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Hundreds Of Human Proteins Exploited By HIV Identified

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The HI virus contains itself just nine genes encoding 15 proteins, which wreak havoc on the human immune system. But this bare bones approach could have a fatal flaw. Lacking robust machinery, HIV hijacks human proteins to propagate, and these might represent powerful therapeutic targets.

In the top panels, HIV (red) infects cultured human cells (cellular DNA is stained blue). In the bottom panels, HIV levels are lower, as researchers interfered with the production of a host protein called TNPO3. (Credit: Image courtesy of Harvard Medical School)

Using a technique called RNA interference to screen thousands of genes, Harvard Medical School researchers have now identified 273 human proteins required for HIV propagation. The vast majority had not been connected to the virus by previous studies.

Drugs currently used to treat the viral infection interact directly with the virus itself, and it’s quite simple for the rapidly mutating virus to avoid destruction by altering how it interacts with these chemicals. Patients use a cocktail of HIV inhibitors because the virus is less likely to evolve resistance to multiple drugs at the same time. But some HIV strains have still managed to evade particular drugs. These could eventually develop resistance to several drugs, especially among patients who don’t adhere to their regimens.

“Antiviral drugs are currently doing a good job of keeping people alive, but these therapeutics all suffer from the same problem, which is that you can get resistance, so we decided to take a different approach centered on the human proteins exploited by the virus,” says Harvard Medical School (HMS) Professor and senior author Stephen Elledge, who holds primary appointments in the Department of Genetics and at Brigham and Women’s Hospital. “The virus would not be able to mutate to overcome drugs that interact with these proteins.”

Labs around the world have made impressive contributions to our understanding of the HIV life cycle. Over the last two decades, they’ve identified dozens of human proteins, or host factors, required for HIV propagation. The new study builds on this work, essentially quadrupling the list of host factors to include proteins involved with a surprising array of cellular functions ranging from protein trafficking to a type of programmed cell death called autophagy.

“The expanded list is a hypothesis generation machine,” explains Elledge, who is also a member of the HMS-Partners Health Care Center for Genetics and Genomics and investigator with the Howard Hughes Medical Institute. “Scientists can look at the list, predict why HIV needs a particular protein, and then test their hypothesis.” He hopes that such research will lead to new therapeutics.

To create the list, postdoctoral researcher and first author Abraham Brass–working with Derek Dyxkhoorn and Nan Yan from HMS Professor Judy Lieberman’s lab–began with a library of short interfering RNAs (siRNAs) targeting specific human genes. Each siRNA disrupts the gene’s ability to produce a particular protein.

With the help of the staff at the Institute of Chemistry and Cell Biology at Longwood (ICCB-L), Brass placed the siRNAs on thousands of human cells, with just one gene being targeted in each well of cells. Thus each well contained cells lacking a particular protein. Next, he unleashed HIV on the cells. If HIV replication was inhibited in a given well, it suggested the missing protein was involved.

Of the 273 proteins he identified, just 36 had been previously implicated in the HIV life cycle. He picked three of the other 237 proteins, and subjected them to a host of careful genetic experiments, proving they too truly play a role in HIV propagation.

Immune cells–the very cells HIV attacks–contain high concentrations of many of the 273 host factors, offering further proof of the list’s validity.

“We’re closing in on a systems level understanding of HIV, which opens new therapeutic avenues,” says Elledge. “We might be able to tweak various parts of the system to disrupt viral propagation without making our own cells sick.”

“This is the first whole genome screen for human proteins required by HIV, and we’re confident that it netted real results,” adds Brass. “Given the method, we missed some proteins, but the majority of the ones we found are highly likely to play a role in HIV propagation.”

