Science Spotlight

How the Gale Lab “RIG”s innate immune responses against hepatitis B

From the Gale Lab, Pathogen Associated Malignancies Program, Cancer Consortium

In the age of COVID-19 we are inundated with information about how our adaptive immune system, comprised of specialized immune cells, patrols our bodies to destroy viruses before they can infect us. But how does a cell that’s already infected fight off a virus? Almost all our cells contain receptors that recognize biological features common to viruses (pathogen-associated molecular patterns, or PAMPs). Viral fragments that bind to these receptors get flagged as “foreign”, triggering the internal machinery known as the innate immune response and causing the cell to switch on antiviral genes. For instance, your own genome is neatly packed away in the nucleus of each cell, so DNA found in the cytoplasm is an anomaly that signals “danger”. However, what about a virus that’s camouflaged so well that your own cells don’t know where they end and where the virus begins?

Hepatitis B virus (HBV) is a “stealth virus”, capable of blending in. It has a DNA-based genome, and in the later stages of infection this genome gets compressed into a long-lived viral mini-chromosome, referred to as covalently closed circular DNA (cccDNA), that hides itself in the nucleus among the host DNA. This vanishing trick allows HBV to maintain a steady-state population within cells without setting off the innate immune response’s alarm bells. This contributes to chronic disease that requires a majority of patients to undergo lifelong antiviral therapy. Current treatments can inhibit viral replication, antigen production, and transcription of the viral genome, but work poorly to eliminate the cccDNA from host cells. The laboratory of Cancer Consortium member Dr. Michael Gale Jr at the University of Washington South Lake Union Campus recently published a study in iScience demonstrating a new method for clearing cccDNA from infected cells.

The Gale Lab found that RIG-I agonists activate IRF3-dependent antiviral genes in hepatocytes, leading to the suppression and active decay of cccDNA, a stubborn component of chronic hepatitis B virus infection.
The Gale Lab found that RIG-I agonists activate IRF3-dependent antiviral genes in hepatocytes, leading to the suppression and active decay of cccDNA, a stubborn component of chronic hepatitis B virus infection.

The innate immune response, as outlined above, relies on cellular receptors to detect foreign materials within cells. One of these receptors, RIG-I, binds to RNA-based PAMPs and activates the transcription factor IRF3, which then shuttles to the nucleus to induce antiviral genes. RIG-I activation has a previously characterized role in restricting and clearing hepatitis C virus (HCV) infection. However, HCV is an RNA virus, unlike HBV, and the Gale Lab did not know if the same pathway had any effect on HBV, and in particular the long-lived cccDNA pool. Led by Dr. Lee, the authors tested two compounds for their ability to activate RIG-I signaling: F7, a small molecule identified in a previous screen, and poly-U/UC, a short segment of the HCV genome. Both compounds activated IRF3 and antiviral genes in a dose-dependent manner, and both compounds appeared to reduce the formation of cccDNA compared to HBV-infected but untreated cells.

This alone is good news for preventing the accumulation of cccDNA in new infections, but what about in the case of existing HBV infections? The authors next treated hepatocytes with F7 or poly-U/UC starting 3 days post-infection with HBV. They compared their results to treatment with the antiviral drug Entecavir (ETV), which is commonly prescribed to patients with chronic HBV and which reduces but does not eliminate cccDNA. Cells treated with F7 or poly-U/UC, on their own or in combination with ETV, harbored significantly less cccDNA when compared to HBV-infected cells treated with ETV alone. Importantly, the amount of cccDNA continued to reduce over time, with cells that received combination therapy of ETV + F7 or ETV + poly-U/UC containing 25% of the amount of cccDNA as ETV-only cells by 20 days post-infection. This suggests that F7 and poly-U/UC are contributing to the active decay of cccDNA in nuclei of infected cells.

To round out their study, the Gale Lab decided to repeat their study in primary human hepatocytes (PHH), which represent a more endogenous system than the immortalized cell lines used in their previous experiments. They found that poly-U/UC activated IRF3 and IRF3-dependent genes in these cells, and that poly-U/UC treatment 24hr after HBV infection reduced the amount of cccDNA by a drastic 75-88.5%, depending on dose. Dr. Michael Gale Jr. summarized the exciting findings: “This work shows that targeting RIG-I with a specific RNA ligand or small molecule activator can serve as therapeutics to induce innate immune activation in HBV-infected cells to direct a response that suppresses HBV infection by blocking cccDNA production.” However, the work also leads to more questions. For instance, innate immune signaling is a tangled web with a large variety of genes involved – so “what are the RIG-I-responsive genes that confer cccDNA suppression?” asked Dr. Gale. The lab likely has many fruitful follow-up experiments ahead of them.

This work was supported by grants from the National Institutes of Health (NIH), NIH contracts, and work under contract with the US Department of Energy.

Cancer Consortium faculty member Dr. Michael Gale Jr. contributed to this research. Dr. Gale thanks the CCG for the cross-institution interactions with liver and cancer experts for scientific consultation.

Lee S, A Goyal, AS Perelson, Y Ishida, T Saito, and MG Gale Jr. 2021. “Suppression of hepatitis B virus through therapeutic activation of RIG-I and IRF3 signaling in hepatocytes”. iScience. 24: 101969.