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Silencing Genes in HIV
One of nature’s oldest tricks thwarts the AIDS virus in cell cultures
Edward R. Winstead

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Nature would never leave something as valuable as genetic code unprotected. One of the security measures protecting DNA in plants and animals is a mechanism that fights viruses by shutting down their genes. In plants the phenomenon is known as gene silencing, and it has been widely used to study plant genes. It has also been used to create transgenic tomato plants that are resistant to plant viruses.

Schematic presentation of the second step of the RNAi pathway. View larger

What works in tomatoes may also work in people. A number of laboratories are investigating ways to exploit the naturally occurring defense mechanism in humans to fight HIV, the virus that causes AIDS. A new study reports success in blocking the virus in cell cultures by silencing genes in HIV and human cells.

Philip A. Sharp, of the Massachusetts Institute of Technology in Cambridge, and colleagues silenced the main structural protein in the virus, p24, and the human protein CD4, which the virus needs to enter the cell. The strategy impairs the virus in infected cells and limits its spread into healthy cells. The study, co-led by Premlata Shankar of the Center for Blood Research at Harvard Medical School in Boston, appears in Nature Medicine.

"We were able to inhibit the production of the virus either by blocking new infections or blocking the production of new viral particles in infected cells," says Judy Lieberman, also of the Center for Blood Research. "That's pretty encouraging."

The concept of silencing genes in HIV is straightforward: Hit the virus where it counts by eliminating a protein it needs to reproduce or cause infection. The human version of gene silencing has a different name—RNA interference, or RNAi—but the mechanism is fundamentally similar to the one in plants and involves some of the same proteins.

In RNAi, short, interfering double-stranded RNA (siRNA) molecules are added to the cell. The cell recognizes and degrades messenger RNA corresponding to the target sequence. As a result, little or no protein is produced.

‘This is a kind of secret weapon inside us that we have not noticed until now.’

The key to RNAi is using strands shorter than about 30 base pairs, because longer strands can cause the cell to commit suicide. "The cell realizes it is infected by the virus and commits hara-kiri to prevent its spread to neighbors," says Carl D. Novina, a member of the research team at MIT.

This self-destruction is known as the interferon response; Sharp's team bypassed the response by injecting siRNAs that were 21 to 23 base pairs long.

What makes RNAi so exciting to many researchers is its potential for knocking out a protein without harming the cell. By comparison, chemotherapy invariably kills tumors by destroying cancerous cells as well as healthy cells nearby.

"This method is extremely specific," says Lieberman. "You can shut off HIV without interfering with any genes other than the ones you target." Cells are basically missing 21 nucleotides—the RNA that is degraded—and the rest of the genome is untouched.

The researchers tested the plausibility of pre-loading human cells with siRNA as a way to protect against infection. They took latently infected human cells, activated the HIV genes and were still able to block the production of new viral particles. After entering a host cell, HIV inserts itself into the genome, where it can reside without expressing its genes.

As with the drug 'cocktails' that patients take to kill HIV at different stages of its life cycle, the most effective RNAi strategy will likely include multiple targets. These could be targets that block entry into the cell and disrupt the virus life cycle inside the cell.

The selection of targets is critical, and HIV mutates so rapidly that a target RNA sequence may become obsolete in a short period of time. This is one reason to silence genes in the host cell.

After identifying short RNA sequences in the target proteins, the researchers screen the sequences against the entire human genome, a process that takes about twenty minutes on a computer. "The critical thing in selecting targets is to chose unique RNA sequences that do not correspond to genes required for normal cell metabolism," says Novina.

"This is just the beginning of a wave of studies on using siRNA to inhibit viral infection," says Thomas Tuschl, of the Max-Planck-Institute for Biophysical Chemistry in Göttingen, Germany. "It's clear that siRNA is basic research at the moment, but the technology can now be evaluated as a potential therapy."

Scanning electron micrograph of HIV-1 budding from cultured lymphocyte.

Even if no therapies are developed using RNAi, the technology will help researchers dissect the biology of HIV infection and design drugs based on the information.

"You can turn off a human gene for a week and then challenge the cell with HIV to see whether the cell gets infected," says Tuschl. "This will help us understand how HIV functions in the cell and will have a big impact on developing new drugs."

"The next big breakthrough will be when we learn that injecting siRNA into an organism does the same thing as it does in tissue cultures," he says, noting that such work will probably happen in mice.

Tuschl led the study last year that helped inspire Novina and Lieberman to test RNAi against HIV. His team reported in Nature that short strands could 'sneak in under the radar' and trigger a silencing mechanism in human kidney cells and other mammalian cells.

The effects of adding siRNA to cells are transient, and finding ways to deliver siRNA to human cells—or engineer cells to express them—remains a primary challenge for the field.

Researchers at the City of Hope Cancer Center in Duarte, California, recently developed a DNA-based delivery system. As reported in Nature Biotechnology, they generated human cells that produced siRNA against the REV protein, which is important in causing human disease.

"Our goal is to engineer human cells to be resistant against attack by the virus and also make them incapable of spreading HIV," says John Rossi, who led the study. The idea is that patients could one day be given a population of modified cells that are highly resistant to viral infection.

"As with all technological advances, it remains to be seen how generally useful it will be," says Rossi, noting that RNAi "is not the universal panacea for antiviral therapy." He points out that some viruses have proteins that allow them to circumvent the mechanism.

RNA silencing of green fluorescent protein (GFP) (center) in leaves from Nicotiana benthamiana is suppressed by an animal (left; B2 protein of flock house virus) or a plant (right) viral suppressor, leading to enhanced GFP expression (lighter green/yellow areas).

The purpose of RNAi in animals has not yet been demonstrated, but some clues to its role were reported recently by scientists at the University of California, Riverside, who study the flock house virus—an animal virus that can infect plants. The researchers discovered that the flock house virus contains a protein that suppresses gene silencing in both plants and flies.

The fact that one suppressor protein works in two kingdoms is strong evidence that the silencing pathways in plants and animals are related, the team concludes in Science. Furthermore, the finding suggests that animal cells have used RNAi to protect themselves from viral attacks in a similar manner as plants.

"We show that RNAi is a natural antiviral defense," says Shou-Wei Ding, who led the study. "This is a kind of secret weapon inside us that we have not noticed until now. And one reason we haven't noticed it is that viruses contain proteins that suppress the weapon."

See related GNN article
»Modified tomato plants silence the genes of invaders

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Novina, C.D. et al. siRNA-directed inhibition of HIV-1 infection. Nat Med. Published online June 3, 2002.
Li, H., Li, W.X. & Ding, S.W. Induction and suppression of RNA silencing by an animal virus. Science 296, 1319-1321 (May 17, 2002).
Lee, N.S. et al. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat Biotechnol 19, 500-505 (May 2002).
Elbashir, S.M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494-498 (May 24, 2001).

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