Single-molecule analysis of influenza virus transcription and replication

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The proposed work involves using special microscopy methods to understand how the flu virus copies its genetic material. The flu (influenza) virus is the microscopic pathogen that causes flu in human and in animals. Naturally, the flu virus is found in wild birds; however, it has the ability through mutations to move to other host organisms, including humans. Such transmission leads to mild annual epidemics as well as severe pandemics, such as the Spanish flu 1918 pandemic, which was one of the deadliest disasters in human history, responsible for over 50 million deaths worldwide. Slightly different versions of the flu virus were also responsible for the swine flu outbreak in 2009 and the H5N1 bird flu. Bird flu was especially devastating for the poultry industry since millions of birds need to be culled, resulting in huge economic losses; it also led to exposure of farmers to bird flu, which increased the danger of bird flu outbreaks in poultry leading to viruses with the ability to be transmitted within human populations. Our research will help the scientific community to understand better how the virus propagates and help in efforts to control and stop its spread.The flu virus has its genetic material ("genome") made of tiny RNA fragments. In order to multiply, the virus copies its RNA using a tiny viral molecular machine called an RNA polymerase. Despite the flu virus being one of the best-studied viruses, we are still unsure about how exactly the genomic copying is achieved and controlled. One of the reasons for this is that it was impossible to study the copying of its RNA genes directly. Direct observation is difficult since the polymerase machinery is very small, it is difficult to generate it in a pure form, and it is difficult to assemble the machinery on the RNA to be copied in an efficient and homogeneous way.Through a collaboration of a laboratory that studies the virus with a laboratory that develops and uses advanced optical microscopes, we can now overcome these difficulties and want to observe the copying of the viral genome directly, as it happens. We plan to achieve this by preparing fluorescent polymerase machines (for example, a protein that fluoresces green) and fluorescent bits of the viral RNA (for example, an RNA that fluoresces red), and use the direct observation of the different colours to understand how the polymerase recognizes the RNA, and copies it. We can observe these changes directly as they occur by anchoring the RNA or the polymerase machine on a glass surface and then recording fast "molecular movies" of the copying of the viral genetic material.This fascinating observation is performed using a special microscope, which we call a "single-molecule fluorescence microscope". This microscope is carefully designed to allow detection and monitoring of individual ("single") fluorescent molecules present in a detection zone (as opposed to conventional microscopes that require thousands or millions of molecules to be present in a detection zone). The single-molecule fluorescence microscope allows one to determine the number of parts that make up a biological machine, to measure how strong the parts bind to each other, how far apart the parts are spaced and at what orientation, and what are the movements of the parts when the tiny biological machine works. We are also very interested in studying how tiny differences in the structure of the copying machine allow the virus to move for a bird host to humans. We anticipate that our work will allow us to understand better how the virus copies its RNA and use this new information and our microscopes to find new ways to combat the disease. Our discoveries should also help understand and control other viruses that cause danger to humans and animals.