The viral replication cycle is crucial for a virus to spread inside the body and cause disease. Focused on that cycle in the hepatitis A virus (HAV), UNC School of Medicine scientists discovered that viral replication requires specific interactions between the human protein ZCCHC14 and a group of enzymes called TENT4 poly(A) polymerases. They also found that an oral compound stopped replication at a key step, making it impossible for the virus to infect liver cells.

The results are the first to demonstrate an effective drug treatment against HAV in an animal model of the disease, the team suggested. “Our research demonstrates that targeting this protein complex with an orally delivered, small molecule therapeutic halts viral replication and reverses liver inflammation in a mouse model of hepatitis A, providing proof-of-principle for antiviral therapy and the means to stop the spread of hepatitis A in outbreak settings,” said senior author Stanley M. Lemon, MD, professor in the UNC Department of Medicine and UNC Department of Microbiology & Immunology, and member of the UNC Institute for Global Health and Infectious Diseases.

Lemon and colleagues reported on their findings in the Proceedings of the National Academy of Science (PNAS) in a paper titled “The ZCCHC14/TENT4 complex is required for hepatitis A virus RNA synthesis,” in which they concluded, “These results reveal requirements for ZCCHC14-TENT4A/B in hepatovirus RNA synthesis, and suggest that TENT4A/B inhibitors may be useful for preventing or treating hepatitis A in humans.”

Outbreaks of hepatitis A have been on the rise since 2016, even though the HAV vaccine is very effective. But not everyone gets vaccinated, Lemon pointed out, and HAV can exist for long periods of time in the environment—such as on our hands and in food and water— resulting in more than 44,000 cases, 27,000 hospitalizations and 400 deaths in the United States since 2016, according to the CDC figures cited by the team.

The researchers noted that several outbreaks have occurred over the past several years, including in San Diego in 2017 driven largely by homelessness and illicit drug use, causing severe illness in about 600 people and killing 20. In 2022, there was a small outbreak linked to organic strawberries in multiple states, leading to about a dozen hospitalizations. Another outbreak in 2019 was linked to fresh blackberries.

Globally, tens of millions of HAV infections occur each year, the investigators further pointed out. Symptoms include fever, abdominal pain, jaundice, nausea, and loss of appetite and sense of taste. Once sick, there is no treatment. “There is no effective antiviral therapy for hepatitis A … Despite excellent vaccines, resurgent outbreaks of hepatitis A have caused thousands of hospitalizations and hundreds of deaths within the United States in recent years.”

Lemon, who in the 1970s and 80s was part of a Walter Reed Army Medical Center research team that developed the first inactivated HAV vaccine administered to humans, said research on HAV tapered off after the vaccine became widely available in the mid-1990s. Cases plummeted in the 2000s as vaccination rates skyrocketed. Researchers turned their attention to hepatitis B and C viruses, both of which are very different from HAV and cause chronic disease. “It’s like comparing apples to turnips,” Lemon said. “The only similarity is that they all cause inflammation of the liver.” HAV is not even part of the same virus family as hepatitis B and C viruses. And, as the team noted in their newly published paper, “Many aspects of the hepatitis A virus (HAV) replication cycle remain to be elucidated.”

In 2013, Lemon and colleagues discovered that the hepatitis A virus changes dramatically inside the human liver. The virus hijacks bits of cell membrane as it leaves liver cells, cloaking itself from antibodies that would have otherwise quarantined the virus before it spread widely through the bloodstream. This work provided insight into how much researchers had yet to learn about this virus that was discovered 50 years ago and has likely caused disease dating back to ancient times.

A few years ago, researchers found that hepatitis B virus required TENT4A/B for its replication. Meanwhile, Lemon’s lab led experiments to search for human proteins that HAV needs in order to replicate, and they found ZCCHC14—a particular protein that interacts with zinc and binds to RNA.

“This was the tipping point for this current study,” Lemon said. “We found ZCCHC14 binds very specifically to a certain part of HAV’s RNA, the molecule that contains the virus’s genetic information. And as a result of that binding, the virus is able to recruit TENT4 from the human cell.”

In normal human biology, TENT4 is part of an RNA-modification process during cell growth. Essentially, HAV hijacks TENT4 and uses it to replicate its own genome. “Here, we show that ZCCHC14 and TENT4A/B are required for viral RNA synthesis following translation of the viral genome in infected cells,” the investigators stated. Their work suggested that stopping TENT4 recruitment could stop viral replication and limit disease.

Lemon’s lab then tested the compound RG7834, which had previously been shown to actively block Hepatitis B virus by targeting TENT4. In the studies reported in the PNAS paper, the researchers detailed the precise effects of oral RG7834 on HAV in liver and feces and how the virus’s ability to cause liver injury is dramatically diminished in mice (Ifnar1-/- animals) that had been genetically modified to develop HAV infection and disease. These mice lack type I interferon receptors and are highly permissive for HAV, developing infection mirrors that of hepatitis A infection in humans. “Our data show that oral delivery of RG7834, a dihydroquinolizinone TENT4 inhibitor, blocks HAV replication and profoundly interrupts pathogenesis in a murine model of hepatitis A,” the investigators wrote.

The research results suggest the compound was safe at the dose used for, and acute time frame of, the reported study. They noted in their paper, “These results reveal requirements for ZCCHC14-TENT4A/B in hepatovirus RNA synthesis, and suggest that TENT4A/B inhibitors may be useful for preventing or treating hepatitis A in humans … Although the mechanism by which TENT4 contributes to HAV replication requires further study, its role as an essential host factor provides an unexpected opportunity for both antiviral therapy and chemoprevention of hepatitis A.”

Lemon further stated, “This compound is a long way from human use. But it points the path to an effective way to treat a disease for which we have no treatment at all.” RG7834 was developed by Roche for use against chronic hepatitis B infections, and has been tested in a Phase I trial. However, the UNC team noted, animal studies suggested the compound may be too toxic for use over long periods of time. “The safety of

“RG7834 was evaluated in a phase I ascending-dose clinical trial involving 49 participants without reported adverse effects (ClinicalTrials.gov NCT02604355),” the investigators noted in their paper. “However, the compound has not progressed further due to toxicity concerns identified in long-term animal exposure studies.”

In contrast, Lemon noted, “The treatment for Hepatitis A would be short term … and, more importantly, our group and others are working on compounds that would hit the same target without toxic effects.” As the authors concluded in their paper, “Experiments in Ifnar1-/- mice suggest that short-term pharmacologic intervention with RG7834 or potentially fewer toxic hepatoselective compounds may have substantial clinical benefit and could be lifesaving in severe hepatitis A.”

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