Pauline Chugh | PubMed Health

PubMed2006

Infection of human immunodeficiency virus and intracellular viral Tat protein exert a pro-survival effect in a human microglial cell line.

The interaction of human immunodeficiency virus type 1 (HIV-1) with CD4+ T lymphocytes is well studied and typically results in virally induced cytolysis. In contrast, relatively little is known concerning the interplay between HIV-1 and microglia. Recent findings suggest that, counter-intuitively, HIV-1 infection may extend the lifespan of microglia. We developed a novel cell line model system to confirm and mechanistically study this phenomenon. We found that transduction of a human microglial cell line with an HIV-1 vector results in a powerful cytoprotective effect following apoptotic challenge. This effect was reproduced by ectopic expression of a single virus-encoded protein, Tat. Subsequent studies showed that the pro-survival effects of intracellular Tat could be attributed to activation of the PI-3-kinase (PI3K)/Akt pathway in the microglial cell line. Furthermore, we found that expression of Tat led to decreased expression of PTEN, a negative regulator of the PI-3-K pathway. Consistent with this, decreased p53 activity and increased E2F activity were observed. Based on these findings, a model of possible regulatory circuits that intracellular Tat and HIV-1 infection engage during the cytoprotective event in microglia has been suggested. We propose that the expression of Tat may enable HIV-1 infected microglia to survive throughout the course of infection, leading to persistent HIV-1 production and infection in the central nervous system.

Cell Line↗

PubMed2006

Modification of human immunodeficiency virus type 1 reverse transcriptase to target cells with elevated cellular dNTP concentrations.

Retroviruses and DNA viruses utilize cellular dNTPs as substrates for their DNA polymerases during viral replication in infected cells. However, because of S phase-dependent dNTP biosynthesis, the availability of cellular dNTPs significantly varies among cell types (e.g. dividing versus nondividing cells and normal versus tumor cells). Here we tested whether alterations in the dNTP utilization efficiency and dNTP binding affinity of viral DNA polymerases can switch viral infection specificity to cell types with different dNTP concentrations. We employed an HIV-1 reverse transcriptase (RT) mutant (Q151N), which is catalytically active only at high dNTP concentrations because of its reduced dNTP binding affinity. Indeed, the modified HIV-1 vector harboring the Q151N mutant RT preferentially transduced tumor cells containing higher cellular dNTP concentrations than primary cells (e.g. human lung fibroblasts (HLFs) and human keratinocytes). Although the wild type HIV-1 vector transduced both HLFs and tumor cells, the Q151N vector failed to transduce HLFs and keratinocytes but efficiently transduced tumor cells. Pretreatment of HLFs with deoxynucleosides, which increase cellular dNTP pools, enabled the mutant vector to transduce HLFs, suggesting that the transduction failure of the RT mutant vector to primary cells is because of inefficient reverse transcription in low cellular dNTP environments. We also observed that the Q151N vector expressing herpes simplex virus-thymidine kinase renders tumor cells sensitive to gancyclovir. This study validates a novel strategy in which modifications of viral DNA polymerases in various vector systems allow the delivery of target genes exclusively to tumor cells exploiting elevated cellular dNTP concentration as a tumor cell-specific host factor.

Cells, Cultured↗