Understanding How Researchers Visualize the HIV Spike

What tools are researchers using to bring HIV’s Envelope protein into focus and what have they learned about its structure?

One of the most exciting developments in AIDS vaccine research in recent years has been the isolation of dozens of broadly neutralizing antibodies (bNAbs) from the serum of a minority of HIV-infected individuals. In the lab, at least, these antibodies are capable of neutralizing many of the HIV strains currently in circulation by binding a three-legged, spike-like protein on the surface of the virus called the Envelope trimer, or Env. Scientists are now applying what they are learning from these antibodies to design vaccine candidates that might elicit similar antibodies in people before they are exposed to HIV, and so block infection (see VAX May 2010 Primer on Understanding if Broadly Neutralizing Antibodies are the Answer).

The bNAbs elicited by any such vaccine would attack HIV before it can invade its target cells. Ideally, they would not only target a broad spectrum of HIV variants, but do so potently—in other words, at very low concentrations.

But eliciting such antibodies isn’t easy. The regions of Env most susceptible to neutralizing antibodies are hidden by a thick coat of sugars. These sugars restrict antibody access to the underlying protein surface. Many of the protein targets—or epitopes—that are accessible to antibodies, meanwhile, do not elicit neutralizing antibodies, and act as decoys that confound the immune response. But perhaps most importantly, large swathes of the trimer change constantly due to HIV’s extraordinary mutability. This allows the virus to continuously evade immune recognition.

Scientists have sought to overcome these challenges by studying how bNAbs latch onto the Envelope trimer (seeVAX March 2011 Primer on Understanding HIV’s Envelope Protein). But determining the shape of the trimer—with or without an antibody bound to it—has been an uphill battle.

One way to do so is by X-ray crystallography, which involves beaming X-rays through a crystal of the purified protein and reading how the X-rays are scattered by that passage. This allows researchers to determine the precise spatial arrangement of atoms that make up the protein molecule. If the molecule—or the relevant part of it—can be co-crystallized in complex with an antibody that recognizes it, crystallography reveals in exquisite detail how the two interact with each other. Researchers attempting to reverse engineer novel HIV vaccine candidates rely heavily on such imaging.

But the HIV trimer poses unique challenges to that approach. Because the complex is structurally dynamic and highly unstable, researchers have had great difficulty crystallizing it in its functional (or “native”) state.

Freezing the trimer
To circumvent these problems, some laboratories employ a newer generation imaging technology called cryo-electron microscopy, or cryo-EM, to study the trimer’s structure. Cryo-EM involves snap-freezing a protein in liquid nitrogen. This freezes Env in its natural state. Scientists then use an electron microscope to capture thousands of images of the protein from different angles. These images are then merged to reconstruct a high-resolution, 3D image of the frozen protein’s fine structure.

Cryo-EM has often been used to study the Env trimer on the virus. Today scientists are also using a more advanced version of cryo-EM—single-particle cryo-EM—to analyze Env in isolation, affording a higher resolution snapshot of its structure. In one recent study, researchers used this approach to describe Env in its earliest native state, before it binds receptor proteins on the target cell. They found that Env, in its unbound state, has a “doughnut hole” in its center and is quite different from the densely packed structure that emerges at the end of the viral entry process. The images also capture an unusual, cage-like architecture, which likely helps HIV evade the immune system, and show how the triangular pyramid structure of Env hampers access by antibodies.

In another recent study, researchers used single particle cryo-EM to take a look at Env at a later stage of the infection process, just after it has docked with its protein receptor on the target cell. They found that Env partially opens up at this stage, exposing parts of its inner surface that it deploys to fuse the virus membrane with the cell membrane. This exposes the inner surface of Env to attack by antibodies.

While these cryo-EM models of Env are not as vivid as those obtained by X-ray crystallography, scientists have found them instructive and suspect they could help in the design of HIV immunogens—the active ingredients of vaccine candidates—that induce bNAbs. Already, they are planning to synthesize molecules that mimic the partially opened trimer, a conformation it assumes after it has bound its cellular receptors. A vaccine bearing such an immunogen might induce antibodies that prevent membrane fusion, which is essential to the viral life cycle.

Meanwhile, researchers are learning more about how some of the bNAbs prevent viral entry by interacting with other regions of the HIV Envelope trimer. In one recently completed experiment, researchers mixed HIV particles with two bNAbs—b12 and VRC01—and used cryo-EM to study if and how the binding of bNAbs changes the structure of the Envelope spike. They found that VRC01 did not require any changes in Env structure to bind its target; b12, however, did. This might explain why the VRC01 antibody neutralizes a broader range of HIV strains than b12.

To learn more about how researchers obtain models of the HIV Envelope glycoprotein, link to this video that details work from the laboratory of Sriram Subramaniam at the US National Cancer Institute.