By Bharat Srinivasa
Infectious disease has undoubtedly been one of history’s greatest killers of human beings. But with the development of vaccines, the discovery of antibiotics and drastic changes in public health, the human life span has increased over the past century. Yet, the threat of infectious disease still haunts us. As an HIV epidemic ravages through Africa and South East Asia, and a global avian influenza pandemic looms, there is an urgent need to understand, and help the immune system in its battle against pathogens. On the other hand, there is also a need to stop an over-active immune response.
One of the most important features of the immune system is its ability to differentiate between self (i.e., human) and non-self (i.e., a pathogen). When a virus enters a cell in our body, a host of immune proteins (such as MDA5, RIG-I) recognize patterns that are common to pathogens but not present in us – double stranded Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), or triphosphorylated RNA, for example.
When these patterns are detected, cellular signaling pathways are initiated, resulting in the secretion of a key cytokine called Interferon. Interferon, in turn activates numerous genes to produce defense proteins, including the IFIT family of proteins in the infected and surrounding cells, creating an anti-viral environment.
This key protein enables the human immune system to detect viruses and prevent infection by acting as foot soldiers guarding the body against infection. They recognize foreign viral RNA produced by the virus and act as defender molecules by potentially latching onto the genome of the virus and preventing it from making copies of itself, blocking infection. The findings are a promising step towards developing new drugs for combatting a wide range of immune system disorders.
Identifying these proteins, and the roles they play has been an intense area of research – especially for Bhushan Nagar of the Department of Biochemistry. “It [the IFIT family of proteins] was discovered a long time ago, but the functional analysis started only 10-15 years ago,” he says.
In 2011, Giulio Superti-Furga’s lab at the Austrian Academy of Sciences published a paper in Nature that used a proteomics approach, which involved fishing in a soup of laboratory cell extract with triphosphorylated RNA as bait. To their surprise, they captured the IFIT proteins. Nagar refers to this work as “the seminal finding.”
Superti-Fagar contacted Nagar, whom he had met during the latter’s post-doctoral studies. This started the collaboration that led to the current work, a structural description of the IFIT5 protein and its interaction with triphosphorylated RNA, which was published earlier this year in Nature.
“The structural biology – technique-wise – is routine,” said Nagar. “We made the protein in bacteria, where serendipitously, some portion of the protein was bound to bacterial triphosphorylated RNA.”
They initially had some difficulty studying this protein, since it bound to RNA of various sizes [heterogenous RNA]. To further study the protein’s interaction with RNA, they had to make RNA in the test tube, which Nagar credits Yazan Abbas, the fourth year PhD student and lead author of the paper for “doing an excellent job in following up on the literature on how to make [large amounts of less heterogenous] RNA and really figuring things out.” Both Nagar and Abbas agree that making the RNA was the hardest part of the project.
The eureka moment, a smiling Abbas says, was “seeing the electron density maps which are used to build atomic models of protein. We had this for IFIT5, but we were not sure of where the RNA goes. When we solved it [the structure] with the RNA, when we had the electron density map with the triphosphate RNA, we got it. According to my boss, he just said ‘we’re in’.”
“The definite proof [of function] is when you have a crystal structure,” says Nagar. “Six months before our work, the structure of RIG-I interacting with RNA was published, the MDA5 structure was published – this is a very hot area of research.”
The IFIT family of proteins consists of a tetratricopeptide repeat domain (TPR domain) that, due to the work of Nagar, is now known to interact with both triphosphorylated RNA and proteins. What this means in the context of anti-viral immunity is still unknown, and is where the project is headed. The next stages of this project will look at “drugs or inhibitors based on these RNA molecules, for when the immune response is too strong,” says Nagar. Other future directions involve understanding the role of this protein in viral infections, such as influenza.
“The virus is attacking, these proteins are defending, what you normally get when you see the flu virus is a balance between the two. If we did not have this defense, we’d have much worse symptoms than what we get,” says Nagar.
Bharat Srinivasa is pursuing a PhD in Immunology at McGill. His research involves studying the role of respiratory virus infections in increasing the risk of asthma.