Duke Human Vaccine Institute (DHVI) investigators used a technique called time-resolved, temperature-jump (TR, T-Jump) small-angle x-ray scattering (SAXS) to capture the spectacularly brief moment during which the HIV-1 envelope (Env) glycoprotein undergoes a structural rearrangement that is essential for the virus to gain entry into human cells. The team’s results, which show how a tiny section of the HIV-1 glycoprotein surface snaps open and shut in a millionth of a second, could help scientists design and develop broadly neutralizing antibodies that would prevent this structure from popping open, as the basis for an AIDS vaccine.

Research lead Rory Henderson, PhD, a structural biologist who is an associate professor of medicine in DHVI, and colleagues, reported on their findings in Science Advances, in a paper titled “Microsecond dynamics control the HIV-1 envelope conformation,” in which they concluded “Our findings show that the microsecond timescale structural dynamics play an essential role in controlling the Env conformation with impacts on vaccine design.”

The HIV-1 envelope glycoprotein mediates viral attachment to the host cell’s CD4 receptor and entry into host cells through a complex series of interactions and structural transitions that ultimately result in virion-host cell membrane fusion and host cell infection, the authors explained. “The Env ectodomain is composed of three hetero-protomers, each consisting of a receptor-binding gp120 domain and fusion machinery containing the gp41 domain. In the prefusion closed state, the gp120 domains surround the gp41 domains, thus sequestering the fusion machinery before CD4 receptor and CCR5/CXCR4 coreceptor binding.”

AIDS researchers have been trying to understand the exact mechanism of viral fusion and entry and transitional structural states, but, as the team wrote, “Although remarkable progress has been made in understanding the structures of various Env conformations, microsecond timescale dynamics have not been studied experimentally.” Knowledge of Env structure and dynamics has nevertheless played an important role in HIV-1 vaccine immunogen design, the team, continued. But, while parts of the envelope are constantly moving to evade the immune system, vaccine immunogens are designed to stay relatively stable. “Preventing transitions from the prefusion, closed confirmation is thought to be essential for selecting functional improbable broadly neutralizing antibody mutations for neutralization induction by vaccination.”

Through their newly reported research, Henderson and colleagues monitored structural rearrangements in the HIV-1 Env with microsecond precision, and were able to capture a previously unknown structural transition. “Everything that everybody’s done to try to stabilize this (structure) won’t work, because of what we learned,” said Henderson. “It’s not that they did something wrong; it’s just that we didn’t know it moves this way.”

Coauthor Ashley Bennett, PhD, further explained, “As the virus feels for its best attachment point on a human T-cell, the host cell’s CD4 receptor is the first thing it latches onto. That connection is what then triggers the envelope structure to pop open, which in turn, exposes a co-receptor binding site “and that’s the event that actually matters.”

Once both molecules of the virus are bound to the cell membrane, the process of injecting viral RNA can begin. “If it gets inside the cell, your infection is now permanent,” Henderson said.  “If you get infected, you’ve already lost the game because it’s a retrovirus,” Bennett agreed.

Postdoctoral researcher Ashley Bennett (left) and Associate Professor Rory Henderson of the Duke Human Vaccine Institute with 3D printed models of HIV surface proteins.
Postdoctoral researcher Ashley Bennett (left) and Associate Professor Rory Henderson of the Duke Human Vaccine Institute with 3D printed models of HIV surface proteins. [Duke University]
The moving structure the team found protects the sensitive co-receptor binding site on the virus.” In two distinct Env variants, we detected a transition that correlated with known Env structure rearrangements with a time constant in the hundreds of microseconds range,” the authors wrote. Henderson added, “It’s also a latch to keep it from springing until it’s ready to spring.” Feasibly, keeping it latched with a specific antibody would stop the process of infection.

To see the viral parts in various states of open, closed and in-between, Bennett and Henderson used an electron accelerator at the Argonne National Laboratory outside Chicago that produces X-rays in wavelengths that can resolve something as small as a single atom. The team was awarded three 120-hour blocks of time with the synchrotron to try to get as much data as they could in marathon sessions. “Basically, you just go until you can’t anymore,” Bennett said.

Earlier research elsewhere had argued that antibodies were being designed for the wrong shapes on the virus and this work shows that was probably correct. “The question has been ‘why, when we immunize, are we getting antibodies to places that are supposed to be blocked?’” Henderson said. Part of the answer should lie in this particular structure and its shape-shifting. “It’s the interplay between the antibody binding and what this shape is that’s really critical about the work that we did,” Henderson said. “And that led us to design an immunogen the day we got back from the first experiment. We think we know how this works.”

The authors further concluded, “In summary, these results show that transient intermediate states observable only on a microsecond scale play an essential role in controlling HIV-1 Env conformation. Blocking these early transitions is likely an important consideration in vaccine development efforts to ensure that maturing antibodies remain on track to develop neutralization breadth.”

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