New Haven, Conn. — For decades scientists have recognized that human immunodeficiency virus (HIV) is a formidable viral pathogen. After years of probing work and extensive experimentation, a Yale research team has unlocked one of the reasons why that is.

In a new study, the lab led by immunologist Grace Chen discovered that HIV produces a circular RNA (circRNA) that helps the virus turn on its genes and replicate more efficiently. The discovery, which the researchers dubbed “circHIV,” could represent a new target for future HIV therapies.

The findings, described in the journal Nature Microbiology, follow a long journey for Chen, an assistant professor of immunobiology and of genetics at Yale School of Medicine. Her lab studies circRNAs, which differ from typical or linear RNAs in that they lack distinct, functional “ends.” Unlike linear RNA, loop-shaped circRNAs are very stable.

Chen began this project in 2019 with Prisca Obi, who at the time was her graduate student, and Lichong Yan, an associate research scientist and postdoc, who are the study’s co-first authors. Given HIV’s genetic makeup, Chen thought it would be a likely candidate to be generating circRNAs. She was right.

After first sequencing RNA from HIV-infected cells, the researchers discovered that HIV encodes circRNAs, including the abundantly expressed one that they name circHIV. They then detected the looped structures in infected human cells and in blood plasma from people living with HIV.

Further experiments showed that reducing levels of the circHIV also lowered HIV gene activity, while adding extra circHIV increased it. Finally, protein-binding experiments revealed how the circHIV binds to the HIV Tat (Trans-Activator of Transcription) protein, explaining how it’s able to boost viral transcription so readily.

They gathered much of this key data in early 2020, before the research came to a halt with the arrival of the COVID-19 pandemic.

“We had four to six months of just brainstorming ideas,” Chen said, “when we were working from home. We couldn’t wait to get back in the lab and test them out.”

When they finally returned to the lab, another thing was needed: control samples of blood not infected with HIV. The lab members rolled up their sleeves. “The team was so committed that every one of us gave our own plasma to be negative controls,” Chen said, “and a doctor down the hall from my lab drew our blood.”

Chen cites her background in chemical biology and molecular biology for leading her to wonder if viruses could produce unusual RNAs. She purposefully assembled a research team with diverse training and expertise and invited specialists from “across the medical school when we ran into a question that extended beyond our lab.”

Colleagues from Yale School of Medicine’s departments of immunobiology, microbial pathogenesis, laboratory medicine, and genetics contributed samples, reagents, and expertise. “This is a truly multidisciplinary group, both within my own lab and with our collaborators, and it really represents the spirit of this work,” said Chen, adding that she is grateful for “the generosity of this community.”

Chen was first drawn to circRNA research when she began her postdoc work in 2013, shortly after two landmark papers revealed that circRNAs are abundant and ubiquitously expressed in eukaryotes (organisms with cells that feature a membrane-bound nucleus containing DNA and specialized organelles).

“The papers found so much rich data that had previously been discarded and really launched a whole new area of RNA biology,” she said. “I was enthralled by the idea that we’d been looking at RNA for so long and we’d missed an entire category. We had assumed that functional RNA was only linear, and circRNAs were useless byproducts.”

“We’ve been trying to understand the biology of HIV for decades,” Chen said. “And now we’ve discovered something new about it. I never bet against RNA. It has a way of doing things that surprise you.”