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
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...
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Published in | Oncogene Vol. 21; no. 22; pp. 3592 - 3602 |
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Main Authors | , , , , , , |
Format | Journal Article |
Language | English |
Published |
Basingstoke
Nature Publishing
16.05.2002
Nature Publishing Group |
Subjects | |
Online Access | Get full text |
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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. |
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AbstractList | 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. |
Audience | Academic |
Author | WEI CHAO PEKARSKAYA, Olga KANG, Dong-Chul FISHER, Paul B SU, Zao-Zhong VOLSKY, David J YINMING CHEN |
Author_xml | – sequence: 1 givenname: Zao-Zhong surname: SU fullname: SU, Zao-Zhong organization: Department of Pathology and Herbert Irving Comprehensive Cancer Center, College of Physicians and Surgeons, Columbia University, New York, NY 10032, United States – sequence: 2 givenname: Dong-Chul surname: KANG fullname: KANG, Dong-Chul organization: Department of Pathology and Herbert Irving Comprehensive Cancer Center, College of Physicians and Surgeons, Columbia University, New York, NY 10032, United States – sequence: 3 surname: YINMING CHEN fullname: YINMING CHEN organization: Department of Pathology and Herbert Irving Comprehensive Cancer Center, College of Physicians and Surgeons, Columbia University, New York, NY 10032, United States – sequence: 4 givenname: Olga surname: PEKARSKAYA fullname: PEKARSKAYA, Olga organization: Molecular Virology Division, St. Luke's-Roosevelt Hospital Center, Columbia University, New York, NY 10019, United States – sequence: 5 surname: WEI CHAO fullname: WEI CHAO organization: Molecular Virology Division, St. Luke's-Roosevelt Hospital Center, Columbia University, New York, NY 10019, United States – sequence: 6 givenname: David J surname: VOLSKY fullname: VOLSKY, David J organization: Department of Pathology and Herbert Irving Comprehensive Cancer Center, College of Physicians and Surgeons, Columbia University, New York, NY 10032, United States – sequence: 7 givenname: Paul B surname: FISHER fullname: FISHER, Paul B organization: Department of Pathology and Herbert Irving Comprehensive Cancer Center, College of Physicians and Surgeons, Columbia University, New York, NY 10032, United States |
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ContentType | Journal Article |
Copyright | 2002 INIST-CNRS COPYRIGHT 2002 Nature Publishing Group Copyright Nature Publishing Group May 16, 2002 Macmillan Publishers Limited 2002. |
Copyright_xml | – notice: 2002 INIST-CNRS – notice: COPYRIGHT 2002 Nature Publishing Group – notice: Copyright Nature Publishing Group May 16, 2002 – notice: Macmillan Publishers Limited 2002. |
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Keywords | Human Immunopathology HIV-1 virus Glycoprotein Retroviridae AIDS Astrocyte Immune deficiency Lentivirus Virus Infection Envelope virus Gene Viral disease Human immunodeficiency virus Dementia |
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SubjectTerms | Acquired immune deficiency syndrome Actinin - biosynthesis AIDS Astrocytes Astrocytes - metabolism Astrocytes - virology Biological and medical sciences Blotting, Northern Brain - cytology Brain - embryology Cell cycle Cells, Cultured Central nervous system Cloning Cloning, Molecular Dementia Dementia disorders Exanthema Fibronectins - biosynthesis Gene expression Gene Expression Profiling - methods Glycoprotein gp120 Glycoproteins HIV HIV Envelope Protein gp120 - pharmacology HIV-1 - pathogenicity Human immunodeficiency virus Human viral diseases Humans Hybridization Infections Infectious diseases Kinetics Macrophages Medical sciences Microglia Nervous system Neurodegeneration Nucleic Acid Hybridization - methods Polymerase chain reaction RNA, Messenger - biosynthesis Surgeons Transcriptional Activation Urology Viral diseases Viral diseases of the lymphoid tissue and the blood. Aids Western blotting |
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 |
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