Unravelling a tangle: how segmented double-stranded RNA viruses package their genome

Viruses are obligate parasites and the viral genome is the most important component in keeping a virus alive. Once viruses infect a host cell (human, animal, plant or fish) they must replicate, i.e. make copies of their components and assemble into viable progeny viruses in order to spread. This process of virus synthesis is only possible if the viral genome is incorporated into the new progeny in the correct form. It is particularly difficult for viruses to incorporate the genome if it is in multiple pieces, which is often the case in many viruses (e.g. influenza virus). Understanding how these multiple pieces of genes, each carrying different messages, are incorporated in the correct order and form is essential to understanding how viruses multiply and spread. The lack of this information has created a bottleneck in the research and understanding of many viruses over the years. Therefore this project will focus on animal viruses, particularly Bluetongue virus (BTV) of sheep, which is primarily characterized by its 10 pieces of genes that are not DNA but RNA molecules or segments, each representing a single gene and each generating specific viral proteins.

We are exceptionally positioned at the forefront of this field of research. In previous BBSRC funded projects, we have developed unique tools, techniques, reagents and a series of novel assay systems that have already defined some of the essential steps of how BTV may recruit their segments and assemble into a viable virus, competent for spreading. The precise nature of selection and packaging, that is acquiring a single copy of each of ten segments rather than multiple copies of one, is a complex and highly regulated process as the virus must differentiate between cellular and viral genes. We will take advantages of our tools and assays as well as some of the cutting-edge methods that have been developed only recently by other scientists. We will define this poorly-defined complex process to provide information on how BTV and related viruses package their genes and utilise our own methods and expertise in combination with the other biological and physical sciences through collaboration with experts.

Long term improvements in animal welfare underpinned by basic research into the pathogen concerned are important. BTV is highly pathogenic in certain livestock and has recently emerged in the UK and Europe. Understanding these vital basic processes in the life cycle of the viral genome will make it possible to develop novel, safer designer vaccines for Bluetongue disease and may be other viral diseases that affect livestock. The data will also allow for the development of novel interventions to improve future disease management.

Understanding viral genome packaging is challenging as it relates to the balance that must be struck between the RNA genome being rigidly encased and so protected yet being flexible enough to be poised for template function. This position, a linchpin in the virus life cycle, is poorly understood, in part, through the lack of suitable assay system with which to interrogate the processes involved. In previous BBSRC funded projects, we have developed a series of novel assay systems that have defined some of the essential steps in the packaging process critical for replication of Bluetongue virus (BTV), an orbivirus and model for segmented, double-stranded RNA viruses. BTV is pathogenic in certain livestock and its recent emergence in the UK and Europe resulted in significant economic loss. Long term improvements in animal welfare underpinned by basic research into the pathogen concerned are therefore important. The precise nature of genome packaging, that is acquiring a single copy of each of ten segments rather than multiple copies of one, is a complex and highly regulated mechanism as the virus must differentiate between cellular and viral RNA. We will define this complex mechanism by using the novel assays developed in our previous studies along with new techniques (SHAPE, Cryo-EM, CLIP-Seq, Single molecule FRET etc) in collaboration with experts in the field, to identify the cis-acting RNA sequences and structures required for packaging. Further, we will define the trans-acting proteins required for recruiting these RNA structures into the subsequent replication cycle. We will clarify a currently ill-defined process to provide information on how genome packaging is brought about for this and related viruses.

Our data will have the potential to slice the Gordian knot of RNA packaging to allow for the development of novel interventions and attenuated vaccines to improve future disease management.

Using BTV as a model system, a significant step forward has been recently made in understanding the assembly pathway and genome packaging of segmented RNA viruses. A combination of in vitro assay systems that we developed during the last BBSRC project, such as a biotinylated-streptavidin-bead coated primer system for recruitment of RNA segments in a sequential manner, a RNA-RNA interaction and RNA complex formation assay to visualize the complexes and their interference by small ORNs, combined with an established cell-free reconstitution of infectious virus system has made it possible to provide some breakthrough insights on RNA packaging mechanism for segmented viruses. Significance of these achievements has been noted with recent two publications in high impact journals (PloS Pathogens and Nucleic Acid Research). The impact on the potential antiviral design has also been recognised by a recent patent application.
The lack of appropriate experimental procedures and tools has been one of the bottlenecks in the understanding of how segmented, in particular segmented RNA, viruses assemble and package their genome. The developments of these new tools described are the first in the field to show the sequential order of genome packaging and detection of RNA networks through specific RNA-RNA interactions, critical to packaging and virus replication. These assays enable identification of the signals associated with packaging and sorting of the genome independently of those involved in replication of the genome and virus. These also allow visualization of the RNA making contacts with other genomic RNA through packaging signals. Validation of these systems has ramifications for understanding the assembly and packaging of other segmented viruses including the related reoviruses and rotaviruses as well as agriculturally significant orbiviruses (AHSV and EHDV). The system may also have implication for other segmented RNA viruses, including Influenza and Rift Valley Fever virus, in adapting the system and providing information on RNA packaging and genome assembly and identification of RNA structures responsible for the interactions and signals important to these processes. An understanding of the process of assembly and key signals that regulate encapsidation of the genome will enable basic fundamental research to be translated to the development of safer novel vaccines. The successful outcome of the proposal will involve identification of specific sequences or signals that regulate the mechanism of the selective packaging of 10 different segments into the genome. One of the implications of this potential breakthrough is that it may lead to the creation of novel therapeutics based on targeting the specific sequences engaged in the genome recruitment and assembly.
The importance of the signals involved in packaging and sorting of the genome segments will be identified, it will then can be validated using the reverse genetic system, which was also developed in our laboratory. The ability to delineate and identify signals and structures involved in packaging and genome replication will be a significant advancement in developing highly attenuated vaccines and targeted therapeutics for the orbiviruses and related rotaviruses.