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BY GENOME RESEARCH
Using sophisticated post-genomic technology, a team of researchers has looked deep within the body’s immune cells and recorded the molecular events triggered by invasion of the human immunodeficiency virus (HIV), creating a detailed account of the devastating progression of cellular injury following HIV infection.
HIV kills critical immune cells called CD4+ T cells, eventually leading to full-scale destruction of the immune system and AIDS. The precise choreography and mechanisms by which HIV causes CD4+ T cells to die has not been detailed until now.
This first-ever sequential record of the multiple steps leading to immune cell death due to HIV infection was created by simultaneously monitoring nearly 7,000 genes at eight points in time over 72 hours, using microarray gene chip technology and a software program designed by UCSD bioinformatics specialists.
The results, published in the July 2001 issue of Genome Research, demonstrate in detail how efficiently HIV commits its cellular coup d'etat, rapidly taking over the cell's DNA machinery and inserting its own viral blueprints for destruction, suppressing vital survival and repair functions, and inducing the cell to kill itself.
|Jacques Corbeil, Ph.D.|
"Better understanding of the steps involved in HIV destruction of immune system cells opens the door to new investigations of methods to potentially block or prevent HIV infection," said Jacques Corbeil, Ph.D., assistant professor of medicine at the University of California, San Diego (UCSD) School of Medicine and the paper's first author.
This study of the specific genetic events in the CD4+ T cell, from infection to cell death, involved medical researchers, bioengineers, and bioinformatics experts from UCSD; the San Diego Supercomputer Center at UCSD; the Veterans Affairs (VA) San Diego Healthcare System, and Affymetrix, the company that develops GeneChips® probe arrays to measure whether individual genes are dormant or active.
Their findings show that HIV suppresses genes vital to immune cell maintenance and repair, while activating a cell-death process called apoptosis.
The scientists utilized UCSD-developed software called 2HAPI (High-density Array Pattern Interpreter, version 2) to analyze the expression of nearly 7,000 genes, the largest number ever studied by HIV researchers.
Daniel Masys, M.D.
“With this technology, our ability to study HIV has moved from observing clinical manifestations of HIV, to studying the molecular machinery of the cell as the virus changes and effects the cell’s living process,” said Daniel Masys, M.D., Director of Biomedical Informatics at UCSD School of Medicine and co-inventor of the HAPI software. This web-based microarray analysis tool was developed by members of the UCSD Center for AIDS Research Genomics core, the San Diego Supercomputer Center, and the UCSD Cancer Center, and is available at http://www.array.ucsd.edu.
To track gene activity in the infected cells, the team used Affymetrix GeneChips®, silicon chips coated with DNA fragments representing known sequences of genes. Simply stated, when these chips are exposed to specially labeled cells, the DNA seeks and binds to active genes within the cell, providing a snapshot of gene expression within the cell at a specific point in time. The chips produce vast amounts of data indicating which genes are activated, which must then be interpreted using computer analysis.
For this study, an HIV infected CD4+T cell line was monitored following infection at several time intervals, from 30 minutes to 72 hours after exposure. For each interval, 10 million infected cells were applied to the microarray gene chips. A control sample of healthy cells was analyzed at the same intervals for comparison.
The 2HAPI software provides automated linkage of data produced by microarray gene chip technology regarding simultaneous expression of thousands of genes, to published information about the expressed genes and gene clusters, allowing investigators to analyze and interpret the relevant gene activity.
The resulting data show how effectively HIV invades and overpowers the host cell's DNA, integrating its own infectious DNA into the host's cellular machinery, poisoning genes and altering the cellular energy source, quashing the cell’s DNA repair mechanism, and setting in motion the cell-suicide process of apoptosis.
Within hours of entering the immune cell, HIV suppresses genes that regulate and maintain a constant and healthy internal environment. The virus cripples enzymes essential for function of the mitochondria, cellular structures that serve as the source of energy needed to sustain life and growth. And, HIV suppresses the genes that ordinarily repair altered cellular DNA, rendering the cell incapable of mending HIV-induced damage as it occurs.
The power of HIV to shut down normal genes and activate others is impressive, and begins immediately. Only 30 minutes after exposure to HIV, more than 500 genes were shut down in the infected cells. Conversely, nearly 200 genes were uniquely activated in the infected cells compared with normal cells. These included genes associated with cellular defense against invasion, and "suicide" genes that normally remain dormant until switched on as part of the normal cycle of cell death.
By shutting down important genes, and activating others programmed to kill the cell, HIV proves to be a swift and deadly predator. Within three days of infection, the HIV-exposed cells had only half the 1,400 active genes normally found within the healthy cell.
“Now that we have the ability to see the specific genes that are modulated by HIV, we’re probing further to find the promoter regions of these genes where activation begins or is suppressed,” Corbeil says. “We want to determine how they are expressed as well as the length of time the genes are turned on or off.”
The research was supported by the National Institute of Allergy and Infectious Diseases, the Center for AIDS Research Genomics Core laboratory, the Universitywide AIDS Research program, the San Diego Veterans Medical Research Foundation, and the San Diego VA Healthcare System.
In addition to Corbeil, authors of the Genome Research paper included Masys; Thomas Gingeras, Ph.D., Affymetrix; UCSD Department of Medicine researchers Davide Genini, Ph.D., Steffney Rought, Ph.D.,Lorenzo Leoni, Ph.D., Pinyi Du, B.S., and Mark Ferguson, B.S.; UCSD Department of Pathology researchers Dennis Sheeter, B.S. and John B. Welsh, M.D., Ph.D.; Lynn Fink, B.S., San Diego Supercomputer Center; Roman Sasik, Ph.D.,UCSD Department of Physics; Affymetrix researchers David Huang, B.S. and Jorg Drenkow, B.S.;and Douglas D. Richman, M.D., UCSD Departments of Medicine and Pathology, San Diego VA Medical Center and Veterans Medical Research Foundation.
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