For people living with human immunodeficiency virus (HIV), lifesaving antiretroviral therapy prevents HIV-infected immune cells from making new copies of the virus, preventing disease and transmission.
Historically, these infected cells were known as “latent” HIV reservoirs, meaning that the HIV within the infected cells was completely inactive.
However, the idea that the entire HIV reservoir is latent is actually a misleading explanation, as some of the HIV reservoirs may still be highly active. Even though antiretroviral therapy prevents the production of a full-fledged HIV virus, some infected cells continue to vomit viral products. ”
Dr. Nadia Lone, Senior Researcher at the Gladstone Institute
That means people with HIV who are undergoing treatment are still dealing with fragments of the virus in their bodies, often leading to long-term inflammation and associated medical conditions, including organ damage and an increased risk of heart attack. Also, the greater the number of such “active” carrier cells in a patient, the faster the HIV recovery will occur if treatment is discontinued for any reason, including inability to receive treatment.
If scientists can better understand how the genes in these cells work, this could point to new possibilities for HIV treatment, such as eliminating these cells or blocking their ability to cough up fragments of HIV. However, existing research methods have not been able to achieve this goal.
Now, in collaboration with a team at the San Francisco Veterans Affairs Medical Center, Roan’s team has developed a new tool called HIV-seq to profile rare HIV-infected cells from people infected with HIV.
“Using our new tool, we found important differences in people’s HIV-infected cells before and after starting antiretroviral therapy,” said Roan, senior author of the study published in 2006. nature communications. “We hope this will help us understand how HIV develops and how long-lived HIV carriers persist for decades among people infected with HIV.”
Capturing the elusive HIV-infected cells
In recent years, there has been an explosion of new biomedical discoveries as a method called single-cell RNA sequencing allows scientists to see which genes are turned on in individual cells. However, studies of active HIV reservoirs in people receiving antiretroviral therapy have been less successful.
“When we applied single-cell RNA sequencing to blood samples from patients receiving treatment, we often detected only one or two cells per person,” said Dr. Julie Frouard, a scientist in Roan’s lab and one of the study’s lead authors. “That’s not enough for meaningful analysis.”
The problem, the researchers reasoned, is that the technique requires a specific piece of RNA, a molecule that carries genetic instructions. Unlike many other RNA fragments in human cells, much of the RNA produced by HIV does not meet the required criteria. Therefore, single-cell RNA-seq is not fully captured and reservoir cells that actively produce HIV may be missed by this method.
To address this obstacle, researchers developed HIV-seq, a new tool for single-cell RNA analysis customized for the virus. It is specifically designed to recognize cells that produce HIV RNA fragments.
“We compared HIV-seq head-to-head with standard approaches to recover and analyze more HIV-infected cells and more HIV RNA within those infected cells,” said Steven Yukl, MD, PhD, a physician-scientist at the San Francisco VA Medical Center and the study’s senior author. “Now, for the first time, we can actually characterize these cells in a meaningful way in people whose HIV is suppressed by antiretroviral therapy.”
Because the storage cells could no longer slip through the cracks, the team recovered 25 such cells from three people undergoing treatment. When HIV-seq was applied to blood samples from patients with active HIV infection who had not yet started treatment, more than 1,000 reservoir cells were recovered from four patients. This is the highest number ever.
“Intense” and quiet cells
The scientists then leveraged HIV-seq to characterize HIV-infected cells in HIV-infected individuals before and after starting treatment and identify proteins present on the surface of these cells.
“Previous single-cell RNA-seq studies have primarily analyzed HIV-infected cells in people who have not yet started treatment,” says Dr Sushama Terwatte, now a research fellow at the Doherty Institute at the University of Melbourne. “We felt that these cells probably looked quite different from the reservoir cells in people on treatment, which can persist for decades while producing HIV RNA fragments.”
In fact, scientists have revealed multiple differences in HIV-infected cells before and after antiretroviral therapy.
Cells taken from people who had not started treatment showed cytotoxic signatures, meaning they had proteins associated with the ability to directly kill other cells. These cells also have lower levels of certain genes associated with HIV suppression, suggesting that HIV may somehow suppress these genes in order to rapidly produce new copies of itself.
“In a general sense, you could say these cells were quite inflammatory, or highly inflammatory,” says Roan, who is also a professor of urology at the University of California, San Francisco (UCSF).
In contrast, HIV-carrying cells in treated people were quieter, had anti-inflammatory effects, and had no cytotoxic effects. They also showed higher levels of genes that help the cells avoid death and achieve long-term survival.
“This is noteworthy because clinical trials are underway for drugs that target pathways that HIV may use to preferentially promote host cell survival,” says Yukl, who is also a professor of medicine at UCSF. “Our data further supports that study.”
Scientists also found higher levels of other proteins in the cells of people undergoing treatment. Some proteins are involved in the ability of cells to proliferate stably over long periods of time, while others are involved in suppressing both HIV production and the immune system. These findings may help explain how active storage cells can fly under the radar for long periods when the immune system should recognize and eliminate them.
“We’re already building on some of our new discoveries by testing in a variety of laboratory models whether targeting these pro-survival pathways can stop the proliferation of HIV-carrying cells,” Roan says. “We hope this is just the beginning of everything we can discover with HIV-seq.”
About research
The paper “HIV-seq reveals differences in gene expression between HIV transcription cells of viremic and suppressed HIV-infected patients” was published in the journal. nature communications March 3, 2026.
Authors are Julie Frouard, Xiaoyu Luo, Natalie Gill, Reuben Thomas, and Nadia Roan of Gladstone. Sushama Terwatte, formerly of San Francisco VA Medical Center. Joseph K Wong and Steven Yukl of the San Francisco VA Medical Center; UCSF’s Douglas Arneson, Atul J. Butte, Rebecca Ho, and Stephen Deeks; Pavitra Roychowdhury of the University of Washington; Seulgi Lee of Zuckerberg San Francisco General Hospital.
This research was supported by the National Institutes of Health, California HIV/AIDS Research Program, UCSF Bay Area CFAR, and the James B. Pendleton Foundation.
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Reference magazines:
Frouard, J. Others. (2026). HIV-seq reveals differences in gene expression between HIV-transcribing cells from viremic and suppressed HIV-infected individuals. Nature Communications. DOI: 10.1038/s41467-026-68797-3. https://www.nature.com/articles/s41467-026-68797-3
