Researchers in the laboratory of Sun Hur provide novel insights into the mechanisms underlying the role of a cellular sensor called MDA5 in alerting the immune system to viral invasion.
Reporting in Proceedings of the National Academy of Science online December 12, 2011, an interdisciplinary team led by Sun Hur (Immune Disease Institute and the Program in Cellular and Molecular Medicine at Children's Hospital Boston) in collaboration with Thomas Walz (Department of Cell Biology, HMS) has characterized a novel mechanism whereby the viral RNA sensor MDA5 binds to the invading double-stranded RNA (dsRNA), forming an ordered filament. The binding then stimulates ATP hydrolysis, which causes the MDA5 to un-bind, or dissociate from the dsRNA. Because the dissociation occurs primarily from the filament ends, longer dsRNA makes the MDA5:dsRNA complex more stable. In the report by first authored post-doctoral fellow Alys Peisley, Dr. Hur and her team suggest that this length-dependent dissociation provides a novel mechanism by which MDA5 regulates antiviral signaling, as the length of viral dsRNA differs dramatically from that of cellular dsRNA.
The Hur Lab and MDA5
dsRNA, like DNA, is composed of two complementary strands; it serves as the genetic material for some viruses and has been shown to stimulate an interferon response, the innate immune defense against viruses. However, the mechanisms by which the immune system identifies invading viral RNA among various other forms of RNA present in the body are still being explored.
Dr. Hur has long been interested in protein/RNA interactions at the molecular and structural levels, specifically how the body distinguishes between self and non-self RNA. Several innate pattern recognition receptors are involved, the most well characterized being RIG-I (retinoic acid-inducible gene I protein). Less studied is its paralog MDA5 (melanoma differentiation-associated protein 5). Previous researchers have identified differences between RIG-I and MDA5, including RNA specificity. While RIG-I recognizes non-self RNA according to chemical makeup, MDA5 does so according to RNA length.
However, the new findings point to other major differences. Upon binding, MDA5 forms a filament along the dsRNA strand, becomes activated, and hydrolyzes ATP, which in turn produces energy and fuels dissociation, or un-binding. Dissociated or free MDA5 re-binds to dsRNA, and MDA5 reaches dynamic equilibrium between filament assembly and disassembly processes coupled to ATP hydrolysis.
As Dr. Hur puts it, "The MDA5 filament is somewhat similar to the actin filament or microtubule, which also continuously alternate between shrinking and growing phases in a manner dependent on nucleotide hydrolysis. The difference is that MDA5 uses this dynamic instability as a mechanism to measure RNA length; it self-regulates the filament turnover cycle according to the underlying dsRNA length. This, I think, is a clever way of converting a binary switch (ATP hydrolysis) to an analog response that reflects RNA length." Thus, Dr. Hur believes that her lab has identified a novel length-dependent mechanism by which MDA5 recognizes non-self RNA and initiates antiviral signaling.
Dr. Sun Hur
Sun Hur's scientific interests and background are eclectic, to say the least. She came to the U.S. from Korea in her fourth year of college as a physics major at UC Santa Barbara, intending to become a theoretical physicist. One interest of hers was the theory behind the instability and chaotic nature of liquid systems. Advised to pursue some combination of biology and chemistry, she began work in computational chemistry and biology.
Her first projects focused on the computational analysis of enzyme structure and energetics, more specifically how an enzyme and its substrate change from one conformation to another during a chemical reaction. "I was fascinated by the fact that an enzyme-really a protein blob-does the sophisticated job of catalyzing a chemical reaction by huge orders of magnitude."
When she went to UC San Francisco to work on crystallography and biochemistry, her interest in protein/RNA interactions began to grow. For example, though many proteins must distinguish between types of RNA, recognition depends on upon correct and incorrect folding of the RNA, rather than the RNA's base pair sequence. This theme of alternate modes of recognition would resurface in her work with MDA5.
Dr. Hur's first project involved RNA modification, which is vital for the correct function of cellular RNA machinery, including that of the ribosome and transfer RNA. In her study of pseudouridine, the most common modification in RNA, she learned that these modifications also serve as a "self" identity mark, preventing their recognition as viral RNAs by immune proteins such as MDA5 and RIG-I. Later, when her lab here in Boston found that so little research (including biochemistry) had been done on MDA5, their work began in earnest.
Future directions with MDA5
The Hur lab is now embarking on a more detailed investigation of the MDA5/dsRNA filament, including the next steps in antiviral signaling. When MDA5 binds to viral dsRNA, the signaling adaptor MAVS (mitochondrial antiviral-signaling protein) becomes involved. MAVS is attached to the exterior of the cellular energy source, the mitochondrion. Dr. Hur suspects that after the MDA5/dsRNA filament forms, it forms a more complex filament with MAVS, triggering antiviral signaling and the involvement of interferon. She elaborates: "Simple RNA binding doesn't explain MDA5's signaling activity. Perhaps ATP hydrolysis causes MDA5 to undergo a dramatic conformational change that allows its signaling domain to interact with MAVS."
The Hur lab is very interested in reconstituting the putative MDA5/dsRNA/MAVS signaling complex, first to characterize how MAVS becomes activated, and then to determine exactly how MDA5 interacts with MAVS. Does it really depend on ATP hydrolysis? A recent report suggests that MAVS itself forms a filament, and that the change between filament and monomeric states is the difference between active and repressed forms of MAVS. Key questions that remain are whether and how the MDA5 filament triggers a transition of MAVS from the monomeric to the filament state. Bin Wu, a postdoc in the Hur lab, has been placed in charge of this project. Though significant progress has been made, much work remains.
Another line of exploration in the Hur lab is how the MDA5/RNA filament is regulated by external elements like LGP2 and DAK, enzymes thought to be positive and negative regulators (respectively) of the innate immune defense against viruses. The common wisdom is that such activity is mediated by blocking RNA binding or other functions, but Dr. Hur and her colleagues suspect that some regulators may actually affect filament formation and dynamics.
Dr. Hur's team also plans to examine the commonly held belief that MDA5 and RIG-I recognize only viral RNA; they suspect that MDA5 can recognize cellular RNA in certain pathological situations. There is some evidence to support their contention, both from their own lab and others. Though not as long as viral dsRNA, many longer dsRNA in cells have not yet been well characterized, including transcripts from short stretches of DNA called Alu elements (originally associated with Alu restriction endonuclease). Often considered "junk" DNA because their function is completely unknown, they appear in many different areas of the genome, and many are transcribed.
Now, having suggested a basis for dsRNA length-dependent antiviral signaling and proposing a novel role for ATP hydrolysis in regulating the interaction of MDA5 and dsRNA, Sun Hur and her colleagues clearly have some complex and fascinating work ahead of them.
Alys Peisley, Cecilie Lin, Bin Wu, McGhee Orme-Johnson, Mengyuan Liu, Thomas Walz, and Sun Hur. Cooperative assembly and dynamic disassembly of MDA5 filaments for viral dsRNA recognition. Proc. Natl. Acad. Sci. USA. 2011 Dec 12; Epub ahead of print.
