Single-molecule analysis of influenza virus transcription and replication

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.

We will study the molecular mechanisms of the influenza virus transcription and replication. The influenza virus belongs to the family of negative-sense, single-stranded RNA viruses, which include many important animal and humans pathogens that can cause severe disease. The influenza A virus genome comprises 8 segments of viral RNA (vRNA) with highly conserved 5' and 3' termini interacting to form promoter-like structure bound by the viral RNA polymerase (RNAP). The viral RNAP is highly conserved and may serve as an excellent target for developing antivirals.

Despite the influenza virus being the best-studied negative-sense ssRNA virus, there are many questions about the molecular mechanisms of its genome transcription and replication, in part due to the lack of single-molecule studies. For example, little is known about the conformational changes in promoter RNA and the polymerase in early transcription and replication. Further, despite the recent discovery that replication of complementary RNPs requires an additional polymerase molecule, the role of that molecule is unclear.

We will address these challenges using a combination of powerful single-molecule fluorescence methods and biochemical assays. Aided by recently published structures of the entire RNAP complex and large amounts of influenza proteins, we will prepare labelled polymerases and RNA, and test models of action, revealing rich mechanistic detail about influenza transcription and replication and enabling new ways to combat the disease. Our main tool will be cutting-edge methods, such as single-molecule fluorescence resonance energy transfer and tethered-fluorophore motion. Our studies will provide direct, real-time views of the viral genome duplication and reveal transient intermediates and structural states difficult to observe using other methods. Our work will also help understand polymerase mutations involved in enhanced avian-to-human transmission, with clear implications for human health.

Our mechanistic analysis, the new single-molecule tools, and the novel fluorescence reagents should reveal rich mechanistic detail about influenza transcription and replication, and enable new ways to combat the disease. These findings should also apply to the study of other members of the negative-sense RNA virus group.

Our work can help substantially in the development of anti-viral drugs against influenza. The limited number of current anti-viral drugs rely on targeting the outer neuraminidase and M2 proteins of the virus. The influenza RNA polymerase, due to its central role in the viral replication and its high degree of conservation, forms an attractive universal target for drug design and vaccine development; antivirals that target viral polymerases exist as treatments for other viral diseases such as AIDS and hepatitis. The structural information we will obtain should also help in silico drug screening using computational approaches.

Drug discovery will also benefit from our real-time functional assays that probe influenza replication and transcription, since they can identify drug candidates and evaluate their effectiveness and mode of action. We will explore ways of engaging with industrial partners to accelerate drug discovery, e.g., by working towards developing tests for the efficacy of drug candidates using a high-throughput portable single-molecule fluorescence microscope recently developed in the Kapanidis group.

Such developments in understanding and controlling influenza, as well as related animal viruses, will be important from an animal-health viewpoint, since such viruses can be harmful to many hosts. Flu diseases such as bird flu can also be economically devastating for food industries such as the poultry industry, since millions of birds needed to be culled in the recent outbreaks, resulting in huge economic losses. The outbreaks also lead to exposure of farmers to bird flu, which increase the danger of bird flu outbreaks in poultry leading, to viruses with established human-to-human transmission.

Many animal viruses, such as the influenza virus, are also important from a human medical perspective. The emergence of the deadly avian H5N1 influenza in the human population, coming from a wild aquatic bird source, highlights the importance of studying animal viruses that can be transmitted to humans. Our mechanistic work can also help understand some of the polymerase mutations involved in enhanced avian-to-human transmission, with clear implications for human health.

Detailed mechanistic information from our experiments, as well as the ability to work with flu genomic RNA will aid in early and sensitive diagnosis of viral infections. An attractive application of such methods would involve rapid detection (within 15 min) of influenza virus strains in the doctor's office, allowing a GP to identify dangerous strains (e.g., swine flu or avian flu) and prescribe appropriate medication during a single patient visit; this unique capability should improve the health of populations by minimizing the spread of infections, and result in significant savings since it can limit the unnecessary prescription of antivirals.

Our results will be communicated through academic channels such as scholarly journals, departmental and group websites, and press releases, as well as non-academic channels that will engage end-users of the biosensing applications (hospitals, GPs, and trade associations). Our single-molecule studies of influenza, coupled with use of a compact fluorescence imager, and motivated by a real-world health challenge can help public engagement by promoting fundamentals of molecules and advanced imaging in schools and science shows. Finally, the researchers involved in this work will gain important transferable skills desirable in interdisciplinary industrial or clinical environments involving demanding image analysis, data mining and data processing.