While the virus'HIV slips out of a human cell to latch onto and eventually inject its deadly payload of genetic code, there's a remarkably brief moment when a tiny piece of its surface pops open to begin the infection process.
Seeing that structure open and close in a few millionths of a second is giving researchers at the Duke Human Vaccine Institute (DHVI) a new approach to the surface of the virus that could lead to the production of broadly neutralizing antibodies for an AIDS vaccine.
The results of the study were published in Science Advances.
HIV: here's what the new discovery means
Being able to attach a specific antibody to this small structure that would prevent it from opening would be critical.
The moving part is a structure called the envelope glycoprotein, and AIDS researchers have been trying to understand it for years because it is a key part of the virus's ability to dock with a T-cell receptor known as CD4. Many parts of the envelope are constantly moving to evade the immune system, but vaccine immunogens are designed to remain relatively stable.
“Everything we've all done to try to stabilize this (structure) isn't going to work, because of what we've learned,” said lead author Rory Henderson, a structural biologist and associate professor of medicine at DHVI. “It's not that they did anything wrong; we just didn't know things were this way.”
Postdoctoral researcher and study co-author Ashley Bennett offers a play-by-play: As the virus seeks its best attachment point on a human T cell, the host cell's CD4 receptor is the first thing it attaches to. This connection is what then causes the envelope structure to open up, which in turn exposes a co-receptor binding site “and that's the event that really matters.”
Once both virus molecules have bound to the cell membrane, the process of injecting viral RNA can begin. “If it gets into the cell, the infection becomes permanent,” Henderson said.
“If you get infected, you've already lost the game because it's a retrovirus,” agrees Bennett.
The mobile structure found protects the sensitive co-receptor binding site of the virus. “It's also a stopper to keep him from shooting until he's ready to shoot,” Henderson said. Keeping it blocked with a specific antibody would stop the infection process.
To see viral parts in various open, closed, and intermediate states, Bennett and Henderson used an electron accelerator at Argonne National Laboratory outside Chicago that produces X-rays at wavelengths capable of resolving something as small as a single atom. But this expensive, shared equipment is in high demand. AIDS researchers were given three 120-hour blocks of time with the synchrotron to try to get as much data as they could in marathon sessions. “Basically, you keep going until you can't anymore,” Bennett said.
Previous research elsewhere had argued that the antibodies were designed for the wrong forms of the virus, and this work shows that was probably correct.
“The question has been 'why, when we immunize, do we get antibodies to places that should be blocked?'” Henderson said. Part of the answer should lie in this particular structure and its changing shape.
“It's the interplay between the binding of the antibody and its shape that's really critical to the work we've done,” Henderson said. “And that led us to design an immunogen the day we returned from the first experiment. We think we know how it works.”
The path to a successful HIV vaccine depends on a critical first step: the activation of specific immune cells that induce broadly neutralizing antibodies.
In a paper published in the journal Cell, a research team led by the Duke Human Vaccine Institute achieved the required initial step in a study using monkeys. The next phase of the work will now move on to testing on humans.
“This study confirms that the antibodies are, at a structural and genetic level, similar to the human antibody that we need as the basis for a protective HIV vaccine,” said first author Kevin O. Saunders, Ph.D. , associate director of the Duke Human Vaccine Institute and associate professor in the departments of Surgery, Molecular Genetics, and Integrative Microbiology and Immunobiology.
“We are on the right track,” he said. “From here, we just need to start putting together the additional components of a vaccine.”
In previous work, the research team had isolated naturally occurring broadly neutralizing antibodies from an individual, and then retraced all the changes undergone by the antibody and the virus to reach a point of origin for the native antibody and its site of HIV link. envelope.
With this knowledge, they designed an antibody-eliciting molecule that mimics the native antibody and its binding site on the HIV envelope.
Four years ago, Saunders and colleagues published a study in Science in which they determined that monkeys produced neutralizing antibodies when vaccinated with the engineered immunogen, but it was unclear whether those antibodies were like the broadly neutralizing antibody needed for a human vaccine .
In the current study, researchers created a new, more potent formulation of the vaccine and administered it to monkeys. This time, their goal was to determine whether the neutralizing antibodies generated in animals were structurally and genetically similar to the antibodies needed in humans. They were.
