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"RNA Interference" Technique Blocks HIV among Potential Targets

Duke University and Howard Hughes Medical Institute researchers Bryan R. Cullen and Glen A. Coburn report that "RNA interference" can prevent HIV-1 from replicating in human cells. In applying the relatively new RNA interference technique to HIV, they join rapidly expanding efforts to understand and apply this natural phenomenon not only to viral diseases affecting humans but also to those affecting agriculturally important animals and crops. In short, RNA interference is catching on "like wildfire" as a tool for blocking expression of specific genes.

"In the next decade, we will see the use of this technique to silence essentially any mammalian gene in any cell, as a tool for asking if the gene is essential, or involved in a particular process," says Phillip Sharp of the Massachusetts Institute of Technology in Cambridge. "It is going to be a tremendously powerful tool for laboratory research, for sure, and the number of papers is going to shoot through the roof."

This fervor was sparked in 1998 when Andy Fire of the Carnegie Institute in Washington, D.C., and Craig Mello of the University of Massachusetts Medical School in Worcester identified RNA interference as a natural way by which plants inhibit the replication of RNA viruses—keying in on, and attacking, characteristic double-stranded RNA molecules that arise during their replication cycle.

"We were curious whether it [RNA interference] would do so in a vertebrate," Cullen says. "Our lab works on HIV-1, so that was the obvious virus to look at. We asked, `is it possible to block replication in human cells by inducing RNA interference?' It turned out that this pathway is conserved between plants and humans. We got very strong interference." Thus, for example, both immortalized and primary human T cells prevent HIV-1 replication in vitro, he and his colleagues report in the September 2002 issue of the Journal of Virology (76:9225-9231).

RNA interference is "a very old mechanism that probably evolved as a defense against viruses." Cullen says. "Many viruses make double-stranded RNAs when they replicate. But, normally, animal and plant cells never see much double-stranded RNA, so [it] is a huge alarm signal."

Eukaryotes have a complex mechanism for processing double-stranded RNA warning signals—cutting them into 21-22 nucleotide-long segments that are absorbed into the "RNA-induced silencing complex," or RISC. Once part of RISC, those double-stranded segments separate into single strands, called small interfering RNAs (siRNAs), that search for their respective homologs within the cytoplasm, where viral messenger RNAs (mRNAs) are being translated. When these siRNAs find and attach to homologous sequences within viral mRNAs, those mRNAs are cleaved rather than translated, thereby "interfering" with viral replication.

Cullen and his collaborators introduce into T cells a synthetic version of the 21-nucleotide double strand of RNA that targets HIV-1. Although this synthetic oligonucleotide is effective against the virus in cultured cells, "We don't envision this as a good way to control HIV-1," he says. One big problem is that, although appropriate siRNAs can be introduced directly into HIV-infected cells in vitro, "cells susceptible to HIV-1 are scattered throughout the body, [making] delivery a real difficulty."

Another looming problem when dealing with HIV-1 is its high mutation rate, which might enable it to escape blocks imposed by RNA interference, assuming that the process can somehow be made to work effectively in AIDS patients. "If you had a cellular target the virus depends on, and you were able to silence that, escape would not be nearly as likely," Sharp says.

In testing this, Sharp and his collaborators find that blocking the CD4 coreceptor for the virus on cell surfaces "caused degradation of the messenger RNA, reducing the amount of CD4 protein coreceptor on the surface of the cell [and] decreasing efficiency of HIV infection in those cells," he says. Although this target is not practical to exploit because it is required for normal immune functions, "the coreceptor for HIV infection is not a poor target," he notes. "In fact, people who have a mutation in that gene are resistant to HIV." However, he adds, "Even with great luck, it will take many years to maybe show benefits in patients."

Applying RNA interference to solve agricultural problems attributable to RNA viruses may be closer to fruition, according to Cullen. "You could in principle make cells completely resistant to a virus by having them constitutively make siRNAs targeted against the virus of choice," he says. For instance, livestock could be rendered immune to common diseases. "I bet in five years a lot of farm animals will have these things in their germ lines," says Frederic Bushman of the Salk Institute in La Jolla, Calif., who is also studying RNA interference.

David Holzman
David Holzman writes from Lexington, Mass.