By chance or design: Defining the genome packaging signals for a multi-segmented RNA virus

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 or plant) they must replicate their genome and multiply in order for them 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 they are 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's main aim is to understand how these multiple pieces are incorporated and dissecting the exact manner and signals involved in the process. The project will focus on animal viruses, particularly Bluetongue virus (BTV) of sheep as well as other related viruses (African horse sickness virus and Epizootic haemorrhagic disease of deer virus). BTV is primarily characterized by its genome of 10 pieces of RNA, each representing single gene and each generating specific viral proteins.

We are exceptionally positioned at the forefront of this field of research and our laboratory has developed unique tools, techniques and reagents over the years that are tailored for researching these viruses. Recently we have pioneered two key technologies for BTV, a cell-free reconstitution (assembly) of infectious virus system and a reverse genetics system that make infectious virus "wholly" from synthetic genes.

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 work may have direct effect on animal wellbeing as well as economic consequences.

The signals for RNA packaging versus genome replication in RNA viruses remain ill-defined due to lack of appropriate experimental methods, particularly for those viruses with segmented RNA genomes. Understanding such signals is important as it relates to a number of properties such as viral fitness and host range. Bluetongue Virus (BTV) is a member of genus Orbivirus within the family Reoviridae, which are characterized by a genome of 10-12 segments of linear, double-stranded RNAs (dsRNA). Many of these viruses are pathogenic to animals and some also infect man. Most genome segments represent single genes, generating a total of 10-13 viral proteins and they readily reassort with closely related strains. BTV which is widely spread in many parts of the world including Europe and UK and is responsible for significant economic loss in livestock, has been the subject of extensive studies and consequently significant progress has been made in sequence, biochemical and structural characterisation. However, due to the lack of appropriate tools and assay systems, certain areas of BTV replication cycle remain unclear including the sorting, packaging and order of the 10 ssRNA segments. Recently we described a new in vitro assembly system for BTV that gives rise to infectious virus particles which offers a unique approach to investigate the functions of cis and trans acting sequences in the capture of RNA segments and their acceptance by the polymerase complex as bona fide templates. We now propose to investigate these unknown areas of the replication cycle using the new system together with our established reverse genetics system which allow identifying the replication-competency in vivo.

A significant step forward in understanding the assembly pathway and genome packaging of segmented RNA viruses has been recently made with the development of the molecular tool to reconstitute an infectious Bluetongue virus particle in vitro in Roy's laboratory. The significance of this development was noted with a publication in a high impact journal (PNAS) and numerous news articles in online science sites. The potential impact of the system on vaccine design was also noted in numerous agricultural/veterinary significant news releases. The lack of appropriate experimental procedures and tools has been one of the bottlenecks in the understanding of how RNA viruses, in particular segmented RNA viruses, assemble and package their genome. The development of this new tool is the first time that an architecturally complex particle with a multiple (10 or more) segmented genome has been assembled outside of a cell. The system is such that it enables the signals associated with packaging and sorting of the genome independently of those involved in replication of the genome and virus. Validation of this system has ramification 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 structure 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 signals that regulate the mechanism of the selective packaging of 10 different segments into the genome. One of the ramifications of this potential breakthrough is that it may lead to the creation of novel vaccines that have specific markers, for DIVA (differentiating infected from vaccinated animals) compliance, incorporated into the genome without affecting the assembly or early stages of replication of the virus.

The importance of the signals involved in packaging and sorting of the genome segments that will be identified can be validated using the reverse genetic system (allows recovering viruses from 10 synthetic single-stranded RNAs), also developed within Prof Roy's laboratory. The ability to delineate and identify important RNA signals and structures involved in packaging and genome replication will be a significant advancement in developing targeted therapeutics for the orbiviruses and related rotaviruses.