Cracking the code of HIV-1 entry into macaque cells

From the Bloom lab (Basic Sciences Division) and Overbaugh lab (Human Biology and Public Health Sciences Divisions).

“There have been as many plagues as wars in history; yet always plagues and wars take people equally by surprise.”

― Albert Camus, The Plague

As the world strives to find a cure for the ongoing SARS-CoV-2 pandemic, another deadly epidemic lurks in the background: the HIV-1 (human immunodeficiency virus) pandemic, which is months short of turning forty. Both pandemics remain unchecked due to the lack of an effective vaccine or a curative drug. Understanding the basic biology of viral infections is essential for the development of curative therapeutics. Researchers in the laboratories of Dr. Jesse Bloom (Basic Sciences Division) and Dr. Julie Overbaugh (Human Biology and Public Health divisions) forged a collaboration to study the molecular determinants that govern HIV-1 entry into host cells.

An outstanding problem in the field of HIV biology is the lack of a robust animal model that recapitulates the hallmarks of HIV-1 infection in humans. HIV-1 almost exclusively infects human blood cells –T lymphocytes, specifically– as they express the primary HIV-1 receptor known as CD4 and the preferred co-receptor CCR5. As the sequence of these receptors varies between closely related species, these differences become major genetic determinants of species-specific susceptibility. For example, a small change in the CD4 receptor sequence in rhesus macaques renders them resistant to HIV-1 infection.

Nonetheless, rhesus macaques remain the gold standard primate model for studying HIV-1 pathogenesis but not without a caveat: since these monkeys cannot be infected with wild type HIV-1, an SIV/HIV-1 hybrid virus encoding the human envelope (Env) protein in place of the simian Env is used to model HIV-1 infection. Dr. Overbaugh, a senior author of the study, said that their work was built on a surprising discovery by Dr. Daryl Humes, a former graduate student in the lab who is now a staff scientist in the lab. Dr. Humes found that the macaque CD4 T cell receptor did not support HIV-1 entry and infection. “Prior studies in the field totally missed the boat on this because they used highly lab adapted HIV-1 strains that did recognize the macaque CD4, leading to a dogma in the field that turned out not to apply to HIV-1 strains that were not adapted in this way,” said Dr. Overbaugh. Dr. Humes was the first to show that HIV-1 strains circulating in humans showed low infectivity in cells expressing the macaque CD4 receptor. “We realized why it was so challenging to develop a biologically relevant HIV-1 animal model in macaques”, she added.

Deep mutational scanning of HIV-1 envelop protein identifies mutations that enhance entry using macaque receptors.
Using deep mutational scanning, the authors can select for HIV envelope proteins with mutations in their transmembrane domain that enhances entry into macaque T cells Image provided by Dr. Julie Overbaugh.

This discovery highlighting the functional differences between macaque and human CD4 was a breakthrough, and it opened the horizon for more questions. Can we mutate HIV-1 Env to allow more efficient entry via macaque CD4 receptors? To answer this question, Dr. Overbaugh collaborated with the Bloom lab, which has developed an assay to investigate the consequences of single point mutations in viral proteins, including HIV ENV using Deep Mutational Scanning (DMS). Dr. Jeremey Roop, a former postdoc in the Bloom lab, and his colleagues used DMS to screen thousands of Env point mutants to select for those that enhance HIV-1 entry via macaque receptors. They published their findings in a recent issue of Viruses.

The study identified specific mutations in the HIV-1 envelope protein that enhance entry to cells expressing the rhesus macaque CD4 and CCR5 receptors. Dr. Roop commented on the significance of their work for the field: “We identified mutations in the HIV Envelope protein that allow the virus to more efficiently enter into Rhesus macaque cells. Knowledge of these mutations expands our understanding of how HIV can evolve to cross species barriers, and may also inform vaccine development studies that use Rhesus macaque as an animal model,” said Dr. Roop.

The authors used an Env variant that was cloned directly from a recently infected individual to make sure it is representative of transmitted/founder, circulating HIV-1 strains. They identified previously uncharacterized amino acid mutations in the N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR) regions of the gp41 subunit of Env. These mutations significantly enhanced HIV-1 entry into cells bearing macaque receptors. “Our work is the first to identify a suite of mutations in HIV Envelope’s highly conserved heptad repeat region that impact entry into macaque cells. Interestingly, viruses bearing these specific mutations are rarely observed during human infection,” said Dr. Roop.

In the future, the researchers will investigate if viruses bearing these mutations could be especially vulnerable to the human immune response. “This may help explain why we rarely observe them in nature,” said Dr. Roop. Other interesting questions remain to be answered. “Can these mutations also enhance infection of other non-human primates? Do they facilitate cross-species transmission?” he wondered.

This study was funded by the National Institutes of Health and the Damon Runyon Foundation.

FHCRC/UW Cancer consortium member Dr. Julie Overbaugh contributed to this work.

Roop, J. I., Cassidy, N. A., Dingens, A. S., Bloom, J. D., & Overbaugh, J. (2020). Identification of HIV-1 envelope mutations that enhance entry using macaque CD4 and CCR5. Viruses, 12(2).