In the fight against infectious diseases, the most daunting foes include viruses that use glycans to fend off antibodies. These glycans form on the outermost spike proteins of HIV and many other viruses, including influenza, Ebola, Lassa, and coronaviruses. Because the glycans are highly mobile, they can seem a blur to antibodies—as well as to scientists hoping to visualize them using conventional atomic-scale imaging technology, that is, conventional cryo-electron microscopy (cryo-EM).

Cryo-EM has been combined with other analytical techniques to visualize HIV’s glycan shield. This work, recently accomplished by scientists at Scripps Research and Los Alamos National Laboratory, captured details that were never seen before, including vulnerabilities that could be exploited by new vaccines. Curiously, the new work suggests that HIV glycans play defense and offense simultaneously. According to the scientists, the glycans not only deflect antibodies, they also reinforce the structure of HIV’s spike protein, keeping it poised for infection.

Details appeared October 22 in the Proceedings of the National Academy of Sciences, in an article titled, “Visualization of the HIV-1 Env glycan shield across scales.” The article describes how the scientists mapped the dense array of N-linked glycans on Env, the HIV-1 envelope glycoprotein.

“Here, we present an integrated approach of single-particle cryo-EM, computational modeling, and site-specific mass spectrometry (MS) to probe glycan shield structure and behavior at multiple levels,” the article’s authors wrote. “We found that dynamics lead to an extensive network of interglycan interactions that drive the formation of higher-order structure within the glycan shield.”

By mapping HIV’s glycan shield, the researchers hope to contribute to a better understanding of why antibodies react to some spots on the virus but not others. The researchers also hope that their findings may guide the design of new vaccines.

“We now have a way to capture the full structures of these constantly fluctuating glycan shields, which to a great extent determine where antibodies can and can’t bind to a virus such as HIV,” said the study’s lead author Zachary Berndsen, PhD, a postdoctoral research associate in the structural biology lab of Scripps Research professor Andrew Ward, PhD, one of the study’s corresponding authors.

The Scripps Research team collaborated with the lab of Gnana Gnanakaran, PhD, staff scientist at Los Alamos National Laboratory and the study’s other corresponding author. Los Alamos is equipped with high-performance computing resources that enabled fresh approaches for modeling the glycans.

The researchers combined cryo-EM with sophisticated computer modeling and a molecule-identifying technique called site-specific mass spectrometry. Cryo-EM relies on averaging tens or hundreds of thousands of individual snapshots to create a clear image, thus highly flexible molecules like glycans will appear only as a blur, if they show up at all.

But by integrating cryo-EM with the other technologies, the researchers were able to recover this lost glycan signal and use it to map sites of vulnerability on the surface of Env.

“This is the first time that cryo-EM has been used along with computational modeling to describe the viral shield structure in atomic detail,” noted Srirupa Chakraborty, PhD, co-lead author and postdoctoral researcher in the Gnanakaran lab at Los Alamos National Laboratory.

The new combined approach revealed the glycans’ structure and dynamic nature in extreme detail and helped the team better understand how these complex dynamics affect the features observed in the cryo-EM maps. From this wealth of information, the team observed that individual glycans do not just wiggle around randomly on the spike protein’s surface, as once was thought, but instead clump together in tufts and thickets.

“There are chunks of glycans that seem to move and interact together,” Berndsen explained. “In between these glycan microdomains is where antibodies apparently have the opportunity to bind.”

Experimental HIV vaccines rely on modified, lab-made Env proteins to elicit antibody responses. In principle, these vaccines’ effectiveness depends in part on the positioning and extent of the shielding glycans on these lab-made viral proteins. Therefore, Berndsen and colleagues applied their method to map the glycans on a modified HIV Env protein, BG505 SOSIP.664, which is used in an HIV vaccine currently being evaluated in clinical trials.

“We found spots on the surface of this protein that normally would be covered with glycans but weren’t—and that may explain why antibody responses to that site have been noted in vaccination trials,” Berndsen said.

That finding, and others in the study, showed that Env’s glycan shield can vary depending on what type of cell is being used to produce it. In HIV’s infections of humans, the virus uses human immune cells as factories to replicate its proteins. But viral proteins used to make vaccines normally are produced in other types of mammalian cells.

In another surprise discovery, the team observed that when they used enzymes to slowly remove glycans from HIV Env, the entire protein began to fall apart. Berndsen and colleagues suspect that Env’s glycan shield, which has been considered merely a defense against antibodies, may also have a role in managing Env’s shape and stability, keeping it poised for infection.

The team expects that their new glycan-mapping methods will be particularly useful in the design and development of vaccines—and not only for HIV. Many of the techniques can be applied directly to other glycan-shielded viruses such as influenza viruses and coronaviruses, and can be extended to certain cancers in which glycans play a key role, the researchers added.

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