Unmasking the villain

CHAPEL HILL, N.C.—Viruses have always been a challenge forpharmaceutical therapies, but HIV has been one of the more frustrating entitiesagainst which researchers pit themselves—given that therapies quickly fall preyto mutations giving rise to resistant strains of HIV, meaning that evenmulti-drug cocktails fail to truly wipe out the infection.

So, the news coming out of the University of North Carolinaat Chapel Hill in early August that researchers have, for the first time,decoded the structure of an entire HIV genome comes as welcome news toresearchers. It means that they may soon understand better the strategies thatHIV uses to infect humans and cause AIDS, may better understand other viruses'tricks as well and will be better equipped to come up with groundbreakingtherapies to use against them.

The HIV genome work, reported in the cover story of the Aug.6 issue of the journal Nature, beganwith work by Dr. Kevin Weeks and other chemists at UNC, whose lab focuses onthe chemical and structural biology of RNA. According to Weeks, he and hiscolleagues thought that technologies created in their lab could help the HIVresearch community. So, they worked out a plan to collaborate with UNCvirologists and the National Cancer Institute (NCI) and began study to solvethe structure of an entire HIV-1 genome.

The new results show that the HIV RNA genome containsnumerous RNA structures that influence how HIV proteins are made and how thevirus hides from the body's defenses. Moreover, the HIV genome structureappears to be so extensive as to constitute "another level of the geneticcode," notes Weeks, a professor of chemistry in UNC's College of Arts andSciences and one of the lead authors of the study.

HIV, just like viruses that cause such other seriousdiseases as influenza, hepatitis C and polio, carries its genetic informationas single-stranded RNA rather than double-stranded DNA. The information encodedin RNA is more complex than in DNA, Weeks explains, with RNA able to "fold intointricate patterns and structures." These structures are created when the ribbon-likeRNA genome folds back on itself to make three-dimensional objects.

Weeks says that prior to this new work, researchers hadmodeled only small regions of the HIV RNA genome, in part because the HIV RNAgenome is so large, with two strands of nearly 10,000 nucleotides each.

"There is so much structure in the HIV RNA genome that italmost certainly plays a previously unappreciated role in the expression of thegenetic code," Weeks says. "We are also beginning to understand tricks thegenome uses to help the virus escape detection by the human host."

Weeks, who is also a member of the UNC LinebergerComprehensive Cancer Center, and Joseph M. Watts, a chemistry postdoctoralfellow supported by the Lineberger Center, used technology developed by Weeks'lab to analyze the architecture of HIV genomes isolated from infectiouscultures containing trillions of viral particles that were grown by Dr. RobertGorelick and Julian Bess of the NCI.

They then teamed up with UNC researchers in the College andthe School of Medicine for further analysis: Christopher Leonard in theDepartment of Chemistry; Dr. Kristen Dang from the Department of BiomedicalEngineering; Dr. Ron Swanstrom, a professor of microbiology and immunology atUNC Lineberger; and Dr. Christina Burch, an associate professor of biology.

They found that the RNA structures influence multiple steps in the HIVinfectivity cycle.

Swanstrom and Weeks note that the study is the key tounlocking additional roles of RNA genomes that are important to the lifecycle ofthese viruses in future investigations.

"One approach is to change the RNA sequence and see if thevirus notices," says Swanstrom, who is also director of the UNC Center for AIDSResearch. "If it doesn't grow as well when you disrupt the virus with mutations,then you know you've mutated or affected something that was important to thevirus."

In a comment about the UNC study in Nature, Hashim M.Al-Hashimi of the Department of Chemistry and Biophysics at the University ofMichigan in Ann Arbor, Mich., notes that the approach of the UNC team wasunique because so many researchers zoom in on

"stems and loops" in viral genomes that contain motifs todirect various steps of viral replication so that they can better understandtheir function. The UNC team instead "zoomed out."

Watts, Weeks and the other researchers used instead atechnique called SHAPE (selective 2ʹ-hydroxylacylation analyzed by primer extension) that provides images of lowerresolution than those traditionally obtained by NMR spectroscopy and X-raycrystallography. But as Al-Hashimi points out, this bird's eye-style view "spana much larger area of the genome. The technique is thus akin to zooming out ona map and getting a broader view of the landscape at the expense of finedetails."

"SHAPE may be generally useful for identifying newregulatory elements in large RNAs. All of these elements represent hypothesesand starting points that we hope will stimulate further detailed examination,"the UNC researchers explain in their Nature article.

Structural biologists will be able to use this kind ofgenomic map to "judiciously" zoom in on pieces of the HIV-1 genome in order todetermine architectural and functional principles at the atomic level,Al-Hashimi indicates.

"Bridging these disparate RNA structure-function scales aswell as moving towards movies of the genome in functional motion will be challengesfor the future," he writes. "But for now, it seems that the quest for ahigh-resolution structure of the entire HIV-1 RNA genome has begun in earnest."