“We thought we were on the right track in 2019 and now we have atomic-level details that confirm those findings,” Saunders said. “It's an important step forward.”
The human immunodeficiency virus (HIV) is a formidable pathogen. It changes rapidly; in fact, estimates suggest that the genetic diversity of the virus in a single person at any given time is equal to the diversity of influenza worldwide for a year. The infection has also developed structures to protect itself from recognition and attack by antibodies and therapies. All these factors contribute to making the virus dangerous and difficult to treat.
The more researchers can understand the biological processes underlying how HIV infects cells, the better they will be able to design treatments to penetrate the virus's defenses and destroy it. Now, Caltech researchers have photographed the elusive structure of the HIV protein at the atomic scale, seeing its details with a resolution of a billionth of a meter.
The work was conducted in the laboratory of Pamela Björkman, the David Baltimore Professor of Biology and Biological Engineering and Merkin Institute Professor. An article describing the study appears in the journal Nature. The study's first authors are Caltech postdoctoral scholars Kim-Marie Dam and Chengcheng Fan.
HIV primarily attacks immune cells called T cells, disabling them so they cannot defend other cells in the body from infection. As an HIV virion prepares to enter a T cell, it undergoes some shape changes. These happen on the so-called virus envelope protein, the protein on the surface of the virus that allows it to enter cells. Because envelope proteins are so important to the virus's infection process, they are good targets for therapies or vaccines.
The HIV envelope protein is “trimeric,” resembling a tripod-like flower with three “stem” portions – each called gp41 – and three “petal” regions called gp120. To initiate the infection, each of the three gp120 proteins docks with a type of receptor on the T cell called CD4. Once three CD4 receptors are attached by the three gp120 proteins, they expose sites that are recognized by a host coreceptor, and then a needle-like structure emerges from the stem regions of the “flower,” allowing the virus to infect and gain entry into the human cell.
What happens if the “petals” of the gp120 protein can only attach to one or two CD4 receptors? Can the envelope protein still open fully so the virus can infect the cell? Understanding this process could have significant implications for designing therapies.
If researchers could prevent just one or two CD4 receptors from being captured by a gp120, would that be enough to thwart the infection? To answer this open question, the team tried to image the envelope protein in these scenarios with only one or two bound CD4s.
“Structurally characterizing the conformations of the intermediate shell is incredibly valuable for understanding how HIV proteins function at a fundamental level,” says Dam.
But imaging these structures is a challenge: Creating “heterotrimers,” or envelope proteins that bind only one or two CD4 receptors, is not easy to do in a test tube for biochemical reasons. Through an innovative engineering approach, the team was able to devise a protocol to create stable heterotrimers. Then, using Fan's expertise in a delicate procedure called cryo-electron microscopy, they were able to capture images of the fragile structures of heterotrimers bound to CD4 receptors.
The structures have shown that if only one or two CD4 receptors are bound, the envelope protein is unable to fully open and undergo the shape-changing process associated with infection.
“One of the main questions emerging from this work is: can envelope proteins that do not fully open still facilitate infection?” Dam says.
The team then shared the results with Walther Mothes' lab at Yale University, which was conducting similar attempts to visualize heterotrimers. Sharing information between the two labs demonstrated that the behavior of engineered heterotrimers free-floating in a test tube (an experimental setup in which proteins are in solution or soluble rather than bound to viral membranes) is remarkably similar to the way proteins wrap around the viral surface and behave in a more “real” infection scenario.
This is an important finding because soluble constructs are used as the basis for the development of new therapies, and it is critical to know whether they accurately mimic natural processes.
Structural biology research like this is not only important for studying HIV but many different types of viruses.
“We learned so much from HIV,” says Dam. “When the COVID-19 pandemic began, we applied what we learned from HIV to SARS-CoV-2.”
“The structures of these previously unknown intermediate envelope conformations offer fascinating insights into structural changes driven by receptor interactions prior to fusion of the host and viral membrane,” says Björkman. “Our research not only opens new avenues for exploring the complexities of HIV infection, but also provides valuable information that goes beyond therapeutic design, improving our overall understanding of viral dynamics.”
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