Published Online January 10, 2008 Science DOI: 10.1126/science.1152725

Science Express Index Research Articles

Identification of Host Proteins Required for HIV Infection Through a Functional Genomic Screen
Abraham L. Brass 1, Derek M. Dykxhoorn 2{dagger}, Yair Benita 3{dagger}, Nan Yan 2, Alan Engelman 4, Ramnik J. Xavier 5, Judy Lieberman 2, Stephen J. Elledge 6*

1 Department of Genetics, Center for Genetics and Genomics, Brigham and Women’s Hospital, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA.; Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
2 Immune Disease Institute and Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA.
3 Center for Computational and Integrative Biology, Harvard Medical School, Boston, MA 02114, USA.
4 Dana-Farber Cancer Institute, Division of AIDS, Harvard Medical School, Boston, MA 02115, USA.
5 Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.; Center for Computational and Integrative Biology, Harvard Medical School, Boston, MA 02114, USA.
6 Department of Genetics, Center for Genetics and Genomics, Brigham and Women’s Hospital, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA.

* To whom correspondence should be addressed.
Stephen J. Elledge , E-mail: selledge@genetics.med.harvard.edu

These authors contributed equally to this work.

HIV-1 exploits multiple host proteins during infection. We performed a large-scale siRNA screen to identify host factors required by HIV-1 and identified over 250 HIV-dependency factors (HDFs). These proteins participate in a broad array of cellular functions and implicate new pathways in the viral life cycle. Further analysis revealed previously unknown roles for retrograde Golgi transport proteins (Rab6 and Vps53) in viral entry, a karyopherin (TNPO3) in viral integration, and the Mediator complex (Med28) in viral transcription. Transcriptional analysis revealed that HDF genes were enriched for high expression in immune cells suggesting that viruses evolve in host cells that optimally perform the functions required for their life cycle. This effort illustrates the power with which RNA interference and forward genetics can be used to expose the dependencies of human pathogens such as HIV, and in so doing identify potential targets for therapy.

Written by huehueteotl

January 12, 2008 at 3:14 pm

Posted in HIV

Tagged with ,

2 Responses

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  1. I was surprised to learn the Harvard study led by Professor Stephen Elledge was conducted in highly aneuploid “HeLa-derived TZM-bl cells”. I’ve worked the past 11 years studying the consequences of aneuploidy and feel I should pass along some of my concerns about the study’s experiments and the authors’ conclusions.

    As I say, HeLa cells are massively aneuploid cervical cancer cells from the 1950s, containing typically 76–80 chromosomes and 22–25 abnormal chromosomes per cell instead of the normal complement of 46 (Macville M, Schrock E, Padilla-Nash H, Keck C, Ghadimi BM, Zimonjic D, Popescu N, Ried T: Comprehensive and definitive molecular cytogenetic characterization of HeLa cells by spectral karyotyping. Cancer Res 1999, 59:141-150).

    No two HeLa cells are genetically alike, in fact no two use the same network of genes, gene products, transcription factors, receptors, etc. Furthermore, the HeLa cells are highly genomically unstable; they change with each cell division.

    Thus, the claimed “273 proteins that the AIDS virus needs to survive in human cells” are probably an artifact of performing the experiments in the HeLa cells and actually have little or nothing to do with what goes on in people.

    Ultimately, the multi-million dollar question is even if one can prevent HIV from replicating in HeLa cells by removing the susceptible human proteins, will the same thing happen in normal human cells in patients? The odds are strongly against it.

    There is also the conceptual problem of administering drugs against human proteins since these drugs will be inherently toxic. I was in the drug design and development business for 20 years. The first principle in coming up with drugs against infectious agents was to target proteins specific to the pathogen and not human in order to minimize potential and actual toxic consequences.

    Researchers should ponder these questions before going down the very expensive road suggested by the Harvard group.

    Sincerely,

    David Rasnick, PhD

    David Rasnick

    January 13, 2008 at 5:54 pm

  2. I thank you for this comment, and agree with you. Alas, if any, that is the real plus value of a blog, to stimulate and provide a fore for controversy, more than any journal really can. Else it would be merely a container of aging papers embroidered with a blog author’s personal opinions. It is ways too seldom though, that someone is ready to render a thoughtful comment, I believe😦

    huehueteotl

    January 13, 2008 at 11:47 pm


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