Protein Cuts Early Infection Down to Size

Researchers have found a crucial step in the early warning system that activates the body’s defenses against influenza, hepatitis C, rabies, and related infections. This recently discovered sensing pathway, a new paper reports, depends in part on signaling by an antiviral molecule that belongs to a large family with recognized but poorly understood antiviral credentials.

“This is a critical link in a multistep pathway that is key to the host response to virus infection,” said virologist John Hiscott of McGill University, an expert in innate immunity who was not involved in the study. “It is an important step in the rapid response mechanism.”

The molecule, TRIM25, helps transmit the alarm early in the biological chain reaction that goes on to flood the cell and surrounding tissue with well-known antiviral and inflammatory chemicals that limit virus replication. These signals help cells attract warriors from the adaptive immune system to the site of infection for a systemic response. A famous relative, TRIM5-alpha, blocks HIV replication after infection.

The study “provides a detailed mechanism for how the host reacts against a viral infection to generate antiviral activity,” said senior author Jae Jung, HMS professor of microbiology and molecular genetics at the New England Primate Research Center. The findings are published in the April 19 Nature.

Ten years ago, immunologists thought they had figured out how cells defend themselves against the initial assault of unfriendly bacteria, viruses, and fungi. Molecules on cell surfaces called Toll-like receptors, a family of 10 proteins in humans, recognize the microbes and start a cascade of signaling that turns on the interferons, cytokines, and chemokines of innate immunity.

Toll-like receptors are not the only game in town anymore, a recent review proclaimed. The fast-moving field has revealed a more extensive innate immunity network that quickly kicks in as soon as microbial infection begins and long before the adaptive immune system gets going.

One recently discovered innate pathway detects influenza, hepatitis C, and similar viruses that can sneak undetected past the Toll-like receptors. The pathway’s key sensor, retinoic acid–inducible gene I (RIG-I), stands guard inside cells and activates the same intracellular virus-clearing chemicals as the Toll-like receptors. RIG-I’s activity was discovered less than three years ago in Japan in the labs of Taskashi Fujita and Shizuo Akira, a co-author on Jung’s paper (and the scientist who gave the 2007 Dunham lectures at HMS).

Earlier this year, other researchers found evidence, but did not prove, that abnormally low RIG-I expression in tissues of monkeys infected with the reconstructed 1918 pandemic influenza virus may have contributed to the deaths of more than 40 million people worldwide.

Graduate student Michaela Gack started the project two years ago. Researchers knew that RIG-I senses certain RNA viruses, such as influenza and other respiratory pathogens, as well as flaviviruses, like hepatitis C, and activates a complex signaling cascade.

“All these important molecules have to be regulated,” Gack said. “They can’t always be turned on. So we said, let’s look for another cellular partner that might regulate the RIG-I function.”

One end of RIG-I binds to the virus, and the other end signals the downstream partner. Gack used the signaling end to pull down anything else in the cell that would bind to it. The strongest results from mass spectometry showed a chain of three ubiquitin molecules attached to RIG-I. Ubiquitin is better known for its part in degrading proteins than activating them. The different functions seem to depend on how and where the ubiquitins are linked.

Further analysis confirmed that the ubiquitins were chained together in a way that would work to send a molecular signal. Ubiquitination increased when the cell was stimulated with a common RNA virus. Gack found the exact site on RIG-I that anchored the chain. A point mutation there disabled the interferon response, apparently by disrupting ubiquitination.

“We found something, but we still had to figure out why it is there and what it does,” Gack said. TRIM25 emerged from two more sweeps in search of the cellular protein that delivered the ubiquitin molecules to RIG-I. Cancer researchers know TRIM25 as estrogen finger protein (EFP). It ubiquitinates and degrades a cell cycle inhibitor, but it can be overexpressed in breast and ovarian cancers, transforming into an oncogene that promotes excessive cell growth.

Jung immediately recognized the delivery protein as a member of the TRIM family.

“I hardly knew anything about the TRIM family, which so far consists of 68 members and is one of the hottest areas in the viral research field,” Gack said. One building over, teams of researchers work on the famous relative, TRIM5-alpha, whose ability to block the very early stage of HIV infection was discovered three years ago by Joseph Sodroski, HMS professor of pathology at the Dana–Farber Cancer Institute (see Research Briefs, Focus, March 5, 2004).

“Now we had to test if this is the E3 ligase that delivers ubiquitin to RIG-I,” Gack said. Sure enough, overexpression of TRIM25 increased RIG-I ubiquitination and produced more interferon, while a gene knockdown assay decreased both. Just to be sure, they tested the antiviral role of TRIM25 in cells from a trim25-knockout mouse developed in Japan. Cells without TRIM25 showed a 100-fold increase in viral replication and were unable to produce interferon-beta.

“It was very straightforward; first we got ubiquitin, then we got the ubiquitin E3 ligase,” Gack said. Now, the remaining question was, “Why was ubiquitination by TRIM25 essential for the signal transduction of RIG-I to induce interferon?” A RIG-I point mutant that took away the TRIM25 ubiquitination site bound poorly to its downstream signaling partner MAVS in comparison with the wild-type RIG-I.

This investigation completes the first part of Gack’s doctoral experiments, conducted at HMS as part of an exchange program with Friedrich–Alexander University in Germany, which will award her doctorate. To follow up, she is seeking the viral protein that can do a runaround of this step in the RIG-I alert system. Hepatitis C, for example, chops up MAVS and thereby disarms the interferon-mediated antiviral pathway at an enzymatic step just after RIG-I.

The strength of this paper comes not just from identifying TRIM25, Hiscott said, but from a variety of techniques and approaches that defined the mechanism of where and how the protein affects the RIG-I pathway and then a number of different ways that showed its operation in the cell and its importance for the antiviral response.