Classic Technique Reveals HIV in Action

REVELATION: The glycoprotein gp120 binds to the CD4 receptor on the T cell, causing the viral molecule to contort in a way that enables it to bind to the nearby chemokine receptor, too. This dual binding of HIV to two co-receptors triggers fusion of the viral and T cell membranes. Infection begins. X-ray crystallography has played a pivotal role in life science research, from providing data crucial to deciphering the DNA double helix, to revealing the structure of HIV protease. Now, the technology has glimpsed the complex choreography that precedes HIV infection: the viral surface glycoprotein gp120 binding the CD4 receptor on a vulnerable T cell. And the dance is more complex than immunologists had thought. "The virus uses a complicated set of mechanisms to target the cell and trigger a reaction to fuse the viral and cell membranes. The crystal structure doesn't completely reveal the mechanism of the conformational change that makes the fusion possible, but we are beginning to get at it," says Wayne Hendrickson, a professor of biochemistry and molecular biophysics and Howard Hughes Medical Institute Investigator at the Columbia University College of Physicians and Surgeons in New York. His group, with collaborators at the Dana-Farber Cancer Institute in Boston, SmithKline Beecham Pharmaceuticals in King of Prussia, Pa., and Tulane University Medical Center in New Orleans, have obtained the long-sought crystal structure of gp120 bound to CD4 and a stabilizing antibody. And it reveals what the researchers call a "silent face," a portion of the topography of the gp120 molecule that the immune system never sees. Dennis Burton Evidence of the import of the work was its debut on the covers of both Science and Nature on the same week in June, an event reminiscent of Bruce Springsteen's simultaneous appearance on the covers of Time and Newsweek years ago (P. D. Kwong et al, Nature, 393:648-59, June 18, 1998; R. Wyatt et al, Nature, 393:705-11, June 18, 1998; C. Rizzuto et al, Science, 280:1884, June 19, 1998; R. Wyatt and J. Sodroski, Science, 280:1984, June 19, 1998). Not only does the structure fill in a major gap in scientists' knowledge of HIV pathogenesis, but it provides information that is almost certain to have clinical impact. "The implications are enormous. The work provides the first glimpse of the interaction of gp120 and CD4 at the molecular level. This type of understanding is likely to prove crucial in HIV vaccine research, as it has been in HIV drug design for the protease inhibitors," says Dennis Burton, an immunologist at the Scripps Research Institute in La Jolla, Calif. Although many research groups have been dissecting gp120 as a route to revealing HIV's secrets, the quest that led to the crystal structure began in Hendrickson's lab in 1987, when graduate student Peter Kwong decided to tackle the daunting task. "I'd been interested not only in gp120, but in the CD4 part too, for quite awhile. We wanted to see HIV caught in the process of binding to the CD4 receptor," explains Kwong, who is currently an associate research scientist at Columbia. Crystallization is as much art as science. Obtaining crystals of bulky proteins is particularly challenging, especially those adorned with carbohydrates, as is gp120. The problem the research team faced was that HIV, courtesy of the gp120 molecules that extend like spokes from its envelope, cloaks itself from the human immune system at the same time that it attacks, like a microscopic Klingon warship pursuing the U.S.S. Enterprise unseen. The "cloaking device" for gp120 consists of sugars atop loops of variable amino acid sequences, which together shield regions of conserved sequences. Overall, the outer loops and sugars render gp120 very flexible--bad news for a crystallographer, Kwong knew. "The mechanisms the molecule has to protect itself from the immune system make it difficult to crystallize. A crystal is an ordered lattice that needs rigid contacts. If it can make rigid contacts, then the immune system can see it. So, HIV made its surface very flexible," he adds. The virus is also a wolf-in-sheep's-clothing. "The sugars are the same kinds that are put on proteins on our own cells, and therefore immune system cells recognize the virus as self," adds Hendrickson. This disguise short-circuits an immune response. Visualizing gp120 as it approaches a human T cell CD4 receptor meant literally stripping away its defenses. And that's where professor of pathology Joseph Sodroski and his co-workers at the Dana-Farber Cancer Institute came in. Over many years, they have scrutinized every part of gp120 with antibody binding experiments and deletion mutations. "A large body of mutagenesis data on gp120 generated in our lab indicated that some of the variable loops could be deleted," says Sodroski. Joseph Sodroski The Columbia researchers used Sodroski's recombinant gp120 molecules minus key variable sequence loops, removed many of the sugars, and added stabilizing antibodies provided by James Robinson at Tulane University. They made many combinations, and one finally worked. "We can use mutational analysis to say the CD4 binding site must be here. But with X-ray crystallography, we actually see it," says Hendrickson. What we can now see--a water-filled cleft that forms as gp120 and CD4 tango--may guide the future of AIDS research. "This provides a handle, a place to stick something that binds with a higher affinity. The cavity is crying out for a therapeutic intervention," says Kwong. Vaccine research will benefit too. Existing gp120 experimental vaccines use the whole molecule in its natural state, shields and all. "Seeing the protein, seeing the defenses built in that allow HIV to evade an immune response, will allow people to devise strategies for molecules that raise antibody responses better than the natural molecules," predicts Sodroski. The new work, however, will not derail the gp120-based vaccine phase III clinical trial set to begin this summer by VaxGen Inc. of South San Francisco, Calif. The image that graces the covers of Science and Nature is only a beginning, the investigators caution. It is but a single, static peek at a dynamic molecule that may assume a number of different forms as it delivers a deadly virus into human cells. But already the research team is onto the next step, systematically adding back bits of gp120's armor, to see if they can catch the molecule in other guises. Sums up Kwong: "What we did doesn't tell the whole story; we hope to provide that with other structures. It is only an initial snapshot--we have a lot more to do." And, with this new view, so do many other researchers.

Classic Technique Reveals HIV in Action

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