title: Identification and cloning of human astrocyte genes displaying elevated expression after infection with HIV-1 or exposure to HIV-1 envelope glycoprotein by rapid subtraction hybridization, RaSH.
abstract: Neurodegeneration and dementia are common complications of AIDS caused by human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system. HIV-1 target cells in the brain include microglia, infiltrating macrophages and astrocytes, but rarely neurons. Astrocytes play an important role in the maintenance of the synaptic micro-environment and in neuronal signal transmission. To investigate potential changes in cellular gene expression associated with HIV-1 infection of astrocytes, we employed an efficient and sensitive rapid subtraction hybridization approach, RaSH. Primary human astrocytes were isolated from abortus brain tissue and low-passage cells were infected with HIV-1. To identify genes that display both early and late expression modifications after HIV-1 infection and to avoid cloning genes displaying normal cell cycle fluctuations in astrocytes, RNAs were isolated and pooled from 6, 12, 24 h and 3 and 7 day uninfected and infected cells and used for RaSH. Temporal cDNA libraries were prepared from double-stranded cDNAs that were enzymatically digested into small fragments, ligated to adapters, PCR amplified, and hybridized by incubation of tester and driver PCR fragments. By subtracting temporal cDNAs derived from uninfected astrocytes from temporal cDNAs made from HIV-1 infected cells, genes displaying elevated expression in virus infected cells, termed astrocyte elevated genes (AEGs), were identified. Both known and novel AEGs, not reported in current DNA databases, are described that display early or late expression kinetics following HIV-1 infection or treatment with recombinant HIV-1 envelope glycoprotein (gp120). For selected AEGs, expression of their protein products was also tested by Western blotting and found to display elevated expression following HIV-1 infection. The comparable pattern of regulation of the AEGs following HIV-1 infection or gp120 treatment suggest that HIV-1 exposure of astrocytes, even in the absence of productive infection, can induce changes in cellular gene expression.
authors.affiliation: Department of Pathology and Herbert Irving Comprehensive Cancer Center, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA.
authors.forename: Zao-Zhong
authors.initials: ZZ
authors.lastname: Su
authors.forename: Dong-Chul
authors.initials: DC
authors.lastname: Kang
authors.forename: Yinming
authors.initials: Y
authors.lastname: Chen
authors.forename: Olga
authors.initials: O
authors.lastname: Pekarskaya
authors.forename: Wei
authors.initials: W
authors.lastname: Chao
authors.forename: David J
authors.initials: DJ
authors.lastname: Volsky
authors.forename: Paul B
authors.initials: PB
authors.lastname: Fisher
mesh_terms: D000185:Actinin / Q000096:biosynthesis; D001253:Astrocytes / Q000378:metabolism / Q000821:virology*; D015152:Blotting, Northern; D001921:Brain / Q000166:cytology / Q000196:embryology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D005353:Fibronectins / Q000096:biosynthesis; D020869:Gene Expression Profiling / Q000379:methods*; D015699:HIV Envelope Protein gp120 / Q000494:pharmacology*; D015497:HIV-1 / Q000472:pathogenicity*; D006801:Humans; D007700:Kinetics; D009693:Nucleic Acid Hybridization / Q000379:methods*; D012333:RNA, Messenger / Q000096:biosynthesis; D015533:Transcriptional Activation
chemical_list: D005353:Fibronectins; D015699:HIV Envelope Protein gp120; D012333:RNA, Messenger; D000185:Actinin