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Unmasking the villain
CHAPEL HILL, N.C.—Viruses have always been a challenge for pharmaceutical therapies, but HIV has been one of the more frustrating entities against which researchers pit themselves—given that therapies quickly fall prey to mutations giving rise to resistant strains of HIV, meaning that even multi-drug cocktails fail to truly wipe out the infection.
So, the news coming out of the University of North Carolina at Chapel Hill in early August that researchers have, for the first time, decoded the structure of an entire HIV genome comes as welcome news to researchers. It means that they may soon understand better the strategies that HIV uses to infect humans and cause AIDS, may better understand other viruses' tricks as well and will be better equipped to come up with groundbreaking therapies to use against them.
The HIV genome work, reported in the cover story of the Aug. 6 issue of the journal Nature, began with work by Dr. Kevin Weeks and other chemists at UNC, whose lab focuses on the chemical and structural biology of RNA. According to Weeks, he and his colleagues thought that technologies created in their lab could help the HIV research community. So, they worked out a plan to collaborate with UNC virologists and the National Cancer Institute (NCI) and began study to solve the structure of an entire HIV-1 genome.
The new results show that the HIV RNA genome contains numerous RNA structures that influence how HIV proteins are made and how the virus hides from the body's defenses. Moreover, the HIV genome structure appears to be so extensive as to constitute "another level of the genetic code," notes Weeks, a professor of chemistry in UNC's College of Arts and Sciences and one of the lead authors of the study.
HIV, just like viruses that cause such other serious diseases as influenza, hepatitis C and polio, carries its genetic information as single-stranded RNA rather than double-stranded DNA. The information encoded in RNA is more complex than in DNA, Weeks explains, with RNA able to "fold into intricate patterns and structures." These structures are created when the ribbon-like RNA genome folds back on itself to make three-dimensional objects.
Weeks says that prior to this new work, researchers had modeled only small regions of the HIV RNA genome, in part because the HIV RNA genome is so large, with two strands of nearly 10,000 nucleotides each.
"There is so much structure in the HIV RNA genome that it almost certainly plays a previously unappreciated role in the expression of the genetic code," Weeks says. "We are also beginning to understand tricks the genome uses to help the virus escape detection by the human host."
Weeks, who is also a member of the UNC Lineberger Comprehensive Cancer Center, and Joseph M. Watts, a chemistry postdoctoral fellow supported by the Lineberger Center, used technology developed by Weeks' lab to analyze the architecture of HIV genomes isolated from infectious cultures containing trillions of viral particles that were grown by Dr. Robert Gorelick and Julian Bess of the NCI.
They then teamed up with UNC researchers in the College and the School of Medicine for further analysis: Christopher Leonard in the Department of Chemistry; Dr. Kristen Dang from the Department of Biomedical Engineering; Dr. Ron Swanstrom, a professor of microbiology and immunology at UNC Lineberger; and Dr. Christina Burch, an associate professor of biology.
They found that the RNA structures influence multiple steps in the HIV infectivity cycle.
Swanstrom and Weeks note that the study is the key to unlocking additional roles of RNA genomes that are important to the lifecycle of these viruses in future investigations.
"One approach is to change the RNA sequence and see if the virus notices," says Swanstrom, who is also director of the UNC Center for AIDS Research. "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 the virus."
In a comment about the UNC study in Nature, Hashim M. Al-Hashimi of the Department of Chemistry and Biophysics at the University of Michigan in Ann Arbor, Mich., notes that the approach of the UNC team was unique because so many researchers zoom in on
"stems and loops" in viral genomes that contain motifs to direct various steps of viral replication so that they can better understand their function. The UNC team instead "zoomed out."
Watts, Weeks and the other researchers used instead a technique called SHAPE (selective 2ʹ-hydroxyl acylation analyzed by primer extension) that provides images of lower resolution than those traditionally obtained by NMR spectroscopy and X-ray crystallography. But as Al-Hashimi points out, this bird's eye-style view "span a much larger area of the genome. The technique is thus akin to zooming out on a map and getting a broader view of the landscape at the expense of fine details."
"SHAPE may be generally useful for identifying new regulatory elements in large RNAs. All of these elements represent hypotheses and 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 of genomic map to "judiciously" zoom in on pieces of the HIV-1 genome in order to determine architectural and functional principles at the atomic level, Al-Hashimi indicates.
"Bridging these disparate RNA structure-function scales as well as moving towards movies of the genome in functional motion will be challenges for the future," he writes. "But for now, it seems that the quest for a high-resolution structure of the entire HIV-1 RNA genome has begun in earnest."