Pitt study outlines new way to tackle HIV

PITTSBURGH — A study out of the University of Pittsburgh outlines a new technology for targeting Human Immunodeficiency Virus, or HIV.

Published Tuesday in the journal Cell Chemical Biology and funded by a grant from the National Institutes of Allergy and Infectious Diseases, it paves the way for a slice of medicine focused not just on allowing people with HIV to live long and happy lives, but ultimately working to clear the virus from their bodies.

More than 1.2 million people live with HIV in the U.S., according to the Centers for Disease Control and Prevention, and nearly 1 in 8 is unaware of their infection. While antiretrovirals have been approved in this country to treat HIV for decades, they do not destroy the virus, only quell viral load so long as a person takes them.

"The field now as a whole is looking at curative research," said Lori Emert-Sedlak, a research associate professor of microbiology and molecular genetics at Pitt and first author on the paper. "There are many drugs that suppress the virus," she said. "We need to get rid of the virus entirely."

One of those approaches involves targeting a key HIV protein — nef — and Pitt researchers were able to destroy it.

The nef protein is made by a gene in the HIV virus. Related viruses, like Simian Immunodeficiency Virus (SIV), the primate version of HIV, also have nef proteins. The protein shows up shortly after a person is infected and helps the virus replicate in the body. It evades immune responses — and allows the virus to slink by undetected — by removing key cell surface structures that would otherwise alert the immune system of their foreign presence.

"You can't cure something the immune system can't see," said Emert-Sedlak.

Studies have shown that certain people with HIV who have nef-defective genes often do not progress to developing AIDs, pointing to the potential that nef may be a good target.

While past research has focused on inhibiting nef and thus stopping replication of HIV in the body, there's a risk of viral rebound if the person stops taking the drug.

"Although (previous technologies) were effective in blocking some functions of nef, it was difficult to shut everything down, because it has multiple functions" said Thomas Smithgall, senior author on the paper and a professor in the Department of Microbiology and Genetics at the University of Pittsburgh School of Medicine. "(Our complex) is not just a blocker, but a destroyer."

Is it a cure for HIV?

"We try to avoid the use of the c-word," joked Smithgall.

They accomplished their results with a little bit of chemistry Legos, if you will. The team took a compound called a nef-inhibitor — which binds to the nef protein, and which they had created and described in past research — and made it into a complex called a PROTAC, with a molecule that links them together.

PROTACS are large molecules that degrade proteins by attaching a "red flag" to them, thus directing them to the proteasome — what Emert-Sedlak refers to as the "garbage disposal of the cell."

That flag, in this case, is a protein called ubiquitin, which the body already produces en masse (hence its name): "We're utilizing the cell's own machinery," said Emert-Sedlak, a Pittsburgh native who joined Smithgall's lab in 2005.

Ubiquitin is abundant. The task for the team was to create numerous different PROTACs in order to find ones that worked well.

So Colin Tice, a research fellow at the Philadelphia-area Fox Chase Chemical Diversity Center, synthesized more than 100 compounds for the team to test out. After months of experimentation, they settled on a dozen promising compounds.

The team grew human T cells in a dish — T cells are a kind of immune cell in the body — with nef, which had a fluorescent tag attached so researchers could see when it was produced. Then, after expressing nef, they fed the cells the dozen PROTACs and measured whether they degraded nef.

They found that a select six or seven PROTACs destroyed nef via flagging it for the aforementioned garbage disposal. The PROTACs also restored key cell surface proteins that are often dangerously low in people infected with HIV and help the body to recognize infected cells.

The entire study took more than two years to complete.

"This study unravels another molecule derived from the virus that can be targeted to inhibit replication of the virus," Kamel Khalili, chair of the microbiology, immunology and inflammation at Temple University's Lewis Katz School of Medicine, said via email statement. Khalili studies HIV but was not involved in this research.

"It is important to note that this strategy is not set up for elimination of the virus, but rather the decrease of viral replication," he noted.

Before PROTACs, the team screened over 250,000 compounds to identify preexisting nef inhibitors, which alone took 18 months and led to a previous paper by Emert-Sedlak.

The lab still wants to learn more about how to effectively destroy nef and plans to scale up to an animal model. They also want to examine any off-target effects of the PROTAC complex — in other words, changes to cells or side effects that are unintended.

While they showed that their PROTAC complex can destroy nef, they have more work to do to link it to full HIV clearance — and before they could link it to a viable treatment.

"We show that you can create molecules that go into cells, hit their target, seemed to destroy it, restore receptors, and have antiretroviral activity," said Smithgall. "But we haven't directly shown that, as a consequence, that will trigger an anti-HIV immune response. And we're actually doing that with another lab here. We're just starting those experiments."

And the team recognizes the need to address the potential of drug resistance, as often occurs with viral treatments.

"This is a huge problem, and it can't be ignored," said Smithgall. "How that is going to play out with nef isn't clear."

Thus far, it's one of the first studies that outlines using a PROTAC complex for infectious disease. PROTACs exist for cancer: The idea is to clear out the proteins that are allowing the bad guys to hide, enabling the immune system to fight back.

"Many viruses have nef-like proteins," said Emert-Sedlak — including the SARS-CoV-2 virus, which causes COVID-19. "This technology could be broadly applicable to other viruses."

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