of almost 100% of cells; this was visible in a QC step in which cells 24 hours after infection were stained for p24 antigen. MOI was set so that this infection rate was achieved after 1 hour incubation of cells with virus. Mock, UV-inactivated virus, and live virus infections were carried out in three (each) separate tubes and infected cells were maintained in separate suspensions (9 samples each). Time point aliquots were then collected in 4-hr intervals from each suspension, cells were pelleted, and lysed in 1 ml Trizol. Lysed samples were immediately frozen on dry ice and moved to -80M-BM-0C until RNA extraction, probe labeling and microarray analysis.

ORGANISM(S): Homo sapiens

SUBMITTER: Michael Katze

PROVIDER: E-GEOD-56484 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Our previous work demonstrated that HIV-1 infection progressively reduces TCR/CD3 expression due to a defect in CD3g gene transcripts. We further found that knocking down expression of the viral tat and/or nef genes was correlated with CD3g transcript and TCR/CD3 surface receptor levels on HIV-1 infected cells. This study was undertaken to investigate the direct effect of HIV-1 Tat expression on the TCR/CD3 machinery. Progressive downregulation from TCR/CD3hi to TCR/CD3lo to TCR/CD3â was observed on Tat expressing cells in a manner that emulated HIV-1 infection, with a lack of CD3g transcripts again responsible for the defect. When Tat cell cultures containing a mixture of TCR/CD3 surface densities were separated into TCR/CD3hi and TCR/CD3lo/â populations, they quickly reverted to a mixed CD3 phenotype. Thus, the progression TCR/CD3hi to TCR/CD3lo to TCR/CD3â is an active, reversible process with receptor levels fluctuating in response to intracellular dynamics. Examination of tat mutants found that the regions involved in Tat-mediated transactivation and TAR binding are required for TCR/CD3 downregulation while the lysine at position 28 and Tat exon 2 are dispensable. Global gene expression, assessed in association with TCR/CD3 downregulation in HIV-1 infected and Tat expressing cells, detected broad suppression of TCR/CD3 signaling, co-stimulation and negative regulatory genes along with target transcription factors, ligands and receptors. A significant subset of the genes altered in HIV-1 infected cells was specifically targeted by Tat in association with TCR/CD3 loss. Our finding that Tat negatively regulates many facets of the TCR/CD3 machinery has important implications for disease pathogenesis. We used microarrays to investigate changes in CD4+ T cell gene expression induced by HIV-1 infection for comparison with the Tat expressing cells. Examine changes in gene expression in TCR/CD3 negative HIV-1 infected cells compared to TCR/CD3 positive uninfected controls.

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Investigation of temporal changes in cellular gene expression upon infection with HIV-1.

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